Sphingolipids and Cholesterol

Abstract

Sphingolipids and cholesterol are two lipid partners on cellular membranes where they form specific microdomains, named lipid rafts, which mediate specific cell functions. Sphingomyelin (SM) is one of the major sphingolipids. SM and free cholesterol are also two key lipids on the monolayer of plasma lipoproteins, including chylomicron, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), which participate in lipid transport in the circulation. Thus, sphingolipids and cholesterol play a fundamental role in cell membrane structure and blood lipid transport. In this Chapter we will discuss the relationship between both lipids, on the cell membrane and in the circulation, as well as the impact of such relationship in the development of metabolic diseases.

Sphingolipids and Cholesterol on Cell Membrane

Sphingolipid biosynthesis occurs via the actions of serine palmitoyltransferase (SPT), 3-ketosphinganine reductase, ceramide synthase, and dihydroceramide desaturase to produce ceramide, which is the central substrate for the production of sphingomyelin through two sphingomyelin synthases (SMS1 and SMS2), sphingosine-1-phospate, and glycosphingolipids (1). SPT is the first enzyme for sphingolipid de novo biosynthesis and the deficiency of both of its subunits causes lethality (2).

There is a relationship between cell membrane SM and cholesterol. It is known for a long time that SM addition to cells increased cholesterol synthesis (3). Moreover, SM removal from plasma membrane of cells by treatment with sphingomyelinase, an enzyme that degrades SM, reduced free cholesterol levels on the membrane, through promoting cholesterol translocation from the plasma membrane to the ER (4). SM and cholesterol interact in biological and model membranes (5). SMS1 or SMS2 overexpression in hepatocytes significantly increased the levels of intracellular SM and cholesterol, and decreases cholesterol secretion in liver cells (6). In a cell membrane mimicking study, Pathak and London showed that SM/cholesterol/palmitoyl 2-oleoyl phosphatidylcholine, a mixture similar to that in the outer leaflet of plasma membranes, forms nanodomains at physiological temperatures (7). They also found that Triton X-100 does not induce those domain formation or increase the fraction of the bilayer in the ordered state, although it does increase domain size by coalescing preexisting domains (7). SM’s ceramide moiety interacts hydrophobically with cholesterol sterol-ring systems through hydrogen bonds and van der Waal’s forces (8).

Plasma membrane lipid rafts, enriched with cholesterol, SM, glycosphingolipids, have proven to be involved in cell signaling, lipid and protein sorting, and membrane trafficking (9–11). Studies showed that cellular membranes are not just homogenous mixtures of lipids and proteins, and there are two different phases on cellular membrane: liquid ordered (Lo) and liquid disordered (Ld) phases (12). In Lo phases, fatty acid chains are stretched, but lipids do not adopt hexagonal lateral arrangement. Lipid rafts are considered Lo, not only enriched in cholesterol and sphingolipids such as SM and glycosphingolipids, but also many specific proteins. Non-raft domains of the membrane are considered Ld, which are enriched with non-saturated phospholipids and non-raft proteins (13). Because of the lack of an appropriate fluorescent probe and the spatiotemporal stability of lipid rafts, the direct observation of lipid rafts has been greatly restricted. However, with the development of new microscopic techniques, more and more evidence of lipid rafts based on the partition and dynamic behavior of SM has been obtained (14). SM deficiency in lipid rafts is accompanied by reduced cholesterol content (15). We separated cell membrane SM and cholesterol into two peaks, lipid rafts and non-lipid rafts (based on specific markers), SM is mainly located in lipid rafts(16), while cholesterol is about evenly distributed in lipid rafts and non-rafts (17).

Alterations in lipid rafts could affect cell functions. We reported that SMS2 deficiency significantly attenuated the concentration of toll like receptor 4 (TLR4)-MD2 complex on the lipid rafts of macrophages after LPS stimulation, and SMS2 depletion reduced TNFα-stimulated lipid raft recruitment of TNF receptor-1 in cells (18). Further, we found that deficiency in macrophage sphingolipid de novo synthesis significantly lowered SM levels in plasma membrane and lipid rafts. This reduction not only impaired inflammatory responses triggered by TLR4 and its downstream NFκB and MAPK pathways, but also enhanced reverse cholesterol transport (19). Recently, we showed that hepatocyte plasma membrane SM level is one of the key factors in regulating hepatocyte polarity (20) and sphingolipid de novo biosynthesis is essential for intestinal cell survival and barrier function (21). Changes in lipid rafts could also affect insulin signaling (22). Cholesterol extraction with β-cyclodextrin disrupted caveolae in cultured cells and resulted in inhibition of insulin signaling (23–25). Skeletal muscle membrane cholesterol accumulation is an early, reversible, feature of insulin resistance (26). We demonstrated that deficiency in SM biosynthesis significantly increased insulin sensitivity and we attributed this primarily to the reduction of SM in plasma membrane SM-rich microdomains, and not to plasma membrane ceramide levels (27).

Glycosphingolipids are another important component of lipid rafts on cell membrane (28). Glucosylceramide (GluCer), one of the simplest glycosphingolipids, plays key roles in physiology and pathophysiology(29,30). The deposition or accumulation of GluCer within cells and certain organs results in Gaucher disease (31). Changes in the level of GluCer are noticed in response to cardiovascular disease and diabetes, as seen in Gaucher disease (32). GluCer reorganizes cholesterol-containing domains in a fluid phospholipid membrane (33). Glycosphingolipids in lipid rafts are also important in blocking tyrosine phosphorylation of insulin receptor and down-stream signaling (34–36). Pharmacological inhibition of glycosphingolipid synthesis had markedly improved insulin sensitivity in rodent models with insulin resistance (37). Among glycosphingolipids, gangliosides (including GM1, GM2, GM3, and so on), are well-known components in lipid rafts (36,38). Changes in ganglioside levels are known to affect the expression of raft-associated proteins on the cell surface and lead to reduced membrane fluidity, resulting in cellular dysfunctions, such as impaired signal transduction (38–40). It was reported that GM1 and GM2 contribute to insulin resistance(41). GM3 deficiency enhanced insulin receptor activation (42), and GM3 dissociated insulin receptor/caveolin-1 complex, thus resulting in insulin receptor dysfunctionality (43).

In normal situation, ceramide levels in cell membranes are low (0.1–1 mol % of total phospholipid), however, its levels can be higher under cellular stress conditions, such as apoptosis(44). Previously, researchers have indicated that ceramide level changes in model or cell membranes can induce membrane permeability(45), lipid flip-flop motion(46), and lateral domain segregation (47). Indeed, ceramide can give rise to highly ordered gel-like domains(48), which could serve as a “membrane platform” clustering receptor molecules and mediating signal transduction processes(49,50). It is known that membrane ceramide can be derived from SM by acid sphingomyelinase (51). Recent two studies indicated the heterogeneity of membrane lipid bilayer, composing cholesterol, SM, and ceramide(52,53).

Another important impact of SM and cholesterol interaction on plasma membrane is for cholesterol homeostasis, which ensures optimal cholesterol levels in cellular membranes by precise regulation of its synthesis and uptake (54). About 80% cellular cholesterol is located on plasma membrane, however, cholesterol synthesizing regulator (Sterol regulatory element-binding proteins, SREBPs) (55) are located in the ER membrane, which contains only about 1% of total cellular cholesterol (56). The regulation of cholesterol transport between plasma membrane to ER allows cholesterol sensors in ER to monitor the cholesterol level of plasma membrane (57). This regulation is determined by plasma membrane lipid rafts, which critically depends on the interaction of cholesterol with SM (58). Recently, Endapally et al utilized ostreolysin A (OlyA), a protein that binds to membranes only when they contain both SM and cholesterol, and clearly indicated that SM adopts two distinct conformations in membranes when cholesterol is present. OlyA binds only one conformation which contains both SM and cholesterol and it cannot bind to another conformation where SM is free from cholesterol. In cells, levels of SM/cholesterol complexes are held constant over a wide range of plasma membrane cholesterol concentrations, enabling precise regulation of cholesterol levels (59).

The same group researchers also studied cholesterol homeostasis in the plasma membranes of animal cells using two cholesterol-binding bacterial toxin proteins, perfringolysin O (PFO) and domain 4 of anthrolysin O (ALOD4). They found that cholesterol in the plasma membrane is present in three different pools. The first is an accessible pool that contains mobile cholesterol. Excess cholesterol is transported to ER to terminate SREBP activation and decrease cholesterol synthesis when the cholesterol concentration surpasses a threshold; The second is a SM-sequestered pool. The plasma membrane cholesterol cannot be transported to the ER but can be liberated for transport by SMase treatment; The third is an essential pool. The plasma membrane cholesterol is sequestered by other plasma membrane factors (60). Hence, SM and the ratio of cholesterol to SM have the potential to markedly alter cholesterol trafficking and homeostasis in cells (61,62).

Certain SM-binding toxin proteins were also used to evaluate SM/cholesterol interaction. Lysenin is an earthworm toxin which strongly binds to SM (63,64). Atomic force microscopy results showed that lysenin assembles into a hexagonal close packed structure by rapid reorganization of its oligomers on an SM/cholesterol membrane (65). Lysenin has been used for the study of cell membrane structure. Recently, He et al utilized nanoscale secondary ion mass spectrometry imaging with ¹⁵N-labeled lysenin in combination with ¹⁵N-labeled cholesterol-binding proteins to visualize SM-rich and cholesterol-rich domains in the plasma membrane of CHO cells. Their results revealed that SM and cholesterol are not distributed evenly, but are enriched on the surface of microvilli (66). Sticholysin II is another SM-binding protein. It belongs to the actinoporin family, a group of low molecular weight pore forming toxins that switch from a soluble form to an integral membrane protein without the aid of chaperones and translocons (67). Sticholysin II is able to discriminate among membrane domains with SM with respect to those enriched with gangliosides (68).

Sphingomyelin and Cholesterol in the Circulation

Epidemiological studies have shown a positive relationship between total cholesterol concentrations and mortality from coronary heart disease (CHD) (69,70). Total cholesterol does not accurately predict the risk of CHD in many patients, because it is the sum of all cholesterol carried not only by atherogenic lipoproteins (i.e., chylomicron, very-low-density lipoprotein [VLDL], low-density lipoprotein [LDL], intermediate-density lipoprotein [IDL]), so called “Bad cholesterol” but also by antiatherogenic lipoproteins (i.e., high-density lipoprotein [HDL])(71), so called “good cholesterol”. SM plays an important role in plasma lipoprotein metabolism including lipoprotein production, cholesterol efflux, cholesterol absorption, reverse cholesterol transport, atherogenic lipoprotein aortic retention, and so on (72). Metabolic regulation of SM and cholesterol appear to be inter-coordinated (5,62). Both SMS1 and SMS2 overexpression in mice increases the atherogenic lipoproteins, which are enriched with SM (73).

Apolipoprotein B (apoB)-containing lipoprotein production:

ApoB is the major protein component of chylomicron and VLDL, which are the precursors of chylomicron remnant, IDL, and LDL. Thus, these lipoproteins are also known as apoB-containing lipoproteins (BLp) (74). Chylomicron and VLDL transport triglyceride (TG) from the intestine and liver, respectively, into the bloodstream (75). ApoB exists in two forms, apoB100 and apoB48 (76,77). Overproduction of BLp is a major cause of accelerated atherosclerosis (78,79). The regulation of BLp secretion, which takes place primarily post-transcriptionally (80), is poorly understood. Accumulating evidence suggests that formation of BLp (81–83) is accomplished sequentially. This two-step model postulates that the initial product is a primordial small, dense particle formed during or immediately after apoB translation in the ER. Bulk lipids, most likely TG, phosphatidylcholine, SM, and free cholesterol, are incorporated into the primordial particle to form mature BLp (84). Multiple factors are involved in BLp maturation. Microsomal TG transfer protein (MTP) is involved in an early phase of lipid addition to apoB (85,86). Phospholipid transfer protein (PLTP) may also be involved the first and second stage of VLDL lipidation (87–89). Studies in hamsters suggest that de novo synthesized SM is secreted via the VLDL/LDL pathway in the liver (23;24). Isolated rat hepatocytes secrete SM as a part of BLp (25). Two major carriers of SM in plasma are VLDL and chylomicrons (20)(90). Despite these facts, how SM is deposited in nascent BLp remains to be determined. Two candidate proteins could play a role in this process. First, MTP can transfer SM (91) for deposition in nascent BLp. Second, PLTP can transfer SM (92) and promote BLp lipidation (93). Alternatively, there may be other yet unidentified proteins that specifically transfer SM. Recently, we found that hepatocyte total SMS blocking can reduce VLDL production. This phenomenon could be related with a reduction of atherogenicity (94).

Cholesterol absorption:

Dietary lipid absorption occurs in the lumen of the small intestine and on the apical surface of enterocytes. Niemann-Pick C1-like 1 (NPC1L1) and the ATP-binding cassette transporters G5/8 (ABCG5/8) are the two major factors mediating net cholesterol uptake. The former mediates cholesterol uptake, and the latter mediates excretion of excess cholesterol into the intestinal lumen (95,96). CD36 also participates in cholesterol uptake at the brush border of enterocytes (97,98). CD36 (98) and fatty acid transport protein 4 (FATP4) (99), are involved in free fatty acid uptake by enterocytes. It is conceivable that ablation of SMS may reduce the incorporation of SM in enterocyte plasma membrane lipid rafts, a platform for the above-mentioned transporters (97,100–104), with consequent perturbation of endocytosis and protein recruitment to the plasma membrane, ultimately leading to defective lipid uptake and secretion. It was reported that SM could affect cholesterol absorption in the small intestine (61) and cholesterol uptake by CaCo2 cells (105). SM in diet affects plasma and tissue levels of cholesterol and reduces cholesterol absorption in rodent small intestines (106–109). Co-administration of cholesterol and SM leads to inhibition of absorption of both lipids in rats (106).

Reduction of blood total cholesterol by SM consumption in rats and mice makes dietary SM as a “functional food” (72). However, Ohlsson et al found no significant changes in plasma cholesterol profile after dietary SM feeding in humans (110,111). A relatively well controlled study also indicated that in humans, 1 g/day of dietary SM does not alter the blood lipid profile except for an increased HDL-cholesterol concentration and has no effect on cholesterol absorption, synthesis and intraluminal solubilization compared to control (112).

Effect of SM on cholesterol efflux:

Promoting cholesterol efflux to extracellular acceptors is of great importance in the maintenance of cellular cholesterol homeostasis (113,114). One of the major functional properties of HDL particles is to mediate cholesterol efflux and consequently promote reverse cholesterol transport (RCT) (115). SM is the second most abundant phospholipid and major sphingolipid component of HDL (116). SM content in HDL varies widely (117,118). Studies showed that SM 24:1 was the second most abundant species both in HDL₂ and HDL₃, and SM 16:0 was main species in human HDL and more elevated in HDL₂ than HDL₃ (116,119). Therefore, SM levels of HDL could affect HDL metabolism. It was reported that SM accelerated the formation of reconstituted HDL by ApoA1, modified the size and stability of HDL particle. Complete degradation of SM could increase the rate of HDL₃ cholesterol oxidation (120–124). Moreover, increased SM levels in HDL inhibit the HDL remodeling enzymes lecithin-cholesterol acyltransferase (LCAT)(125) and PLTP activity (126).

ATP-binding cassette (ABC) transporters, including ABCA1 and ABCG1, are involved in HDL-mediated cholesterol efflux (127). Importantly, plasma membrane SM levels influence cholesterol efflux through regulating ABCA1 and ABCG1 (128,129). ABCG1 mediates the efflux of SM and cholesterol from cell membranes depending on their level and distribution in the membrane (130,131). We found that SPT subunit 2-haploinsufficient (Sptlc2^+/−) macrophages have significantly lower SM levels in plasma membrane and lipid rafts. This reduction enhanced reverse cholesterol transport mediated by ABC transporters (19). We also found that SMS1 or SMS2 deficiency promotes cholesterol efflux (128,132). However, there is debate about the role of SM-associated cholesterol efflux (133). This vague understanding is partially caused by the absence of an approach to determine the interaction of membrane SM and cholesterol in one living cell. Recently, by using of luminol electrochemiluminescence (ECL), Huang et al., revealed the codetermination of SM and cholesterol in cholesterol efflux in one living cell (134).

SM and atherosclerosis:

Atherosclerosis progression is initiated by atherogenic lipoprotein accumulation and oxidation (135,136), monocyte recruitment, macrophage foam cell formation and inflammation (137,138). Atherosclerosis regression is mediated by lipid lowering, macrophages cholesterol efflux and form cell effective efferocytosis from the atherosclerotic plaques (138,139). Much evidence indicate that SM content in the aortic wall and in plasma is closely related to atherosclerosis. It is well established that SM accumulates in atheroma formed in humans and animal models (140–145). LDL extracted from human atherosclerotic lesions is much richer in SM than LDL from plasma (146–149). The ratio of SM to phosphatidylcholine (PC) is increased 5-fold in VLDL from hypercholesterolemic rabbits (150,151). We found that plasma SM level in Apoe KO mice, is 4-fold higher than in wild-type (WT) animals (152), and this hypersphingomyelinemia together with hypercholesterolemia contribute to increased atherosclerosis (153,154). We also found that human plasma SM level is an independent risk factors for coronary heart disease (155,156). SM increases from 10% at birth to 48% in patients who had undergone a coronary artery bypass grafting and to 60% in patients who had plaques in their coronary arteries, and SM contributes to atherosclerosis and sudden dearth (157). SM is also associated with increased risk of myocardial infarction (158) and human atherosclerotic plaque inflammation (159). Given that SM is the major sphingolipid in atherogenic BLp, SM biosynthesis should have important impact on BLp production as well as atherogenesis. Our laboratory and others have also discovered that chemical inhibition of sphingolipid biosynthesis significantly decreases plasma SM levels, thus lessening atherosclerotic lesions in Apoe KO mice(160,161).

As suggested by Williams and Tabas, subendothelial retention and aggregation of atherogenic lipoproteins also play a very important role in atherosclerosis (162,163). SM-enriched LDL retained in atherosclerotic lesions is acted on by an arterial wall SMase that promotes aggregation and retention, initiating the early phase of atherogenesis (148). We prepared Sms2 and Apoe double knockout (KO) mice. We found that atherogenic lipoproteins from the double KO mice showed a reduction of their retention in aortas, compared to controls. Importantly, the double KO mice showed a significant reduction in atherosclerotic lesions of the aortic arch and root, compared with controls (164).

Conclusion

Both cholesterol and sphingolipids are essential lipids in mammalian cellular membranes and in the circulation. It is well known that dysregulation of both cholesterol and sphingolipids is related with metabolic diseases, such as insulin resistance, metabolic syndrome, and atherosclerosis. There are a lot of studies investigating the relationship between cholesterol and metabolic diseases or sphingolipids and metabolic diseases. As we discussed in this Chapter, cholesterol prefers to interact with sphingolipids, affecting the structure and function of cell membranes and lipoproteins. On the other hand, sphingolipids, especially SM, are involved in cholesterol metabolism, such as cholesterol absorption and RCT. The investigation of the interaction between cholesterol and sphingolipids, and the impact of such interactions in the development of metabolic diseases should be the direction in the future.

Abbreviation

SM

sphingomyelin

SMase

sphingomyelinase

SPT

serine palmitoyltransferase

SMS

sphingomyelin synthase

BLp

apolipoprotein B-contained lipoprotein

VLDL

very low-density lipoprotein

LDL

low-density lipoprotein

HDL

high-density lipoprotein