Investigating Sitosterolemia to Understand Lipid Physiology

Summary

The cholesterol molecule is at the center of the pathophysiology of many vascular diseases. Whole-body cholesterol pools are maintained by a balance of endogenous synthesis, dietary absorption and elimination from our bodies. While the cellular aspects of cholesterol metabolism received significant impetus from the seminal work of Goldstein and Brown investigating LDL receptor trafficking, how dietary cholesterol was absorbed and eliminated was relatively neglected. The identification of the molecular defect a rare human disorder, Sitosterolemia, led to elucidation of a key mechanism of how we regulate the excretory pathway in the liver and in the intestine. Two proteins, ABCG5 and ABCG8, constitute a heterodimeric transporter that facilitates the extrusion of sterols from the cell into the biliary lumen, with a preference for xenosterols. This mechanism explained how dietary xenosterols are prevented from accumulating in our bodies. In addition, this disease has also highlighted the potential harm of xenosterols; macrothrombocytopenia, liver disease and endocrine disruption are seen when xenosterols accumulate. Mouse models of this disease suggest that there are more dramatic alterations of physiology, suggesting that these highly conserved mechanisms have evolved to prevent these xenosterols from accumulating in our bodies.

Introduction

Management of lipids is vital to normal mammalian physiology. As a major class of circulating lipids, elevated serum cholesterol levels have been shown to be a major risk factor for the development of atherosclerosis and cardiovascular disease. Understanding of sterol metabolism dates back to observations of endogenous cholesterol synthesis in herbivores consuming solely non-absorbable plant sterols [1]. These animals, despite a diet of only plant sterols and negligible cholesterol, show no evidence of accumulation of these ‘xeno’ sterols in their bodies, yet contain substantial amounts of cholesterol. Decades later, we now know that cholesterol homeostasis is regulated by the interplay of dietary cholesterol absorption, endogenous cholesterol synthesis, and biliary cholesterol excretion, and the exclusion of non-cholesterol sterols (especially plant sterols) from the body. The rare human genetic disorder of Sitosterolemia (OMIM #210250) has allowed critical insights into these biochemical pathways [2]. The disease is caused by mutations in one of two genes, ABCG5 or ABCG8, on human chromosome 2p21 [3]. Affected individuals may suffer from premature cardiovascular disease, hematologic disorders, endocrine disruption, and possibly liver cirrhosis [3]. Through studies of this disorder, it is now known that the two proteins, ABCG5 and ABCG8, function together as key regulators of sterol trafficking and their normal function is critical for the excretion and elimination of xenosterol and excess cholesterol from the body. This review will focus on Sitosterolemia and how investigations of this disease have increased our understanding of lipid and sterol metabolism.

Background: Dietary Sterols

Sterols are important components of the most eukaryotic cell membranes. The 2-part chemical structure of the sterol molecule consists of a cyclopentane phenanthrene ring nucleus that is similar to steroid hormones and bile acids, and a side chain. Primarily found on the plasma membrane lipid bilayer, sterols interact with phospholipid molecules to maintain cell membrane structure and regulate membrane fluidity [4].

In mammals, cholesterol is the major sterol and is the essential molecular precursor for steroid hormones and bile acids and may be involved in cell signaling. Plant sterols (xenosterols) are unique to plants and are not synthesized in mammalian species [5]. Structurally, plant sterols resemble cholesterol in the same ring nucleus, 3β-hydroxy group, and 8-carbon side chain. However, plant sterols differ in their side chain configuration with an extra methyl constituent on carbon 24 in campesterol (structure not shown), an extra ethyl group on carbon 24 in sitosterol, and an extra ethyl group on carbon 24 along with a double bond between carbons 22 and 23 in stigmasterol (Fig. 1). The alkylated side chains of plant sterols make them more effective at ordering phospholipids and, thus cells containing plant sterols have reduced membrane fluidity compared to those containing cholesterol. There are likely >20 different plant sterol species and >40 xenosterols our diets may expose us to [5].

Figure 1.

A representative set of sterol structures.

The first structure shown is cholesterol, and all other structures are sterols that are xenosterols.

The latter are not synthesized in the human body and most are also considered to be toxic, if they accumulate in the body. The structure of campesterol is not shown.

The relative amounts of cholesterol and plant sterols consumed in the diet depend on their composition in animal fat and vegetable oils. A cholesterol:plant sterol ratio of ∼1 is seen a typical Western diet with daily intakes of approximately 250-400 mg of cholesterol and 200-400 mg of plant sterols [6-8]. Despite comparable amounts of ingested cholesterol and plant sterols, circulating plant sterol levels are less than 0.5 mg/dL as compared to cholesterol levels of ∼42.5-208 mg/dL. Given that humans do not synthesize plant sterols (and have a very limited ability to metabolize them) [9], the substantially lower plasma concentrations of plant sterols is due primarily to a much lower absorption of plant sterols in humans that is approximately one-tenth that of cholesterol [10].

Although the majority of total body cholesterol is derived from de novo cholesterol synthesis [11], it is now evident that dietary absorption significantly contributes to maintaining total body sterol homeostasis [12]. All sterols need to be solubilized in micelles for absorption into enterocytes. Plant sterols effectively compete with, and displace, cholesterol from micelles and can thus decrease cholesterol absorption [10]. The influx of dietary cholesterol regulates the its de novo synthesis. The liver is a key organ in maintaining this balance. Excess body cholesterol is eliminated exclusively by the liver, either by direct excretion as free cholesterol into bile or by its conversion into bile acids for excretion in the bile. The small amounts of dietary plant sterols allowed entry into the body are rapidly excreted by the liver into bile. Thus, humans are equipped with an efficient, discriminating mechanism to retain cholesterol and exclude xenosterols that may be deleterious to the body [13]. Research of the molecular defects leading to the disrupted sterol physiology seen in Sitosterolemia has identified the proteins involved in the selective absorption of cholesterol in the intestine and elimination of plant sterols, as well as excess cholesterol, by the liver.

Sitosterolemia

Sitosterolemia was first reported by Bhattacharyya and Connor in 1973 [2]. They identified two sisters with tendon xanthomas and elevated plasma concentrations of the plant sterols sitosterol, campesterol, and stigmasterol but non-significant elevations in plasma cholesterol. The disease was named β-sitosterolemia due to the finding that sitosterol was the most plentiful plant sterol. The ‘β’ is not necessary as there is no natural ‘α’ enantiomer of sistosterol. Further investigation would identify an autosomal recessive inheritance pattern [14] and a highly-developed mechanism important in dietary sterol trafficking [15-17]. More than 100 cases of the disease have since been reported world-wide, so it a genuine rare disorder [18]. There are more than 20 different known species of plants sterols along with other non-cholesterol sterols found in non-plant species such as shell-fish and these are all retained in subjects with Sitosterolemia [19]. The finding that all non-cholesterol sterols are effectively excluded by the human body highlights an extremely effective mechanism designed to prevent absorption of xenosterols. Discoveries in the study of Sitosterolemia have transformed our understanding of sterol-trafficking conducted by intestinal and hepatobiliary exporters. We now know that a significant amount of sterols are pumped out of the body by ABCG5 and ABCG8, and that xenosterols are preferentially eliminated along with excess cholesterol [20, 21].

Clinical Features and diagnosis

The clinical features that may be seen in Sitosterolemia include premature atherosclerosis, tendon xanthomas, arthralgia/arthritis, endocrine insufficiency, and liver dysfunction [3]. In contrast to adults who typically have mildly elevated total cholesterol, young children affected by the disease may present with markedly elevated plasma cholesterol levels [22]. Hematologic abnormalities, such as hemolysis, thrombocytopenia, and stomatocytosis, are frequently seen in affected individuals and are believed related to the high plasma levels of plant sterols [3]. Of significance, sitosterolemic patients exhibit accelerated aortic atherosclerosis and symptomatic coronary artery disease that can lead to life-threatening myocardial infarction in adolescence and young adulthood [23, 24]. Cases of death from acute myocardial infarction have been reported in children as young as 13 years, with autopsy findings revealing extensively atherosclerotic coronary arteries [23, 25]. There appears to be no sex difference in the predilection for premature obstructing atherosclerosis of the aorta or coronary and carotid vessels in sitosterolemic patients, and aortic valve involvement is not infrequent.

Diagnosis of the disease is confirmed by identifying elevated plasma and tissue levels of plant sterols. Normal humans have barely detectable plant sterol levels that are < 0.5 mg/dL. In contrast, individuals with Sitosterolemia have plant sterol levels greater than 10 mg/dL. Laboratory testing for the accumulation of plant sterols requires GC or HPLC techniques to distinguish between plant sterols and cholesterol. No other known disease has been associated with elevations in plasma plant sterol levels. In some situations, increased plant sterol levels have been seen in patients receiving intravenous soybean based lipid emulsions [26]. Post-mortem tissue accumulation can be seen in almost all tissues with the exception of the brain, demonstrating the important role of the blood-brain barrier in protecting the brain [23]. However, in animal models of sitosterolemia, plant sterol accumulation has been demonstrated [27-29], albeit at much lower levels; the clinical significance of this remains to be elucidated.

Genetics

Sitosterolemia is an autosomal recessive disease [17]. The disease was mapped to the STSL locus on human chromosome 2p21. The STSL locus is comprised of two highly homologous genes, ABCG5 and ABCG8, arranged in a head-to-head organization [30-32]. Only 140 bp separate the initiation sites of the two genes, each gene is made up of 13 exons and 13 introns, are evolutionary conserved and a primordial gene duplication event is hypothesized to have led to the creation of two genes (Fig. 2). Since these genes have been identified from fish to man, their role in a conserved function of regulating whole-body sterol stores seems well-supported [33]. Although transcriptional factors such as LXR are known to regulate expression of these genes, the details of this regulation remain to be elucidated.

Figure 2.

Gene structure of the STSL locus.

The Sitosterolemia locus, STSL, comprises of two highly homologous genes, ABCG5 and ABCG8, that are thought to have arisen by a gene duplication even very early in evolution as this organization is present and conserved from fish to man. There is no classical promoter, the genes being transcribed in an opposite direction and are separated by no more than 140 bp of sequence. Figure adapted from reference [22].

The proteins encoded, sterolin-1 (ABCG5) and sterolin-2 (ABCG8), belong to the ATP-binding cassette family G and act as obligate heterodimers. They act as mutual chaperones during protein synthesis to help attain a final mature stable structure; if one subunit is mutant, the expression of the other is affected, highlighting the obligatory nature [34-36]. Thus, mutations leading to two defective copies of ABCG5 or two defective copies of ABCG8 cause the Sitosterolemia. Parents of affected individuals are obligate carriers for one defective allele, but sufficient normal ABCG5/ABCG8 function (theoretically 50% loss) results in a normal phenotype, although transient rises in plant sterols can be seen when fed margarines fortified with them [37]. The crucial feature of Sitosterolemia is the disturbance of normal pathways that prevent the accumulation of plant sterols and other xenosterols. Discovery of the molecular defect causing Sitosterolemia has identified the long sought-after mechanism that underlies sterol excretion into bile and elucidated the requirement of normally functioning ABCG5 and ABCG8 for sterol elimination from the body. The absence of functioning ABCG5 or ABCG8 leads to hyper-absorption of all no-cholesterol sterols in the intestines and failure to excrete sterols into bile in the liver.

New insights into the pathophysiology of dietary sterol trafficking

Prior to the investigation of this rare disease, the literature and mainstream textbooks either ignored or glossed over how dietary cholesterol was absorbed; the importance of bile salts, emulsification and micelle formation was well-recognized, but how this allowed for the sterols to enter the enterocyte was never elaborated. At the other end, the link between bile acid, phospholipid and cholesterol excretion/secretion into bile was also well-studied, but the mechanism(s) of how cholesterol traversed the membrane and whether this was an active or passive movement was never established, despite the centrality of biliary excretion as a major means to loose whole body cholesterol.

Intestinal Dietary Sterol Trafficking

Once the physiology of the defect in Sitosterolemia came into better focus, at least the role of gene product(s) encoded by the STSL locus was hypothesized, at the very least, to be involved in preventing dietary xenosterols from staying in the body. Early studies of bile from affected subjects hinted that the defect may also affect biliary sterol secretion, but it was clear that clearance of xenosterols was greatly affected [15]. Once the genetic defect was elucidated (with a contemporaneous advance made from studies of rare cholestasis disorders), the picture of a triad of ABC transporters that excreted bile salts (ABCB11), cholesterol (ABCG5/ABCG8) and phospholipid (ABCC4) into bile became much clearer [38], and ultimately supported by studies involving knock-out mouse models. However, how cholesterol was absorbed, as opposed to xenosterols/cholesterol excreted via ABCB5/ABCG8 still remained to be explained. The discovery of a novel drug, Ezetimibe by Davis and his colleagues at Schering-Plough Inc., opened up an avenue; although the molecular target of Ezetimibe (Zetia®) was not known, one hypothesis was that ABCG5/ABCG8 could be its target. This led to a study design to test if the drug was as efficacious in subjects with Sitosterolemia at lowering cholesterol, as it was in normal subjects in phase III studies [39]. Remarkably, not only did the drug work, it lowered plant sterols, and led to an FDA-approved indication for treatment (probably the only ‘blockbuster’ drug approved for a rare indication!). Since the target of Ezetimibe remained elusive, efforts were re-doubled at Schering and in a remarkably innovative effort, using multiple pathways and creativity, they identified another novel protein, the Niemann-Pick C1-like 1 (NPC1L1) [40, 41]. In the space of 4y, how sterols are absorbed and excreted by the mammalian body to regulate whole-body sterol balance protein was established with bona fide molecular targets. Subsequent work has helped solidify these concepts and extended these to include the role of the intestine to also directly excrete cholesterol [42]. Current concepts now embody a scenario whereby dietary sterols are digested by pancreatic cholesterol esterase to yield free sterols, which then compete with each other to gain entry into micelles formed by mixing with biliary cholesterol, phospholipids and bile salts. Failure to form micelles will prevent all subsequent dietary sterol absorption and represents an obligatory step. Mediated by NPC1L1, the micelle content (sterols but seemingly not the bile salts) gain entry into the enterocyte. The exact molecular details of this process are also unclear, but may involve vesicular trafficking involving NPC1L1 [43-45]. Once inside the enterocyte, cholesterol is preferentially esterified via ACAT-2 [46], whereas non-cholesterol sterols remain primarily unesterified, and presumably are pumped back into the intestinal lumen via ABCG5/ABCG8 (See Fig. 3, left-hand panel). A small amount of free xenosterols do end up getting incorporated into chylomicrons (estimated to be <5% of dietary intake) and can be thus secreted into lymph [47]. It should be noted that intestinal ABCG5/ABCG8 may also be responsible for some net excretion of dietary cholesterol at the enterocyte level, thus diminishing intestinal absorption; in Abcg8 knockout mice, studies involving thoracic duct cannulation who that there is increased dietary cholesterol flux (presumably because there is less enterocyte excretion) [48].

Figure 3.

Sterol trafficking pathways.

The process of absorption of dietary sterol absorption begins with secretion of bile containing bile salts, free cholesterol and phospholipids that then mix with dietary sterols to form micelles. These interact with NPC1L1, allowing sterol entry into the enterocyte. ACAT-2 esterifies cholesterol allowing the bulk of this to proceed into chylomicrons and any plant sterols absorbed remain unesterified and are excreted by ACBG5/ABCG8 into the lumen. However, a small amount of plant sterols can make it into chylomicrons. In the blood, chylomicrons are metabolized with progressive loss of triglycerides, relative enrichment of sterols and these remnants are cleared by the liver. Any excess cholesterol, as well as any of the xenosterols are excreted by the liver ABCG5/ABCG8 into bile for elimination. ApoB; apolipoprotein B, ApoE; apolipoprotein E, ACAT-2; Acyl-CoA:cholesterol acyl-transferase, BSEP; bile salt export protein (also ABCB12), HDL: high density lipoproteins, LDL-R; low density lipoprotein receptor, MDR2/3; Multiple drug resistance protein (also ABCB4), MTP; microsomal triglyceride transfer protein, NTCP; sodium/taurocholate co-transporting polypeptide, PL; phospholipid, SR-B1: Scavenger receptor B1. Figure adapted from reference [67].

Hepatic Dietary sterol trafficking

Chylomicrons, containing dietary lipids, are progressively metabolized in the capillary endothelial beds, which remove primarily triglycerides [49] and the sterol-enriched remnants are rapidly cleared by the liver. In the liver, dietary cholesterol can enter the metabolic pathway (re-esterification, re-secretion in VLDL, broken down to bile salts, or can be stored), but xenosterols are preferentially diverted to biliary secretion, via ABCG5/ABCG8 (see right-hand panel, Fig. 3). The net resultant effect is an average daily dietary cholesterol absorption of 55%, but <1% xenosterol absorption [50]. Animal models to mimic sitosterolemia have now been reported and have expanded our understanding of hepatic dietary sterol metabolism. A phenotype in mice that resemble human disease is seen when both ABCG5 and ABCG8 are disrupted [27]. ABCG5/ABCG8 knockout mice demonstrate sterol hyper-absorption and the inability to excrete sterols into bile. Mice with over-expression of human ABCG5 and ABCG8 exhibit super saturation of cholesterol in bile, excretion of large quantities of neutral sterols, reduced plasma plant sterol levels, and a compensatory up-regulation of the cholesterol synthesis rates [51]. These animals also showed lower cholesterol absorption rates and lower plasma HDL cholesterol levels. In addition, over-expression of hepatic ABCG5 and ABCG8 is associated with reduced production of apoB-lipoproteins and atherosclerosis. The liver is also the center of integration of whole-body cholesterol trafficking [52]. Excess whole-body cholesterol, from synthesis in the periphery is sent back to the liver, where it can be directly excreted into bile via ABCG5/ABCG8, or converted into bile salts and excreted into bile via ABCB12. In humans, but not rodents, NPC1L1 is also expressed in the liver and presumably is responsible for re-uptake of biliary cholesterol (perhaps evolved to retain cholesterol in the body). The reader is directed to a recent review of this subject [53].

Xenosterols and the Hematopoetic system

Erythrocytes were known to be able to take up and partition plant sterols into their membranes and hemolysis was a known associated feature of Sitosterolemia (together with splenomegaly). However, much more focus and attention was drawn to this aspect by Rees et al who showed that mutations at the STSL locus were responsible for a clinical condition known as Mediterranean stomatocytosis/macrothrombocytopenia [54]: affected subjects had macrothrombocytopenia. Interestingly, dietary plant oils were shown to be toxic to a well-known in-bred rat line [55], the spontaneously hypertensive rat-stroke prone (SHR-SP), and plant sterols affected the rate of hemolysis of blood isolated from these animals [56]. Both SHR and SHR-SP lines are mutant for Abcg5 and were sitosterolemic when raised on standard rodent chow [57, 58]. Thus plant sterols seem to lead to increased red cell fragility by partitioning preferentially in the outer leaflet of the red blood cell membrane, though the effects on platelets physiology is more complex. Artificially raising plant sterols, as in children receiving total parental nutrition, elevated plant sterols were associated with cholestasis (see below) and thrombocytopenia [26]. Macrothrombocytopenia was further recapitulated in two mouse models; in the Abcg5 knockout mouse line [59] and a naturally-occurring mutation affecting Abcg5 in the trac mouse line [60]. In both of these reports, the effect of phytosterolemia on the megakaryocyte membrane demarcation was felt to be responsible for the phenotype of macrothrombocytopenia, with the trac mouse also exhibiting cardiomyopathy and leukopenia. In both cases, the platelet phenotypes could be rescued by lowering plant sterols, in the case of the Abcg5 knockout mice by the use of ezetimibe [59], and in the trac mouse by using transgenic complementation [60]. Thus plant sterols affect megakaryocytes at the later stages of maturation and in a reversible manner. More recently, phytosterolemia was shown hyperactivate platelets, with constitutive binding of fibrinogen to its αIIbβ3 integrin receptor and internalization, increased generation of platelet-derived microparticles, and changes in the quantity and subcellular localization of filamin [61]. What is not clear is how plant sterols effect these changes; is it by altering cholesterol levels in the membranes, or is it via specific plant sterol species? It is also not clear if there is a particular plant sterol level that needs to be attained to bring about these toxic changes.

Xenosterols and other organ dysfunction

We reported a case of ‘idiopathic liver cirrhosis’ who was diagnosed because of screening of clinical samples who had elevated plant sterols, and DNA analyses showed he had mutations in ABCG8 [62]. Liver transplantation significantly ameliorated his phytosterol levels [62]. Traditional total parental nutrition (TPN) formulae contain significant amounts of phytosterols and while adult use of TPN is rarely associated with liver dysfunction, hemolysis or thrombocytopenia, pediatric use of TPN, especially prolonged use has been reported to cause these conditions [26]. It is not clear if the phystosterols are directly causative, whether these changes are an epiphenomenon or whether the liver damage is multi-factorial with xenosterols as one potential factor.

Case reports indicate that phystosterols can also affect endocrine function [63], supported by in vitro data that the adrenal gland may have decreased function when exposed to plant sterols [64]. Although humans with sistosterolemia are not noted to be infertile, mice with mutations of Abcg5 or Abcg8 exhibit significant infertility, despite normal endocrine organ development [60, 65]. Although these mice also exhibit severely decreased life-spans [60, 65, 66], the cause of death is not clear. Histological changes in cardiac structure have been documented [60, 66], though whether this translates to cardiac dysfunction has not been demonstrated.

Additionally, a profound loss of fat stores in Abcg8 knockout mice has been reported [65]. Infertility, as well as fat loss can be mitigated by blocking dietary sterol entry using ezetimibe or diets low in plant sterols, suggesting all of these toxic effects are reversible [65].

Conclusions

The molecular mechanisms that regulate whole body sterol homeostasis are essential for maintenance of normal health. Investigations of Sitosterolemia, a rare, autosomal recessive sterol storage disorder, have led to the identification of two key molecules, ABCG5 and ABCG8 (sterolins), critical in sterol-regulating pathways. The loss of these proteins allows the accumulation of all of the dietary sterols, but more importantly xenosterols. Clinical, genomic, and animal studies of this disease have greatly increased our understanding of physiological processes involved in regulating dietary sterol absorption and excretion. When this process is defective, xenosterols accumulate (as does cholesterol) and leads to premature atherosclerosis, macrothromobocytopenia, and rarely liver and endocrine dysfunction, highlighting the key concept that we need to regulate sterol pools, and defend against xenosterol accumulation.

Many questions remain regarding how xenosterols lead to pathophysiology; it is not clear if this is a direct toxic effect of one or more xenosterol species, or whether the pathophysiology is an indirect effect by altering cholesterol trafficking. It is also not clear if low level exposure has any cumulative effect on pathophysiology; we are exposed via the diet to xenosterols on a daily basis and, despite normal sterolin function, there is some variation in xenosterols in the plasma. What is clear is that these sterol-regulating proteins have established a key link between diet, genetics, and environment, and the hope is that continued research will bring us closer to uncovering the molecular basis for the connection between diet, atherosclerosis and heart disease.

Executive summary.

Sitosterolemia

• Autosomal recessive rare disorder

• Mutation in ABCG5 or ABCG8 are causative

• Diagnostically elevated plant sterols in blood

• Classically associated with tendon xanthomas and premature atherosclerosis

New Insights

• Xenosterol accumulation affects all organ systems

• Pure Macrothrombocytopenia as only presenting feature

• Can cause liver dysfunction

• Xenosterol can accumulate in the CNS

• May also act as endocrine disruptors