Biological membranes are lipid bilayers that are formed by amphipathic molecules that are highly organized in one dimension only. Indeed, the hydrophilic (polar) moieties are always oriented toward an aqueous medium, and the hydrophobic moieties stay together to form the interior of the membrane. However, in the other two dimensions membranes are mostly fluid. The simplest lipids (Fig. 1), such as fatty acids and sterols cannot form membranes by themselves because they are not sufficiently amphipathic, in particular they do not have a sufficiently polar moiety. The simplest lipids capable of forming membranes are phospholipids, which are esters of glycerol with two fatty acids and one molecule of phosphate (Fig. 1). The complexity of the hydrophilic moiety of membrane lipids can be increased by attaching amino acids and sugars to form sphingolipids, cerebrosides, and gangliosides. The complexity of the hydrophobic side can be increased by attaching the fatty acids differently to the glycerol (e.g., as ethers rather than esters) but mostly by varying the nature of the fatty acids that form the side chains: varying their lengths, their pattern of desaturation, or even their branching (Fig. 1). In eukaryotes membranes can also contain sterols, which are rigid amphipathic molecules. Sterols interfere with the packing of the phospholipids and thus contribute to membrane fluidity. Membranes can also harbor bio-active lipid-like molecules such as the ubiquitous ubiquinone (Fig. 1), which is formed by a hydrophilic quinone head and a long isoprenoid side chain, and which is both a co-factor for some essential enzymatic systems, such as the electron transport chain, and a membrane-specific antioxidant. Membranes can contain the isoprenoid side chain (such as farnesyl) of lipidated proteins that are attached to the membrane by their lipid moieties (Fig. 1).
As suggested above, lipids contain a lot of information in their very diverse structures (Sud et al., 2007), but this information is not coded in the same way DNA encodes proteins. Instead, the information is coded into the structure of lipids by the presence, absence, or level, of the biosynthetic enzymes that make them, or that modify dietary lipids. Which brings us to a further characteristics of lipids, which is that they often keep much of their information content after being absorbed and assimilated from the diet. This is true for the pattern of desaturation in polyunsaturated fatty acids (PUFAs), but also for sterols. As long as these molecules are not fully broken down they will retain all or part of their bioactive properties. This seems to be true to a lesser degree for the constituents of other classes of bio-macromolecules such as amino acids, sugars or the constituents of nucleic acids. This characteristic allows for interesting experimental manipulations using the diet to alter the specific lipid content of tissues, or to rescue biosynthetic deficiencies. Of interest, some PUFAs, such as linoleic acid (Fig. 1), are essential nutrients for humans, and must be obtained from the diet.
Many of the interesting functions of lipids stem from the effects lipid composition has on the properties of membranes. For example, the composition and structure of membranes will affect the mechanical properties of cells, such as their shape and their motility. In addition, membranes are the site of many or even of most intra- and inter-cellular signaling processes: receptors and channels in membranes transduce signals from the outside to the inside, protein signaling molecules dock on membranes or move from membranes to the nucleus, the plasma membrane invaginates in various ways to internalize extracellular messages or fuses with intracellular vesicles to release messages into the extracellular space. All of these processes can be influenced by the lipid composition of membranes, which influences membrane curvature (McMahon and Gallop, 2005), or the formation of microdomains (Lajoie et al., 2009), such as lipid rafts.
Independently of their effects on membrane properties, the complexity of lipids allows them to be good signaling molecules themselves. In fact many important hormones, or hormone-like substances, are lipids or are derived from lipids. Notable examples from mammals include the derivatives of arachidonic acid, a 20-carbon PUFA (Fig. 1), which is the precursor of eicosanoids such as prostaglandins, prostacyclins, thromboxanes, leukotrienes, and lipoxins, which are crucially involved in inflammation (Shimizu, 2009). More recently, sphingolipids such as sphingosine, ceramide, and sphingosine-1-phosphate have been recognized as having multiple roles in the cell, including in the regulation of cell growth, apoptosis, inflammation, and intracellular trafficking (Hannun and Obeid, 2008).
Most aspects of lipid biology are conserved in C. elegans. However there are some interesting differences with respect to the biosynthetic pathways (reviewed in Kurzchalia and Ward, 2003; Ashrafi, 2007; Watts, 2009). Similar to mammals, C. elegans contains a wide range of fatty acids. Fatty acids are either obtained from the bacterial diet or are synthesized de novo from acetyl CoA. The enzymes acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS) are sufficient for the production of the 16-carbon palmitic acid (16:0), which, through the activity of seven fatty acid desaturases (FAT-1 to FAT-7), one 3-ketoacyl-CoA reductase (LET-767), and two fatty acid elongases (ELO-1 and ELO-2), can be modified into a wide range of polyunsaturated fatty acids (PUFAs). Unlike mammals, C. elegans is capable of synthesizing linoleic acid (18:2n6) and linolenic acid (18:3n3). In addition, monomethyl branched chain fatty acids (mmBCFAs) are synthesized de novo from a branched chain CoA primer using specialized elongases (ELO-5 and ELO-6). Sphingolipids in C. elegans contain an unusual branched-chain sphingoid base, which is likely synthesized from these mmBCFAs. In contrast to the situation with fatty acids, C. elegans are auxotrophic for sterols as they lack the enzymes for de novo sterol synthesis. Thus, they require cholesterol (or similar sterols such as sitosterol or ergosterol) in their growth medium. However, C. elegans is capable of modifying sterols and even possess sterols with a unique, nematode-specific methylation modification of the carbon ring. Lipid synthesis and modification occurs mostly in the intestine, which is also the main site of lipid storage.
The genetics of fat storage has recently become an area of intense research in C. elegans. In vertebrates, fats are stored in a specialized tissue, adipose tissue. Although worms do not have a tissue dedicated to fat storage, many aspects of regulating fat storage have been found to be conserved. Given that obesity, the presence of excess adipose tissue, predisposes people to a great variety of diseases, and is currently on the rise, this is an important field of study with potential medical applications. We refer the reader to recent reviews on this topic (Ashrafi, 2007; Jones and Ashrafi, 2009; Watts, 2009). Here, we will only discuss a few recent publications where mobilization of fat stores is linked to the survival of the organism. For the remainder of the study, we will be focusing on lipid transport within the organism, and its relevance to human disease, as well as on the diverse roles lipids play in signaling.
In the presence of sufficient food and low population density, C. elegans proceeds through four larval stages before becoming a reproductive adult. During periods of stress, such as high temperature, low food abundance, and in particular overcrowding, C. elegans can enter an alternative developmental stage, the dauer stage, until favorable conditions are encountered (reviewed in Hu, 2007). Dauer larvae have increased fat reserves, are nonfeeding, stress-resistant, and capable of surviving for several months. The shift to dauer is regulated by several conserved signaling pathways, including an insulin-like signaling pathway and a transforming growth factor-beta (TGF-β) pathway. Mutations that severely decrease signaling through these pathways lead to dauer formation even under favorable conditions and milder reductions lead to animals with some dauer-like characteristics, and increased fat reserves. As the shift to dauer is accompanied by a metabolic shift, which includes a shift to fatty acid oxidation and glyoxylate based metabolism, survival in the dauer stage likely depends on being able to use stored lipids.
A recent study shows that in fact the ability to regulate lipase activity to maintain lipid stores is also crucial for dauer survival (Narbonne and Roy, 2009). Dauer larvae with reduced AMP-activated kinase (AMPK) signaling do not exhibit long-term survival and rapidly hydrolyze their lipid stores. Reducing the expression of the adipose triglyceride lipase (ATGL) homologue by RNAi, blocks the abnormally fast lipid hydrolysis and restores the dauer lifespan. While we typically think of the intestine as the main site of fat storage in C. elegans, both the AMPK signaling pathway and ATGL exert their effects by acting in the hypodermis, another site of fat storage. However, the ability to hydrolyze lipids in the intestine appears to be crucial for survival during another condition: starvation of worms that have undergone the normal, nondauer, developmental pathway. Indeed a recent study identified two lipases (named fil-1 and fil-2), which are related to ATGL and which are induced during starvation (Jo et al., 2009). These lipases are regulated by a pathway involving IRE-1, an ER protein known to be involved in the unfolded protein response, and its chaperone (BIP/HSP-4). In the absence of IRE-1 or HSP-4 activity, fil-1 and fil-2 are not induced and fat stores in the intestine are not mobilized during starvation. This leads to decreased motility and decreased long-term survival. Thus, these two studies suggest that survival during nonfeeding conditions depends on maintaining a fine balance of storing and hydrolyzing lipids in the hypodermis in one case and in the intestine in another, which is regulated by the controlled activity of distinct lipases.
In addition to playing a specific role for survival during nonfeeding conditions, lipases may also regulate longevity under more normal circumstances. A recent study found that the C-terminal binding protein (CtBP), a conserved NAD(H)-dependent transcriptional corepressor, modulates lifespan in C. elegans, by means of the lipase lips-7 (Chen et al., 2009). Reduction of ctbp-1 activity leads to an increase in lifespan as well as an increase in lips-7 transcript levels and a decrease in TAG levels. RNAi knockdown of lips-7 in ctbp-1 mutants restores TAG levels to that of the wild-type and suppresses the increased lifespan, suggesting that ctbp-1 regulates lifespan, at least in part, by regulating TAG levels. Altered lipase activity has also been linked to longevity in another recent study (Wang et al., 2008). Here, the authors found that mutants with defects in germline stem cell proliferation had decreased fat stores in the intestine and increased longevity. Both effects appear to be achieved by the action of yet another lipase (K04A8.5) acting in the intestine. Although it is not yet clear why reducing fat stores leads to an increase in lifespan, it is possible that the presence of germ cells signals for fat to be stored because developing oocytes will require fat reserves in the form of yolk (see below). These studies suggest that regulating lipases may be a very general mechanism for coordinating fat metabolism with energetic needs.
LIPID TRANSPORT THROUGH THE ORGANISM
Lipoproteins have been extensively studied in mammals, where they are the means by which lipids, phospholipids, triglycerides, free cholesterol, cholesterol esters, and lipid-soluble vitamins, are transported from the gut where they have been absorbed to other tissues (Blasiole et al., 2007). Lipoproteins also serve to distribute lipids, in particular cholesterol, from the liver to peripheral tissues, and from peripheral tissues back to the liver in a process termed reverse cholesterol transport (RCT; Duffy and Rader, 2009). This process has been studied thoroughly because of the relationship between the levels of some circulating lipoproteins and atherosclerosis (see below; Lusis, 2000). The best known lipoproteins in C. elegans are the yolk particles. The protein moieties of yolk particles are vitellogenins, conserved proteins that fulfill this function in both invertebrates and vertebrates. In C. elegans there are 5 genes that code for vitellogenins (vit-2 to vit-6; Spieth and Blumenthal, 1985). In mammals, which do not provision eggs with yolk, there are no vitellogenins. However, apo-lipoprotein B (ApoB), a distant homologue of vitellogenins (Smolenaars et al., 2007), is the major protein in three types of lipoprotein particles: chylomicrons, which are secreted from the gut, very low density lipoprotein (VLDL), which are secreted from the liver, and low density lipoprotein (LDL), which is what VLDL become after they have released some of their lipid cargo (Blasiole et al., 2007). In vertebrates there is also another class of lipoproteins, the high density lipoproteins (HDL), which participate in RCT (Duffy and Rader, 2009). However homologues, or analogues, of the apo-lipoproteins of HDL, such as apo-lipoprotein A (ApoA), have not yet been found in C. elegans.
Yolk Secretion and Uptake
In C. elegans, cholesterol, fatty acids, and possibly other nutrients are transported to developing oocytes by means of yolk particles (Grant and Hirsh, 1999; Matyash et al., 2001; Kubagawa et al., 2006; Fig. 2). Yolk is assembled in the intestine of hermaphrodites, secreted into the body cavity, transported into the gonad and then taken up by late stage oocytes (Kimble and Sharrock, 1983). Although nothing is known about the process of yolk assembly and transport, the process by which it is taken up by oocytes has been well studied. Using a vitellogenin protein tagged with gfp, Grant and Hirsh showed that yolk is taken up by oocytes by a conserved pathway of receptor-mediated endocytosis. They identified RME-2, a member of the lipoprotein receptor superfamily, as the yolk receptor. In the absence of endocytic proteins and in particular, of RME-2, yolk accumulates in the body cavity rather than in oocytes. This results in abnormal oocytes, low production of embryos and very low viability of embryos (Grant and Hirsh, 1999). Until relatively recently, this was the only lipid transport system described in C. elegans. However, several observations hinted that there must be other transport systems. For example, hermaphrodites are capable of transporting cholesterol before the vitellogenins are expressed and males do not express vitellogenins yet accumulate cholesterol in developing sperm (Kimble and Sharrock, 1983; Blumenthal et al., 1984; Schedin et al., 1991; Matyash et al., 2001).
Secretion of Other Lipoproteins
One of the activities required for producing and secreting lipoproteins in mammals is the microsomal triglyceride transfer protein (MTP; Shoulders and Shelness, 2005). MTP is an ER protein that helps both to fold the very large ApoB protein and to load it with lipids, before its travels through the rest of the secretory pathway. The MTP homologue in C. elegans is encoded by the gene dsc-4 (Shibata et al., 2003). DSC-4/MTP does not seem to be required for yolk production as mutants have no defects in oocyte maturation or embryo production, in contrast to rme-2 mutants. Yet, disruption of dsc-4 by mutation or RNAi has several phenotypic effects, including on the rate of development of the germline and on the defecation cycle length, a behavioral rhythm that is driven by the physiology of the gut (Branicky and Hekimi, 2006). This suggests that DSC-4/MTP is required for the secretion of a type of lipoprotein that is distinct from yolk and that might resemble mammalian VLDL/LDL in that it would serve to transport lipids between tissues (Fig. 2). It remains unclear, however, what the core apoprotein of this hypothetical lipoprotein might be. One possibility is that DSC-4 folds and lipidates vitellogenins, but into a particle that is distinct from yolk. Indeed human MTP has been found to be able to act upon a Xenopus vitellogenin (Sellers et al., 2005). Moreover, we have previously reported that RNAi knockdown of some vitellogenins, in particular vit-5, has phenotypic effects on the germline that are similar to those of dsc-4 mutations (Shibata et al., 2003). Alternatively, as DSC-4/MTP is evolutionarily related to vitellogenins and ApoB (Smolenaars et al., 2007), it might be the apo-protein itself. One way to explore this possibility in the future, would be to determine whether dsc-4 has some role in males and larval hermaphrodites, which transport cholesterol, yet do no express vitellogenins.
In contrast to the yolk particle, nothing is known about how the MTP-dependent lipoprotein particle may be taken up by cells. However, in addition to RME-2, the worm expresses several receptors that resemble vertebrate LDL receptors, including LRP-1, LRP-2, and others. It also has receptors that resemble the vertebrate scavenger receptor SR-BI, which also recognizes lipoproteins (see below). Thus, there could be one or several receptors involved in the MTP-dependent pathway.
dsc-4 was originally identified as a mutation that suppresses the slow defecation and the slow germline development of clk-1 mutants (Branicky et al., 2001). CLK-1 is a mitochondrial enzyme that is necessary for the biosynthesis of ubiquinone, and clk-1 mutants are auxotrophic for ubiquinone. Several observations suggest that dsc-4 suppresses clk-1 by reducing the level of secretion of a type of lipoprotein. For example, reducing dietary cholesterol levels in mammals reduces the plasma levels of ApoB-based lipoproteins. Similarly, reducing cholesterol intake in worms mimics the effects of dsc-4 on both germline development and defecation (Shibata et al., 2003; Hihi et al., 2008).
Why would a reduction in lipoprotein secretion suppress the phenotypes of clk-1 mutants? In both worms and mice, clk-1 mutants appear to have low cytoplasmic oxidative stress (Kayser et al., 2004; Stepanyan et al., 2006; Yang et al., 2007; Lapointe and Hekimi, 2008). In mammals, increasing cytoplasmic oxidative stress by knockout of the CuZn cytoplasmic superoxide dismutase SOD1 leads to decreased ApoB secretion (Uchiyama et al., 2006), because oxidatively damaged lipoprotein particles are recognized, retained, and eliminated before secretion (Brodsky and Fisher, 2008). In clk-1 mutants, increasing oxidative stress by knocking down SOD-1 suppresses the same phenotypes as dsc-4 mutations and low dietary cholesterol (Shibata et al., 2003). This leads to a model in which the low cytoplasmic oxidative stress in clk-1 mutants increases the secretion of DSC-4/MTP-dependent lipoproteins, and any intervention, e.g., low dietary cholesterol, decreased MTP function, or increased oxidative stress, that can reduce the secretion of MTP-dependent lipoproteins, suppresses clk-1 mutants (Fig. 3). However, it is not currently known why high levels of lipoprotein slow down germline development and defecation rate in C. elegans.
It has been suggested that the beneficial health effects of consuming dietary omega-3 PUFAs might result in part from an elevation of oxidative stress in the Golgi that results in lower VLDL secretion (Pan et al., 2004). Of interest, alterations in PUFA biosynthesis in C. elegans resulting from mutations in fat-3, in elo-1, and in elo-2 also affect the defecation cycle (Kniazeva et al., 2003; Watts et al., 2003). It is tempting to speculate that these mutations act in part by affecting lipoprotein levels by affecting their susceptibility to oxidative damage.
A C. elegans Model of Dyslipidemia
As mentioned above, lipoprotein metabolism has been studied in great details in mammals and in particular in people because of its importance for health. Indeed, pathologically high levels of circulating LDL (dyslipidemia) are a serious risk factor for the development of atherosclerosis (Lusis, 2000). One of the mechanisms at play is the uptake of LDL by macrophages in the wall of blood vessels where they initiate a deleterious processes that leads to the formation of atherosclerotic plaque. The process of LDL-cholesterol uptake by macrophages appears to be a scavenging process by which LDL particles that have been damaged by oxidation or other processes are recognized and removed from the circulation. High intake of fat and cholesterol boosts the level of LDL, whereas reducing cholesterol intake, or pharmacologically reducing synthesis or transport can lower circulating LDL and have beneficial effects. After MTP was found to be necessary for VLDL formation pharmacological MTP inhibitors have been developed but have not yet reached the market as drugs (Burnett and Watts, 2007).
The other type of lipoprotein that is important for human health is HDL. High levels of HDL help to transport cholesterol from peripheral tissues to the liver where HDL is taken up by means of the scavenger receptor SR-BI, and where cholesterol can be eliminated through metabolization and excretion (Duffy and Rader, 2009). Increased HDL levels in people are associated with better prognosis for atherosclerosis, although the best ways and the potential benefits of elevating HDL pharmacologically are still being debated. One type of drug that is being studied for this purpose are agonists of certain nuclear hormone receptors such as LXR or PPARα that can alter the balance of the entire process to favor reverse cholesterol transport, resulting in lowered LDL and increased HDL (Duffy and Rader, 2009).
There appears to be a parallelism between the pathway from dietary cholesterol to atherosclerosis in people and the pathway from dietary cholesterol to effects on defecation rate and germline development in C. elegans (Fig. 3). As described above, alterations in MTP activity, cholesterol uptake and oxidation all affect LDL levels in people and defecation rate and germline development in C. elegans. Furthermore, the regulation of lipid transport appears to be sufficiently well conserved between humans and C. elegans that drugs that have been developed for humans can act as suppressors of the slow defecation rate of clk-1 (Hihi et al., 2008). Indeed, by testing a collection of drugs we found that classic LDL-lowering drugs such as statins, gemfibrozil, and others, could suppress clk-1 mutants. Moreover, previously uncharacterized compounds that were found to suppress the slow defecation rate of clk-1 in a high throughput compound screen were subsequently found to reduce ApoB secretion from cultured human cells and reduce plasma lipoprotein levels in mice (Hihi et al., 2008). It is interesting to note that agonists of LXR and PPARα, and an inhibitor of SR-BI that elevates HDL levels in rats (Nieland et al., 2007), were all capable of suppressing clk-1. This suggests that a process that is antagonistic to VLDL secretion, and that is akin to reverse cholesterol transport, also exists in worms (Fig. 3).
Statins are currently the main treatment used for lowering cholesterol in people. Statins inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which converts HMG-CoA into mevalonate, the rate limiting step in the biosynthesis of cholesterol. Mevalonate is converted into isoprenoids, which, in addition to cholesterol, are precursors for several important molecules such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which prenylate proteins, as well as heme, dolichol, and ubiquinone. Statin treatment leads to a dramatic decrease in circulating LDL-cholesterol in part because the liver cells react to decreased endogenous cholesterol synthesis by up-regulating the expression of the LDL receptor on liver cells (Ma et al., 1986). Statins may also have beneficial effects which are independent of their effect on cholesterol biosynthesis (Albert et al., 2001; Ray and Cannon, 2004). Although C. elegans lacks the enzymes required for the branch of the pathway that converts isoprene into cholesterol (Entchev and Kurzchalia, 2005), we found that statin treatment was effective at suppressing clk-1 (Hihi et al., 2008). As HMG-CoA reductase is present in C. elegans, we speculated that the effect could be due to some other mevalonate-dependent process. A recent study examining in detail the effects statins have in C. elegans (Morck et al., 2009) reports that statin treatment leads to larval arrest as a consequence of a reduction in protein prenylation. It also induces an unfolded protein response in the ER, suggesting that the lack of prenylation leads to an accumulation of unfolded proteins. As these phenotypes overlap with those produced by RNAi knockdown of other enzymes involved in protein prenylation and can be rescued by mevalonate, they appear to be caused by inhibition of HMG-CoA reductase. Importantly, Morck et al. do not observe a decrease in lipid content. Possibly then, statin treatment suppresses clk-1 by affecting LDL secretion only indirectly or by affecting an entirely different process.
Other Pathways of Lipid Transport
It is worth mentioning here that additional pathways of lipid transport have been identified in specific contexts. For example, ndg-4, nrf-5, and nrf-6 mutants have pale eggs, suggesting decreased lipid stores, are defective in transporting fatty acids, and are resistant to the effects of fluoxetine, a drug that inhibits re-uptake of released serotonin (Choy and Thomas, 1999; Choy et al., 2006; Watts and Browse, 2006). ndg-4 and nrf-6 are expressed in the intestine and encode novel multipass transmembrane proteins; nrf-5 encodes a secreted lipid binding protein similar to mammalian cholesterol-ester binding proteins (Choy and Thomas, 1999; Choy et al., 2006). These proteins have been proposed to act in a pathway required for the transport of small lipophilic molecules such as fatty acids and fluoxetine (Watts and Browse, 2006).
The worm homologues of the Neimann-Pick type C protein (NPC1) appear to function in an intracellular lipid transport pathway. NPC1 has been implicated in intracellular cholesterol transport in humans. Mutants of the C. elegans NPC1 homologues, ncr-1 and ncr-2, have dauer phenotypes caused by a lack of dafachronic acid production (Li et al., 2004; Motola et al., 2006; Patel et al., 2008; see below), and ncr-1 mutants are sensitive to cholesterol deprivation and progesterone, an inhibitor of intracellular cholesterol transport (Sym et al., 2000; Li et al., 2004). In addition, it has recently been shown that NPC1 can functionally substitute for NCR-1 in C. elegans, suggesting that these proteins indeed carry out the same function in intracellular transport (Smith and Levitan, 2007). Possibly, ncr-1 and ncr-2 act intracellularly to distribute cholesterol derived from the extracellular MTP-dependent lipoprotein, as suggested by findings in vertebrates (Chang et al., 2006), but this has not been tested in C. elegans.
C. elegans uses a variety of lipid-based signaling molecules. The two best studied classes of molecules are derived from lipids: dafachronic acid, which is derived from a sterol (Held et al., 2006; Motola et al., 2006), and which acts as a hormone to promote reproductive growth (Gerisch et al., 2007), and the dauer pheromone, which promotes entry into the dauer stage (see below; Fig. 4). Work from a large number of studies has elucidated a signaling pathway that acts downstream of the insulin-like and TGF-β signaling pathways to promote reproductive growth. Under favorable conditions dafachronic acid (DA) is produced and binds to the nuclear hormone receptor DAF-12 (which is related to the vertebrate vitamin D and LXR nuclear hormone receptors [Antebi et al., 2000]), which promotes transcription of target genes required for reproductive growth (Shostak et al., 2004) and correct developmental timing (Bethke et al., 2009). Under unfavorable conditions dafachronic acid is not produced and unliganded DAF-12 associates with its co-repressor DIN-1 (a homolog of the mammalian SHARP [SMRT/HDAC1-Associated Repressor Protein] corepressor), which promotes entry into the dauer stage (Ludewig et al., 2004).
Although it was known from genetic and cholesterol deprivation experiments that the ligand for DAF-12 was sterol-derived and required the activity of the cytochrome P450 DAF-9 (Gerisch et al., 2001; Jia et al., 2002; Gill et al., 2004; Matyash et al., 2004), the exact identity of the ligand was described only recently (Held et al., 2006; Motola et al., 2006). Two oxidized derivatives of cholesterol that differ in the position of an unsaturated double bond at C-4 or C-7 of the steroid nucleus (named Δ4- and Δ7-Dafachronic Acid; Fig. 1) were shown to bind to and activate DAF-12 and to bypass the requirement for DAF-9. Two other enzymes that are involved in this pathway in addition to DAF-9 have now also been described: DAF-36 (Rottiers et al., 2006), which is related to “Rieske” oxygenases of plants and bacteria, and HSD-1 (Patel et al., 2008), which is homologous to 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase, a key steroidogenic enzyme in vertebrates. Interestingly, it appears that the different enzymes might be required in different tissues, DAF-9 and HSD-1 in the XXX cells, a pair of neuron-like cells in the head of the animal (Ohkura et al., 2003; Patel et al., 2008) and DAF-36 in the intestine (Rottiers et al., 2006; Fig. 4). Rottiers and colleagues suggest that the distributed biosynthetic steps might allow for the different organs involved, maybe the gut in particular, to register their physiological state for the crucial decision to engage or not in dauer larva development.
Although the identity of the hormone is not known, there are many observations that suggest that molting in C. elegans, the process by which a new cuticle is synthesized and the old cuticle shed, is also regulated by a steroid hormone. In insects, molting is regulated by the hormone ecdysone. Although no clear ecdysone receptor exists in C. elegans, cholesterol deprivation produces a molting defect similar to that observed by disruption of the nuclear hormone receptor nhr-23, the C. elegans homolog of a Drosophila orphan nuclear hormone receptor that is induced by ecdysone (Kostrouchova et al., 1998, 2001). Mutation of another nuclear hormone receptor, nhr-25, also result in molting defects (Gissendanner and Sluder, 2000). Mutation of let-767, which encodes a 17-β-hydroxysteroid dehydrogenase homolog, results in molting defects which are enhanced by cholesterol deprivation, suggesting that it could be directly involved in producing a steroid hormone required for molting (Kuervers et al., 2003).
Of interest, a pathway involving lipoprotein receptors also appear to be required for molting. For example, mutation of lrp-1, the worm homolog of megalin/gp330, a mammalian protein with homology to the LDL receptor, results in a molting defect, which is enhanced by cholesterol deprivation (Yochem et al., 1999). RNAi knockdown of dab-1 (Disabled) and imp-2 (an IMPAS family member), two other genes that appear to act in a pathway with lrp-1, result in similar molting defects (Kamikura and Cooper, 2003; Grigorenko et al., 2004). Possibly, the hormone for molting is synthesized from lipoprotein-derived sterols, or molting itself requires lipoprotein-derived sterols.
Monomethyl Branched Chain Fatty Acids
Although monomethyl branched chain fatty acids (mmBCFAs) can be found in eukaryotic membranes, their role is not known. C. elegans synthesizes two mmBCFAs (the 15-carbon C15ISO and the 17-carbon C17ISO; Fig. 1) de novo using two fatty acid elongation enzymes: elo-5, which is required for production of both C15ISO and C17ISO, and elo-6 which is required for synthesis of C17ISO (Fig. 1). Knocking down elo-5 by RNAi or by mutation leads to a lack of C15ISO and C17ISO and developmental arrest at the first larval stage. Supplementation with C17ISO rescues the developmental arrest, suggesting that C17ISO is essential for growth In C. elegans (Kniazeva et al., 2004, 2008). Similarly, knocking down let-767 by RNAi, leads to an L1 arrest phenotype that can be rescued by mmBCFAs (Entchev et al., 2008). This suggests that let-767 has an essential role in mmBCFA synthesis in addition to the possible role in hormone production described above.
What essential function could mmBCFAs be fulfilling in C. elegans? To address this question, Seamen et al. carried out a screen to find mutations that could suppress the growth arrest of elo-5 mutants (Seamen et al., 2009). Surprisingly, they identified mutations in tat-2, a P-type ATPase. Even more surprisingly they found that tat-2 mutants suppress the growth arrest without restoring C15ISO and C17ISO synthesis. In addition, they found that tat-2 mutations could suppress the phenotypes caused by RNAi knock-down of sptl-1, which disrupts sphingolipid biosynthesis. Sphingolipids in C. elegans contain an unusual branched-chain C17 sphingoid base, which requires the C17ISO precursor synthesized by elo-5 and let-767. Thus, the authors propose that TAT-2 antagonizes the function of C17ISO by affecting the localisation of lipids that mediate the function of C17ISO, such as sphingolipids. Sphingolipids play numerous roles in the cell, and affect multiple signaling pathways (Hannun and Obeid, 2008). Possibly tat-2 mutations suppress C17ISO and sphingolipid deficiency by affecting compensatory signaling pathways.
Most worm researchers are familiar with dauer pheromone, which regulates entry into the dauer stage. C. elegans constitutively releases dauer pheromone, which serves as a signal of population density: high concentrations of dauer pheromone signal high population density and promote entry into the dauer stage (Fig. 4).
Although the existence of the dauer pheromone has been known since the 1980s, it took more than two decades to purify and characterize it biochemically. The first small molecule compound identified was named Daumone (Jeong et al., 2005). Daumone is an ascaroside (also referred to as ascr#1), composed of a glycone moiety (the 3,6-dideoxy sugar ascarylose, named after Ascaris, from which it was initially isolated) and a short fatty acid chain (Fig. 1). Two additional, more potent dauer-inducing ascarosides were subsequently identified (ascr#2 and ascr#3; Butcher et al., 2007). From the work of the Paik, Clardy, and Schroeder laboratories, more than ten ascarosides (named ascr#1- ascr#9) have now been isolated (reviewed in Edison, 2009). The ascarosides differ in the lengths of carbons in the fatty acid chains as well as in modifications to the fatty acid chain and the glycone moiety. Of interest, in addition to acting as dauer pheromones, ascarosides also act in other aspects of inter-worm signaling.
Pheromones are well-known for their roles in sexual attraction in many species. It has been shown that C. elegans hermaphrodites produce a signal to which males are attracted (Simon and Sternberg, 2002). However, the nature of the signaling molecules was unknown. A recent study suggests that certain subsets of the ascarosides in fact fulfill this role (Srinivasan et al., 2008). Srinivisan and colleagues showed that ascr#2, ascr#3 and an additional ascaroside, asc#4, act synergistically as a potent male attractant at picomolar concentrations. At much higher concentrations, ascr#2 and ascr#3 promote dauer entry and are no longer male attractants.
Pheromones also serve as a way for hermaphrodites to perceive the presence of other hermaphrodites. Natural isolates of C. elegans can be classified as either aggregating (i.e., they form aggregates of worms in the presence of food) or nonaggregating. Different variations of the neuropeptide Y-like receptor are largely responsible for this difference (de Bono and Bargmann, 1998). Of interest, nonaggregating strains have been found to be repelled by ascarosides, whereas aggregating strains are attracted to them. Three ascarosides (ascr#2, ascr#3, and ascr#5) appear to directly activate the neurons that promote aggregation (Macosko et al., 2009).
Although ascarosides play diverse roles in signaling the presence of other worms, a recent study suggests that the process of ascaroside biosynthesis itself plays an important physiological role (Joo et al., 2009). Two enzymes involved in the peroxisomal β-oxidation of very long chain fatty acids (VLCFAs), DAF-22 and DHS-28 (homologous to D-bifunctional protein and sterol carrier protein x, respectively) are necessary for ascaroside production as they are required for producing the fatty acid side chain (Butcher et al., 2009; Joo et al., 2009). daf-22 and dhs-28 mutants are defective in peroxisomal β-oxidation, and therefore in the production of short chain fatty acids (SCFA) derived from the VLCFAs. They accumulate massive amounts of VLCFAs and very long chain acyl CoAs, which Joo and co-workers show to be toxic. These authors go further by suggesting that the later steps of daumone biosynthesis, the conjugation of the SCFA to ascarylose, might be a necessary detoxification process. Although it is not presently clear why it would be necessary or advantageous to conjugate these SCFAs to ascarylose for excretion rather than to metabolize them further, this hypothesis raises some interesting possibilities about the primary role of ascarosides. In some of the examples above, it appears as though C. elegans release ascarosides to attract other worms. But in most circumstances it is not clear why a self-fertilizing hermaphrodite would want to release signals to attract other worms, which may then compete for resources. Thus, worms may indeed be using ascarosides to detect other worms, even though these molecules are not released by worms for the purpose of attraction. Moreover, if ascaroside excretion is necessary for detoxification, ascaroside release would not easily be regulated by the worms that produce them, and they would thus serve as reliable indicators of the presence of other worms. This seems like an important property for an environmental cue that brings about dramatic physiological changes, and fits well with the role of ascarosides in dauer formation. In this light, the primary role of ascarosides seems to be less like that of pheromones and more similar to that of bacterial molecules that allow for quorum sensing. The secretion of these molecules, which are sensed by other bacteria that in turn respond by changing their physiology, might also be primarily secreted for the benefit of the cells that secrete them, independently of the use other cells make of the signal (Schertzer et al., 2009).
Polyunsaturated Fatty Acids (PUFAs)
Molecules derived from polyunsaturated fatty acids can also serve as signaling molecules. A recent study shows that in C. elegans PUFA-derived signals are required in oocytes to attract sperm (Kubagawa et al., 2006; Fig. 2). During mating, C. elegans males inject sperm into hermaphrodites. The amoeboid sperm crawl through the uterus to reach the spermathecae, the organ where sperm is stored and where fertilization of oocytes occurs. Loss of specific PUFA biosynthetic enzymes in the hermaphrodite, such as FAT-2 and FAT-3 (the Δ12-desaturase and Δ6-desaturases, respectively), which are required for generating 18- and 20-carbon PUFAs, results in decreased motility of sperm in the reproductive tract. Loss of the LDL receptor RME-2 also results in decreased motility of sperm, suggesting that receptor mediated endocytosis is required to transport PUFAs from the intestine into maturing oocytes, by means of the yolk particle. Presumably, PUFAs are then modified in the oocyte into a signaling molecule which upon release attracts sperm toward the site of release (the oocyte) and into the spermathecae. This is reminiscent of some signaling events in the regulation of mammalian immunity and inflammation where lipid mediators are pervasive in signaling, including to motile cells (Shimizu, 2009). For example, during immune responses in asthma (pulmonary allergic inflammation), the 20-carbon PUFA-derived signaling molecules eicosanoids, leukotriene B(4), and prostaglandin D(2), are generated at sites of inflammation and act to direct T cells to the airways (Luster and Tager, 2004). Thus, PUFAs may act generally as precursors of signaling molecules involved in the attraction of motile cells.
PUFA-derived signaling molecules may also have other functions in the germline. Watts and Browse have reported that supplementing the diet of C. elegans with a specific PUFA, the long chain omega-6 polyunsaturated fatty acid dihommogamma-linolenic acid (20:3(n-6), DGLA) causes sterility (Watts and Browse, 2006). In larvae it induces germ cell degeneration and in adults in induces excessive programmed cell death. Although it is not clear why this particular PUFA has such detrimental effects, possibly it is converted into a signaling molecule that acts to suppress germ cell proliferation.
Some Less Direct Effects of Lipids on Signalling
In addition to acting as signaling molecules themselves, lipids can also affect signaling by modifying other signaling molecules. As mentioned in the introduction, some proteins are attached to membranes by means of lipid moieties such as farnesyl. As in other systems, C. elegans RAS requires farnesylation for its attachment to the membrane, and hence for its activity. Interventions that prevent farnesylation, such as treatment with manumycin A, an inhibitor of farnesyl transferase, decrease Ras signaling (Hara and Han, 1995). Manumycin A treatment also induces the UPR response, similar to the statin treatment that we mentioned above (Morck et al., 2009). This suggests that at least part of the effects of HMG-CoA reductase inhibition result from a decrease in the prenylation of proteins such as RAS.
The activity of membrane channels can also be affected by lipids. In some cases, lipids may affect channel activity by altering the membrane environment of the channel. For example, touch sensitivity in C. elegans requires cholesterol (Huber et al., 2006). Touch is transduced by a multi-subunit complex that includes the pore forming subunits, MEC-4 and MEC-10, which are degenerin/epithelial Na+ channel (DEG/ENaC) proteins, and auxiliary subunits such as MEC-2, a prohibitin homology (PHB)-domain protein (Goodman, 2006). MEC-2 binds cholesterol directly, and its binding to cholesterol is required for channel activation and for touch sensitivity (Huber et al., 2006; Brown et al., 2008). The authors suggest that the role of MEC-2 may be to regulate the activity of MEC-4/MEC-10, possibly by directly recruiting cholesterol to the membrane near the channel or by localizing the channel to regions of the membrane high in cholesterol.
In other cases, lipids may directly modulate channel activity. For example, TRPV channel activity in C. elegans is affected by subsets of PUFAs (Kahn-Kirby et al., 2004). Mutants defective in PUFA biosynthesis, such as fat-1, fat-3, and fat-4 exhibit defects in chemotaxis and nociception which are similar to those mediated by the TRPV channel proteins OSM-9 and OCR-2. Subsets of exogenous PUFAs can rescue the various defects. Moreover, the 20-carbon PUFA eicosapentaenoic acid can directly activate a neuron that mediates nociception, in a manner that is dependent on OSM-9/OCR-2, suggesting that TRPV channels may be directly activated by certain PUFAs.
PUFAs have also been shown to be required for neurotransmission in C. elegans. fat-3 mutants exhibit multiple behavioral defects due to decreased neurotransmitter release. EM studies have revealed that fat-3 mutants have a decreased number of synaptic vesicles at release sites and fat-3 mutants were recently shown to have defects in synaptic vesicle recycling (Marza and Lesa, 2006; Marza et al., 2008). UNC-26 (synaptojanin), which is also required for synaptic vesicle recycling is mislocalized in fat-3 mutants suggesting that the role of PUFAs in neurotransmission may be mediated by localisation of UNC-26, although the exact mechanism is not currently known (Marza et al., 2008).
Specialized membrane microdomains, such as caveolae can also influence signaling. In addition to signaling, caveolae have been implicated in multiple cellular processes including endocytosis, lipid homeostasis, and even mechanosensation (Parton and Simons, 2007). Caveolae are membrane microdomains enriched in sphingolipids, cholesterol and lipid-anchored proteins, that are stabilized by caveolin proteins. Although the roles of caveolae have not been intensively studied in C. elegans, the caveolin protein CAV-1 affects at least one signaling pathway in the germline (RAS; Scheel et al., 1999) and CAV-2 affects various aspects of lipid trafficking in the intestine (Parker et al., 2009), suggesting that caveolae may also be involved in multiple processes in C. elegans.
Part of the power of C. elegans as a model system stems from the tendency in the field to move toward exhaustive descriptions of various aspects of the biology of its subject. Although the study of lipid biology may be a newer field of study in C. elegans there has been rapid progress, including in the study of biochemical pathways that have previously been studied mostly in other organisms. The interest of studying these problems in an animal model like C. elegans is the possibility to use its unique genetic tools and ease of manipulation to quickly relate changes in very basic biochemical pathways to whole animal phenotypes. Indeed, it is possible to relate biochemical changes to phenotypes such as the choice of developmental program, behavior, and life history traits like reproduction and aging. We believe that the recent advances in the lipid field that we have described here exemplifies how well this approach can work.
Because of their ease of handling and the economic advantage that this affords, it would be very useful if invertebrate models like C. elegans could be used for studying human diseases and for helping to identify and characterizing new drugs. One doubt that is often voiced about this approach is whether one can really model a human disease in an animal with a very different body plan, physiology and life style. We believe that the lipoprotein studies that we have described offer an example of how this is indeed possible. On the one hand, it would be difficult to define “atherosclerosis” in C. elegans as it has no obvious arteries, or heart whose arteries risk clogging, or even “dyslipidemia” as we do not yet know where and how to measure nonyolk lipoprotein levels. On the other hand, many of the interventions, including drugs, that impact on human dyslipidemia seem to impact on the phenotype of C. elegans in a way that is consistent with their known roles in mammals (Fig. 3). This is likely due to the conservation of relevant cellular processes and a high degree of conservation at the molecular level. This demonstrates that even complex disease-causing processes that involve several molecular players in distinct organs can be studied in C. elegans, even when the pathology itself cannot be directly modeled.
S.H. is Campbell Chair of Developmental Biology. His work on lipid biology in C. elegans is supported by a grant from CIHR. R.B. is supported by fellowships from EMBO and the MRC. D.D. is supported by a scholarship from NSERC.