Peroxisomes: a Nexus for Lipid Metabolism and Cellular Signaling

Cell Metabolism Review

Peroxisomes: A Nexus for Lipid Metabolism and Cellular Signaling

Irfan J. Lodhi1,* and Clay F. Semenkovich1,2,* 1Division of Endocrinology, Metabolism and Lipid Research; Department of Medicine 2Department of Cell Biology and Physiology Washington University, St. Louis, MO 63110, USA *Correspondence: [email protected] (I.J.L.), [email protected] (C.F.S.) http://dx.doi.org/10.1016/j.cmet.2014.01.002

Peroxisomes are often dismissed as the cellular hoi polloi, relegated to cleaning up reactive oxygen chemical debris discarded by other organelles. However, their functions extend far beyond hydrogen peroxide metabolism. Peroxisomes are intimately associated with lipid droplets and mitochondria, and their ability to carry out fatty acid oxidation and lipid synthesis, especially the production of ether lipids, may be critical for generating cellular signals required for normal physiology. Here, we review the biology of peroxisomes and their potential relevance to human disorders including cancer, obesity-related diabetes, and degenerative neurologic disease.

Introduction arow and De Duve (1976) hypothesized that peroxisomes in an- Peroxisomes are multifunctional organelles present in virtually all imal cells were capable of carrying out fatty acid oxidation. This eukaryotic cells. In addition to being ubiquitous, they are also was confirmed when they showed that purified rat liver peroxi- highly plastic, responding rapidly to cellular or environmental somes contained fatty acid oxidation activity that was robustly cues by modifying their size, number, morphology, and function increased by treatment of animals with clofibrate. In a series of (Schrader et al., 2013). Early ultrastructural studies of kidney experiments, Hajra and colleagues discovered that peroxisomes and liver cells revealed cytoplasmic particles enclosed by a single were also capable of lipid synthesis (Hajra and Das, 1996). Over membrane containing granular matrix and a crystalline core (Rho- the past three decades, multiple lines of evidence have solidified din, 1958). These particles were linked with the term ‘‘peroxi- the concept that peroxisomes play fundamentally important some’’ by Christian de Duve, who first identified the organelle in roles in lipid metabolism. In addition to removal of reactive oxy- mammalian cells when enzymes such as oxidases and catalases gen species, metabolic functions of peroxisomes in mammalian involved in hydrogen peroxide metabolism cosedimented in equi- cells include b-oxidation of very long chain fatty acids, a-oxida- librium density gradients (De Duve and Baudhuin, 1966). Based tion of branched chain fatty acids, and synthesis of ether-linked on these studies, it was originally thought that the primary function phospholipids as well as bile acids (Figure 1). b-oxidation also of these organelles was the metabolism of hydrogen peroxide. occurs in mitochondria, but peroxisomal b-oxidation involves Characterization of peroxisomes (also called ‘‘microbodies’’ in distinctive substrates and complements mitochondrial function; the early literature) was greatly facilitated by the development of the processes of a-oxidation and ether lipid synthesis are unique a cytochemical staining procedure using 3,30-diaminobenzadine to peroxisomes and important for metabolic homeostasis. (DAB), which permits visualization of these organelles based on Here, we highlight the established role of peroxisomes in lipid the peroxidative activity of catalase at alkaline pH (Fahimi, 1969; metabolism and their emerging role in cellular signaling relevant Novikoff and Goldfischer, 1969). Using this staining method, to metabolism. We describe the origin of peroxisomes and Novikoff and colleagues observed a large number of peroxisomes factors involved in their assembly, division, and function. We in tissues active in lipid metabolism, such as liver, brain, intestinal address the interaction of peroxisomes with lipid droplets and mucosa, and adipose tissue (Novikoff and Novikoff, 1982; Novik- implications of this interaction for lipid metabolism. We consider off et al., 1980). Peroxisomes in different tissues vary greatly in fatty acid oxidation and lipid synthesis in peroxisomes and their shape and size, ranging from 0.1–0.5 mM in diameter. In adipo- importance in brown and white adipose tissue (WAT) (sites rele- cytes, peroxisomes tend to be small in size and localized in the vant to lipid oxidation and synthesis) and disease pathogenesis. vicinity of lipid droplets. Notably, a striking increase in the number Finally, we review what is known about the mechanisms under- of peroxisomes was observed during differentiation of adipogenic lying human peroxisomal disorders. cells in culture (Novikoff and Novikoff, 1982). These findings suggest that peroxisomes may be involved in lipid metabolism. Peroxisomal Biogenesis Beevers and colleagues implicated peroxisomes in lipid meta- Despite two decades of research on the process, the origin of bolism by demonstrating that enzymes involved in fatty acid peroxisomes remains controversial (Dimitrov et al., 2013). In oxidation are colocalized in plant peroxisome-like organelles principle, organelles can be derived from (1) growth and fission called glyoxysomes, which are capable of converting fatty acids of preexisting organelles, (2) templated assembly using an exist- to metabolic intermediates for carbohydrate synthesis (Cooper ing copy of the organelle, or (3) de novo generation (Lowe and and Beevers, 1969). Based on the finding that the fibrate class Barr, 2007). For peroxisomes, two alternative theories of biogen- of hypolipidemic drugs promotes peroxisome proliferation, Laz- esis have been proposed (Figure 2A). According to the classical

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Figure 1. Structure and Functions of Peroxisomes The peroxisome is a single membrane-enclosed organelle that plays an important role in metabolism. The main metabolic functions of peroxisomes in mammalian cells include b-oxidation of very long chain fatty acids, a-oxidation of branched chain fatty acids, synthesis of bile acids and ether-linked phospholipids, and removal of reactive oxygen species. Peroxisomes in many, but not all, cell types contain a dense crystalline core of oxidative enzymes.

Fujiki, 1985), recent studies suggest that preperoxisomal vesicles, precursors of peroxisomes, bud off of the ER, thus obvi- ating the need for luminal continuity to allow transport of peroxisomal contents (van der Zand et al., 2012). Using a bimolecular fluorescence complementation assay, these authors monitored peroxisomal assembly in real time. This assay is based on reconstitution of a functional fluorescent protein mediated by interactions between two peptides fused to two different nonfluorescent halves of the fluorescent protein (Kerppola, 2008). van der Zand and colleagues used this technology model of peroxisome biogenesis, peroxisomes are autonomous to show that two distinct preperoxisomal vesicles, each carrying organelles that arise from preexisting peroxisomes through half of a peroxisomal translocon complex, undergo heterotypic growth and division (Lazarow and Fujiki, 1985). This model fusion, forming a functional translocon that permits uptake of (Figure 2A, top) was supported by the observation that some matrix proteins from the cytosol (van der Zand et al., 2012). peroxisomes in histologic sections of liver are visualized as Factors Involved in Peroxisomal Biogenesis dumbbell-shaped interconnected structures. Such structures Regardless of their origin, peroxisomes require a group of pro- were more abundant following induction of peroxisome prolifer- teins called peroxins for their assembly, division, and inheri- ation by pharmacological means or by partial hepatectomy tance. The budding yeast Saccharomyces cerevisae is a (Legg and Wood, 1970; Reddy and Svoboda, 1971; Rigatuso particularly apt model for studying peroxisomal biogenesis due et al., 1970). This model was generally accepted for over 20 to its ease of genetic manipulation and its robust peroxisomal years. However, subsequent studies suggested that peroxi- proliferation induced by oleate-containing medium (Subramani, somes could arise de novo. In yeast cells lacking detectable 1998). Over 30 peroxins, encoded by Pex genes, have been peroxisomes due to a single gene mutation, reintroduction of identified in yeast (Dimitrov et al., 2013). At least a dozen of these the wild-type version of the gene complements the defect and proteins are conserved in mammals, where they regulate various restores peroxisome formation (Baerends et al., 1996; Ho¨ hfeld aspects of peroxisomal biogenesis, including factors that control et al., 1991; Wiemer et al., 1996), consistent with the idea that assembly of the peroxisomal membrane, factors that interact peroxisomes may not be exclusively autonomous. with peroxisomal targeting sequences (PTSs) allowing proteins An alternative model (Figure 2A, bottom) holds that peroxi- to be shuttled to peroxisomes, and factors that act as docking somes are semiautonomous organelles deriving their matrix pro- receptors for peroxisomal proteins. teins from the cytosol and their membrane proteins and lipids from At least three peroxins (Pex3, Pex16, and Pex19) appear to be the endoplasmic reticulum (ER) (Tabak et al., 2013). A potential ER critical for assembly of the peroxisomal membrane and import of origin for peroxisomes is supported by classic morphology peroxisomal membrane proteins (PMPs) (Figure 2B). Pex19 is a studies showing peroxisomes as clusters surrounded by smooth soluble chaperone and import receptor for newly synthesized ER in absorptive cells of guinea pig intestine (Novikoff and Novik- PMPs (Jones et al., 2004). Pex3 buds from the ER in a preperox- off, 1972). Numerous contacts between peroxisomes and smooth isomal vesicle and functions as a docking receptor for Pex19 ER were observed, suggesting that peroxisomes arise from (Fang et al., 2004). Pex16 acts as a docking site on the peroxi- dilated regions of ER. These regions were thought to contain somal membrane for recruitment of Pex3 (Matsuzaki and Fujiki, high concentrations of peroxisomal proteins, such as catalase 2008). Peroxisomal matrix proteins are translated on free ribo- (Novikoff and Novikoff, 1972; Reddy and Svoboda, 1971). somes in the cytoplasm prior to their import. These proteins Although the existence of direct contact and luminal continuity be- have specific PTSs located either at the carboxyl (PTS1) or amino tween peroxisomes and ER has been challenged (Lazarow and (PTS2) terminus (Gould et al., 1987; Swinkels et al., 1991).

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A B

Figure 2. Potential Pathways to Peroxisomal Biogenesis (A) Peroxisomes are generated autonomously through division of preexisting organelles (top) or through a de novo process involving budding from the ER followed by import of matrix proteins (bottom). (B) Peroxisomal membrane protein import. Peroxisomal membrane proteins (PMPs) are imported posttranslationally to the peroxisomal membrane. Pex19 is a soluble chaperone that binds to PMPs and transports them to the peroxisomal membrane, where it docks with a complex containing Pex16 and Pex3. Following insertion of the PMP, Pex19 is recycled back to the cytosol.

Recently, cryptic PTS1 motifs were identified in several glycolytic Green, 1990). Issemann and Green (1990) used reporter assays enzymes, such as glyceraldehyde-3-phosphate dehydrogenase to demonstrate that peroxisome proliferating chemicals acti- (GAPDH) and 3-phosphoglycerate kinase (PGK), in various fungal vated a PPAR (now referred to as PPARa). Subsequently, two species. These motifs are activated by posttranslational pro- related nuclear receptors, PPARg and PPARd were cloned cesses and may direct bona fide cytoplasmic proteins to (Dreyer et al., 1992; Kliewer et al., 1994). peroxisomes under certain metabolic conditions (Freitag et al., PPARs are recognized as fundamentally important regulators 2012). The import receptor for PTS1-containing peroxisomal of lipid metabolism (Wang, 2010). They act as ligand-activated matrix proteins is Pex5 (Dodt and Gould, 1996), whereas Pex7 transcription factors that form obligate heterodimers with reti- functions as the import receptor for PTS2-containing proteins noid X receptors (RXR) and bind to specific DNA sequences (Braverman et al., 1997). These receptors bind cargo in the cyto- known as PPAR response elements (PPREs) located in promoter plasm and transport it to docking sites at the peroxisomal mem- regions of target genes. In the absence of ligand, PPARs are brane. Once their cargo is delivered to the peroxisomal lumen, the associated with corepressors. Ligand binding promotes a receptors recycle back to the cytoplasm (Liu et al., 2012b). The conformation change in PPARs, resulting in replacement of re- recycling of Pex5 requires ubiquitination (Platta et al., 2007). pressors with coactivators and subsequent activation of target Peroxisome Proliferator Activated Receptors in gene expression. The three members of the PPAR family mani- Peroxisome Biogenesis and Function fest distinct tissue distribution patterns and target gene expres- Peroxisome proliferator activated receptors (PPARs) are relevant sion profiles. PPARa is the target of fibrate drugs used to treat to peroxisomal lipid oxidation and synthesis and how these pro- lipid disorders in humans. It is highly enriched in liver and brown cesses impact disease pathogenesis. Fibrates, used to lower adipose tissue (BAT), where it is a key regulator of fatty acid lipids in humans, and certain xenobiotics promote proliferation oxidation. PPARd has significant functional overlap with PPARa of peroxisomes in cells presumably due to de novo biogenesis. but is more widely expressed. PPARg is the target of glitazone This property, as well as the capacity of these compounds to drugs used to treat diabetes in humans. It is enriched in adipose modulate gene expression of the peroxisomal fatty acid oxida- tissue and is absolutely required for adipogenesis (Ahmadian tion machinery (Reddy et al., 1986), led to the hypothesis that et al., 2013; Tontonoz and Spiegelman, 2008). these chemicals activate a nuclear receptor by a mechanism PPARa is known to be important for peroxisome proliferation. analogous to that used by steroid hormones (Issemann and PPARa activation increases not only the expression of fatty acid

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oxidation genes but also the abundance of peroxisomes in liver animal cells is unknown. However, several studies have reported (Schrader et al., 2012). Its activation induces expression of the close associations between peroxisomes and lipid droplets in peroxin Pex11 (Cornwell et al., 2004), which may be involved in various mammalian cells. Live cell imaging in Cos-7 and HepG2 peroxisomal biogenesis by promoting peroxisome division (Li cells expressing GFP-PTS1 revealed tubulo-reticular clusters of and Gould, 2002), as depicted in Figure 2A. Less is known about peroxisomes in close association with lipid droplets (Schrader, how PPARd and PPARg affect proliferation. Recently, high-fat 2001). In 3T3-L1 adipocytes and mouse epididymal WAT, immu- feeding of mice was shown to increase PPARg activation and nogold staining with an antibody against catalase showed peroxisome proliferation in the hypothalamus. Increased perox- numerous small dumbbell-shaped peroxisomes at the periphery isome abundance was thought to decrease levels of neuronal of lipid droplets (Blanchette-Mackie et al., 1995), confirming the free radicals, which modulate feeding behavior through effects observation of Novikoff and colleagues using DAB staining (Novik- on the melanocortin system (Diano et al., 2011). PPARg activa- off and Novikoff, 1982; Novikoff et al., 1980). Recently, several tion may be involved in cold-induced proliferation of peroxi- protein-protein interactions involving lipid droplet resident pro- somes in BAT (Guardiola-Diaz et al., 1999). The transcriptional teins and peroxisomal markers were identified using a bimolecular coactivator PGC1a, critical for thermogenesis (Puigserver fluorescence complementation assay (Pu et al., 2011). et al., 1998), promotes cold-induced peroxisome biogenesis Because peroxisomes and lipid droplets each control lipid in BAT in a PPARa-independent manner, implicating PPARg metabolic flux, the close interaction between the two organelles or PPARd in this effect (Bagattin et al., 2010). Our preliminary suggests a coordinated regulation of metabolism and lipid studies indicate that treatment of 3T3-L1 preadipocytes with trafficking across their boundaries. When an organism requires rosiglitazone substantially increases the expression of several energy, very long chain fatty acids stored as triglycerides in lipid Pex genes, suggesting that PPARg promotes peroxisomal droplets can be mobilized and rapidly transported to peroxi- biogenesis in adipocytes (I.J.L and C.F.S, unpublished). Collec- somes for initiation of fatty acid oxidation. However, it is unlikely tively, these findings suggest that PPARs coordinate both perox- that lipid trafficking between droplets and peroxisomes is unidi- isomal biogenesis and integrative lipid metabolism. rectional. As discussed below, peroxisomes are important in the synthesis of alkyl ether equivalents of acylglycerols. These glyc- Crosstalk between Peroxisomes and Lipid Droplets erides may be differentially compartmentalized as compared to Intracellular lipid storage provides protection against toxic those generated by acylation of glycerol-3-phosphate in the effects of excessive lipid concentrations, a source of energy for ER. Mass-spectrometry-based lipidomic analyses show that cellular growth and metabolic processes, and a survival advan- up to 20% of neutral lipids in lipid droplets are present as an ether tage during starvation. Lipid droplets are omnipresent subcellular lipid equivalent of triglyceride, monoalk(en)yl diacylglycerol organelles that store lipids. Their structure resembles that of (MADAG). MADAG is undetectable in lipid droplets from cells circulating lipoproteins in mammals, consisting of a phospholipid lacking peroxisomes (Bartz et al., 2007). These studies support monolayer surrounding a core of neutral lipids such as triglycer- the existence of bidirectional lipid trafficking between peroxi- ides and cholesteryl esters. Although most prominent in adiposomes and lipid droplets, constituting crosstalk between the cytes, which store excess energy as triglyceride, lipid droplets two subcellular compartments to balance energy synthesis are present in most cell types. Lipid droplets vary greatly in and utilization depending on metabolic context. size, ranging from less than 1 mmto100 mm in diameter. Mature white adipocytes have large unilocular lipid droplets that occupy Peroxisomal Fatty Acid Oxidation the majority of the cytosol. Brown adipocytes have small multi- Peroxisomes in all eukaryotic organisms are thought to have the locular lipid droplets. In most other cell types, lipid droplets are ability to oxidize fatty acids. In plants and most fungi, peroxi- less than 1 mm in diameter (Reue, 2011). Like peroxisomes, lipid somes are the only site of fatty acid oxidation (Poirier et al., droplets are thought to be derived from the ER. According to 2006). In animal cells, fatty acid oxidation takes place in both the prevailing model of lipid droplet biogenesis, neutral lipids mitochondria and peroxisomes. It is becoming increasingly clear are incorporated into the interspace of the bilayer leaflets of the that peroxisomes have a role in fatty acid oxidation that cannot ER membrane, where several enzymes involved in triglyceride be replaced by mitochondria. Peroxisomes in animal cells were synthesis are localized. Once a certain threshold of triglyceride originally thought to play only an ancillary role in fatty acid oxida- accumulation is reached, the outer leaflet of the ER membrane tion, participating in oxidation only when the mitochondrial probably bulges into the cytosol through a budding process, oxidative capacity was exceeded. This notion was challenged forming a new lipid droplet that is released from the ER (Farese by the observation that patients with certain peroxisomal and Walther, 2009). Cytosolic lipid droplets increase their volume disorders (discussed below) have increased serum levels of through localized de novo lipogenesis, transport of extracellular very long chain (>22 carbon atoms) and branched chain fatty lipids into lipid droplets, or fusion with other lipid droplets. acids (Brown et al., 1982; Poulos et al., 1986). Subcellular frac- Several lines of evidence suggest that peroxisomes are closely tionation studies with liver tissue confirmed that b-oxidation of associated with lipid droplets. In yeast grown in oleate, peroxi- very long chain fatty acids preferentially occurs in peroxisomes somes stably adhere to lipid droplets (Binnset al., 2006). This inter- as compared to mitochondria (Singh et al., 1984). The accumu- action appears to be more than a simple juxtaposition; peroxi- lation of branched chain fatty acids in patients with peroxisomal somes extend processes, referred to as pexopodia, into the lipid disorders suggested a requirement for peroxisomes in a-oxida- droplet core. Proteomic analysis of these cells shows that lipid tion (Wanders et al., 2001). Branched chain fatty acids, such as droplets are enriched in peroxisomal b-oxidation enzymes (Binns phytanic acid, have a methyl group on the third carbon atom et al., 2006). Whether peroxisomes and lipid droplets intermingle in (g position), which prevents b-oxidation. These fatty acids first

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undergo oxidative decarboxylation (a-oxidation) in peroxisomes acids of 26 carbon chain length or greater are thought to be to remove the terminal carboxyl group as CO2. This shortened oxidized exclusively in peroxisomes, although the number of fatty acid now has a methyl group on the second carbon and peroxisomal oxidation cycles utilized for these molecules is un- can be further oxidized in peroxisomes or mitochondria. Unlike known. Shorter fatty acids can be oxidized in either organelle 3-methyl branched fatty acids, 2-methyl fatty acids can undergo (Wanders et al., 2010), but the quantitative contributions of b-oxidation with release of a propionyl CoA group, instead of each are not well defined, since metabolism varies depending acetyl CoA, after the first round of b-oxidation (Verhoeven on substrates and physiological context. et al., 1998). Mitochondrial and peroxisomal b-oxidation systems also differ Most fatty acids are metabolized by b-oxidation, which in- in terms of fatty acyl CoA transport from the cytosol. Uptake of volves the removal of two carbons at the carboxyl terminus of fatty acyl CoAs across the mitochondrial membrane requires a the molecule and is carried out in peroxisomes and mitochon- carnitine exchange system, consisting of two carnitine palmitoyl- dria. As noted above, branched chain fatty acids are subject to transferases (CPT1 and CPT2) and the transporter protein carni- a-oxidation, which involves the removal of one carbon at the tine-acylcarnitine translocase (CACT). CPT1 is associated with carboxyl terminus. This process is thought to occur in peroxi- the outer mitochondrial membrane. It exchanges carnitine for somes but not in mitochondria; the adaptive rationale for lacking CoA, resulting in a fatty acid carnitine, which is transported across a-oxidative capacity in mitochondria is not known. Long chain the inner mitochondrial membrane by CACT. Once inside the and very long (> 22 carbons) chain fatty acids can be metabo- matrix, fatty acid carnitine is converted back to fatty acyl CoA lized by u-oxidation, which involves the removal of one carbon by CPT2. Carnitine does not appear to be required for fatty acyl at the end of the molecule farthest from the carboxyl terminus. CoA uptake into peroxisomes. However, peroxisomes do have u-oxidation, thought to be a minor pathway under most physio- carnitine acyltransferase activity, which may be required for con- logical circumstances, occurs in the smooth ER in a process verting acyl CoAs to acylcarnitines so that they can be transferred dependent on the cytochrome P450 CYP4A subfamily. Dicar- to mitochondria for further oxidation (Wanders, 2013). Fatty acyl boxylic acids, the product of u-oxidation, can then be metabo- CoA uptake into peroxisomes requires three ATP binding cassette lized by b-oxidation in peroxisomes (Reddy and Hashimoto, transporter D subfamily proteins that are localized to the peroxi- 2001), suggesting a link between peroxisomal and microsomal somal membrane: ABCD1, ABCD2, and ABCD3 (Wanders et al., fatty acid metabolism in addition to the well-characterized link 2007). Each can form a functional homodimeric or heterodimeric between peroxisomal and mitochondrial fatty acid metabolism. transporter by partnering with another ABCD protein. Each sub- Similarities and Differences between Peroxisomal and unit contains a transmembrane domain as well as an ATP binding Mitochondrial Fatty Acid Oxidation cassette. ABCD1 (also called ALDP) is mutated in the human Although there are some fundamental differences in the disease adrenoleukodystrophy. As a homodimer, ABCD1 is biochemistry of mitochondrial and peroxisomal b-oxidation involved in transporting very long chain fatty acids from the (Wanders and Waterham, 2006), the overall mechanism is cytosol to the peroxisomal lumen for b-oxidation (van Roermund essentially the same and involves four consecutive enzymatic et al., 2008). ABCD2 is particularly abundant in adipose tissue, steps: (1) dehydrogenation that removes two hydrogen mole- where it may import dietary erucic acid (C22:1) into peroxisomes cules and introduces a trans double bond between the a and b (Liu et al., 2012a). ABCD3 (also called PMP70) may import long carbons of the fatty acyl CoA molecule; (2) hydration across chain fatty acids and branched chain acyl CoAs into peroxisomes the double bond, forming a 3-L-hydroxyacyl-CoA; (3) dehydro- for fatty acid oxidation (Imanaka et al., 1999). genation of the 3-L-hydroxyacyl-CoA, forming 3-ketoacyl CoA; Although PPARa is required for transcriptional regulation and (4) thiolytic cleavage of the terminal acetyl CoA group, form- of both pathways (Aoyama et al., 1998), acute regulation of ing a new acyl CoA molecule that is shorter by two carbons. mitochondrial fatty acid oxidation is different from that of perox- Despite these similarities, there are several differences be- isomes. Unlike peroxisomal fatty acid oxidation, the mitochon- tween mitochondrial and peroxisomal fatty acid oxidation (Wan- drial pathway is exquisitely regulated by malonyl-CoA at the level ders and Tager, 1998). First, the enzymes involved in the two of CPT1 (McGarry et al., 1978; McGarry et al., 1977). Whether pathways are entirely different. For example, the first step of mito- there are analogous mechanisms for rapid control of peroxi- chondrial b-oxidation is catalyzed by the flavoenzyme acyl CoA somal fatty acid oxidation is unknown. dehydrogenase to produce FADH2, whose electrons are utilized Physiological Significance of Peroxisomal Fatty Acid to produce two molecules of ATP. In contrast, the first step of Oxidation peroxisomal b-oxidation reaction is catalyzed by a different fla- It is likely that peroxisomal fatty acid oxidation is more complex voenzyme, acyl CoA oxidase. Because peroxisomes lack a respi- than simply shortening chains of very long chain fatty acids that ratory chain, electrons from FADH2 are transferred directly to O2 cannot be directly oxidized in mitochondria. The biochemical (creating H2O2), and energy is released as heat, instead of being basis underlying the metabolism of very long chain fatty acids used to generate ATP. Thus, peroxisomal fatty acid oxidation is in peroxisomes, but not in mitochondria, is known. Peroxisomal less favorable in terms of energy storage than mitochondrial fatty acyl CoA oxidases and mitochondrial acyl CoA dehydrogenases acid oxidation. Because fatty acid oxidation in peroxisomes is not manifest different chain length specificities. However, the phys- carried to completion (Osumi and Hashimoto, 1980; Vanhove iological basis for this organelle-specific functional difference is et al., 1993), this process is thought to shorten very long chain not known. Perhaps mitochondrial toxicities associated with fatty acids, which can then be further oxidized in mitochondria. the presence of very long chain fatty acids favored the evolu- Consistent with this concept, the substrate specificities of perox- tionary selection of peroxisomes for very long chain metabolism isomal and mitochondrial b-oxidation systems are different. Fatty in mammals. Alternatively, peroxisome-dependent generation of

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Figure 3. Chemical Structures of Diacyl- and Ether-Linked Phospholipids In conventional diacyl phospholipids, fatty acyl side chains are linked to the sn-1 and sn-2 positions of the glycerol backbone by ester bonds. Ether-linked phospholipids are a special class of glycerophospholipids that have an alkyl chain attached to the sn-1 position by an ether bond. The sn-2 substituent of ether lipids is generally an ester-linked acyl chain as in diacylphospholipids. The head group of ether phospholipids is usually choline or ethanolamine. Some of the ether linked phospholipids have a cis double bond adjacent to the ether bond and are referred to as alkenyl-acylphospholipids, or more commonly as plasmalogens. According to convention, plasmalogen form of phospholipids have the prefix ‘‘plasmenyl,’’ as in plasmenylcholine. Alkyl-acylphospholipids have the prefix ‘‘plasmanyl.’’ heat (see below) may provide tissue-specific benefits such as lipid synthetic pathway (Hajra and Das, 1996), while others found maintenance of membrane structure, preservation of regional that enzymes involved in bile acid synthesis were also localized to blood flow to ensure oxygenation, or control of optimum temper- peroxisomes (Pedersen et al., 1997). These studies firmly estab- ature to ensure enzymatic functions required for tissue regener- lished peroxisomes as a participant in lipid biosynthesis. ation or repair. Synthesis of Ether Phospholipids Ether lipids (which encompass alkyl-acylphospholipids and Ether phospholipid synthesis requires a series of enzymes asso- alkenyl-acylphospholipids, also known as plasmalogens) (Fig- ciated with the peroxisomal membrane (Figure 4). This pathway is ure 3) have unique physical properties that impact many aspects an alternative route for lysophosphatidic acid (LPA) synthesis (as of cell biology (discussed below). Several studies suggest that the opposed to direct acylation of glycerol 3-phosphate) and is oblig- synthesis of ether lipids is linked to peroxisomal b-oxidation in a atory for synthesis of precursors of all ether lipids in mammals, manner that is physiologically relevant. Injection of [1-14C]ligno- including platelet activating factors (PAFs) and plasmalogens ceric acid (C24:0) into clofibrate-treated rats results in incorpora- (Hajra and Das, 1996). The pathway uses dihydroxacetone phos- tion of the radiolabel into ethanolamine plasmalogens, suggesting phate (DHAP), produced by dehydrogenation of glycerol 3-pho- that peroxisomal fatty acid oxidation may preferentially channel phate (G3P), as a building block for the synthesis of phospho- acetyl CoAs toward ether lipid synthesis (Hayashi and Oohashi, lipids. Fatty acids derived from FAS-mediated de novo 1995). Consistent with this possibility, humans and mice with lipogenesis or other sources are activated to fatty acyl CoA by mutations in ABCD1 have decreased levels of ethanolamine plas- a peroxisome-membrane-associated acyl CoA synthetase malogens, coupled with accumulation of very long chain fatty (ACS). Acyl CoA is used by DHAPAT to acylate DHAP or is acids in brain white matter (Khan et al., 2008). ABCD1 was reduced to a fatty alcohol by fatty acyl CoA reductase 1 (FAR1) recently shown to interact with several lipogenic enzymes, in an NADPH-dependent reaction. A fatty alcohol is used by alkyl including ATP citrate lyase (ACLY), acetyl CoA carboxylase DHAP synthase (ADHAPS) to convert acyl DHAP to alkyl DHAP. (ACC), and fatty acid synthase (FAS) (Hillebrand et al., 2012). Acyl DHAP or alkyl DHAP can be reduced to 1-Acyl G3P (LPA) FAS is a large multifunctional enzyme that primarily synthesizes or 1-O-Alkyl G3P (AGP), respectively, by Acyl/Alkyl DHAP reduc- palmitate (C16:0) using malonyl CoA as a two carbon source tase (ADHAPR). ADHAPR was purified from guinea pig liver and (Lodhi et al., 2011). We recently demonstrated increased fatty biochemically characterized (LaBelle and Hajra, 1974), but the acid oxidation in subcutaneous white fat in mice with adipose- gene encoding this protein was not known in mammals (McIntyre specific knockout of FAS (Lodhi et al., 2012). It possible that this et al., 2003). We recently identified this gene and renamed the increased fatty acid oxidation was primarily peroxisomal since protein it encodes PexRAP (for Peroxisomal Reductase Acti- FAS inactivation results in accumulation of malonyl-CoA, which vating PPARg)(Lodhi et al., 2012). Subsequent steps of phospho- inhibits mitochondrial fatty acid oxidation (McGarry et al., 1977). lipid synthesis, such as acylation of the sn-2 position and addition of the head group (e.g., CDP-choline or CDP-ethanolamine) take Lipid Biosynthesis in Peroxisomes place in the ER (Hajra and Das, 1996). In 1977, Amiya Hajra and colleagues discovered that dihydroxyacetone phosphate acyltransferase (DHAPAT), an enzyme Properties and Functions of Ether Phospholipids required for the synthesis of acyl DHAP, a precursor for glycerol Ether lipids, such as plasmalogens, make up approximately 20% ether lipids, was localized to peroxisomes (Jones and Hajra, of the total phospholipid mass in humans. Tissue levels of these 1977). This was a surprising discovery because it suggested lipids vary greatly. Ether phospholipids are particularly abundant that peroxisomes were not only involved in catabolic reactions, in the brain, heart, and white blood cells. Up to two thirds of the but also the synthesis of phospholipids. In the more common di- ethanolamine phospholipid in the brain and 12% of the total acyl phospholipids, fatty acyl side chains are linked to the sn-1 myelin phospholipid pool is plasmalogen (Nagan and Zoeller, and sn-2 positions of the glycerol backbone by ester bonds. In 2001). In neutrophils, up to 46% of the phosphatidylcholine ether lipids (such as alkyl ether phospholids and plasmalogens), pool is in the plasmanyl form (Bra¨ utigam et al., 1996; Mueller the sn-1 substituent is attached to the glycerol backbone by an et al., 1982, 1984). In contrast, intracellular ether lipids are scant ether bond (Figure 3). In the subsequent 20 years, the Hajra labo- in the liver (Braverman and Moser, 2012). This is surprising, ratory identified several other components of a peroxisomal ether because many of the enzymes in the ether lipid synthetic

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Figure 4. Acyl DHAP Pathway of Phospholipid Synthesis This pathway is obligatory for synthesis of ether-linked phospholipids and is also an alternative route for synthesis of diacylphospholipids. Phospholipid synthesis begins in peroxisomes and is completed in the ER. The pathway uses dihydroxacetone phosphate (DHAP), generated by glycerol 3-phosphate dehydrogenase (G3PDH)-mediated dehydrogenation of G3P, as a building block for the synthesis of phospholipids. Fatty acyl CoA produced by de novo lipogenesis is used by DHAPAT (DHAP acyltransferase) to acylate DHAP or is reduced to a fatty alcohol by a peroxisomal membrane-associated fatty acyl CoA reductase in an NADPH- dependent reaction. The fatty alcohol is used by alkyl DHAP synthase (ADHAPS) to convert acyl DHAP to alkyl DHAP. Acyl DHAP or alkyl DHAP can be reduced to 1-acyl G3P (lysophosphatidic acid) or its ether lipid equivalent, 1-O-alkyl G3P (AGP), respectively, by acyl/alkyl DHAP reductase (also called PexRAP). The subsequent steps of phospholipid synthesis, including acylation at the sn-2 position, occur in the ER. pathway were originally purified from liver. Studies by Vance and nonbilayer structures at much higher temperatures (68C). colleagues suggest that liver synthesizes ether phospholipids This property of plasmalogens is thought to increase membrane but does not store them. Instead, hepatocytes preferentially permeability and promote membrane-membrane fusion (Lohner, secrete ether phospholipids lipids in the form of lipoproteins. 1996). Plasmalogens are also important for the organization of Up to 30% of phosphatidylethanolamine in lipoproteins is in detergent resistant microdomains, cholesterol-rich membrane the form of plasmalogen (Vance, 1990). Others have confirmed regions that are thought to be involved in cell signaling. Plasmal- significant levels of plasmalogens in human serum (Bra¨ utigam ogen-deficient mice lacking DHAPAT (encoded by gnpat) show et al., 1996). The precise molecular function of intracellular and evidence of disruption of these membrane microdomains; circulating ether lipids is unknown. In brain, several lines of evi- cholesterol is inappropriately localized to a perinuclear compart- dence suggest that ether lipids in myelin may be critical for ment, and flotillin-1 is decreased in fractions of neurological modulating ion transport, and similar effects may be at play in tissue from these mice (Rodemer et al., 2003). heart (Ford and Hale, 1996). In white blood cells such as neutro- Ether lipids may be involved in cancer pathogenesis. It has phils, ether lipids may create a sink for polyunsaturated fatty been known for decades that cancer cells have high levels of alkyl acids such as arachidonic acid utilized for inflammatory signaling ether lipids (Albert and Anderson, 1977; Howard et al., 1972; through the generation of eicosanoids (Nagan and Zoeller, 2001). Roos and Choppin, 1984; Snyder and Wood, 1969). Roos and Regardless of the precise processes driven by ether lipids, dra- Choppin (1984) determined the tumorigenecity of mouse fibro- matic developmental abnormalities in humans and mice with blast cell lines differing in ether lipid content. One such line, F40, defective ether lipid synthesis suggest that these lipids are with 30-fold higher ether lipid content as compared to its parental responsible for fundamental cellular tasks that cannot be carried line, required 1,000-fold fewer cells to form tumors in irradiated out by diacyl phospholipids. mice, and these tumors were aggressive. The expression of the Phospholipids are the predominant component of plasma ether lipid synthetic enzyme ADHAPS (also called alkylgycerone- membranes and intracellular membranes. The presence of phosphate synthase [AGPS]) is increased in various cancer cell ether-linked alkyl chains in phospholipids alters their physical lines and primary tumors (Benjamin et al., 2013). AGPS knock- properties and modulates membrane dynamics. This effect is down impaired experimental cancer pathogenesis, including presumably due to the lack of a carbonyl oxygen at the sn-1 po- cell survival, migration, and invasion. Conversely, AGPS overex- sition, which affects hydrophilic head group interactions (Lohner, pression increased ether lipids and promoted tumor growth. 1996; Nagan and Zoeller, 2001). Model semisynthetic mem- While its precise role in cancer progression is unclear, AGPS branes enriched in ethanolamine plasmalogens form nonlamellar may generate structural and signaling lipids that promote carci- structures at around 30C, but diacylphospholipids form these nogenicity (Benjamin et al., 2013).

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Figure 5. Relationship between Peroxisomes and PPARg PPARg is a key regulator of adipocyte differentiation as well as function and is activated by multiple endogenous ligands, including alkyl ether phospholipids, which are synthesized in peroxisomes. PPARg exists as a heterodimer with RXR and regulates expression of a large number of genes harboring PPAR response elements (PPRE), including genes involved in adipogenesis, lipid metabolism, and glucose homeostasis. Emerging studies indicate that PPARg also regulates the expression of genes involved in peroxisomal biogenesis, suggesting a feed-forward mechanism of PPARg activation.

Emerging evidence suggests that alkyl ether phospholipids mobilized in times of need, BAT transforms the chemical energy may be important in cellular signaling. Recent studies with in food into heat through uncoupled respiration. Because of their DHAPAT knockout mice demonstrate that ether lipids likely contribution to lipid metabolism, peroxisomes may regulate serve as self ligands to activate invariant natural killer T (iNKT) adipose tissue development and function (Figure 5). cells (Facciotti et al., 2012). As discussed below, results from Peroxisome Proliferation in Brown and White our laboratory and others suggest that peroxisome-derived Adipocytes lipids may also be involved in activating the nuclear receptor Peroxisomes in adipocytes are small in size and belong to a PPARg. subclass of the organelle referred to as microperoxisomes. Microperoxisomes are difﬁcult to detect by electron microscopy, Peroxisomes in Adipose Tissue Development and because they lack a crystalline nucleoid found in larger peroxi- Function somes, such as those in hepatocytes (Novikoff and Novikoff, Adipose tissue is an important metabolic organ that regulates 1982). Nevertheless, these small peroxisomes are abundantly whole-body energy balance. Two major types of adipose tissue present in adipocytes and are closely associated with lipid drop- are found in mammals, white fat and brown fat. Both types store lets. Using histochemical or immunogold staining for catalase, energy as triglyceride in intracellular lipid droplets and secrete a a large number of microperoxisomes were observed in 3T3-L1 host of hormones, called adipokines, which inﬂuence metabolic adipocytes and mice epididymal WAT (Blanchette-Mackie homeostasis. Whereas WAT primarily stores fat, which can be et al., 1995; Novikoff et al., 1980).

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A large number of catalase-positive particles were also ob- manner. Treatment of cells with one of these lipid species pro- served in rat BAT and increased dramatically with cold exposure, moted PPARg-dependent luciferase reporter activity, suggest- suggesting the presence of physiologically relevant microperox- ing that it could activate PPARg (Lodhi et al., 2012). As noted isomes in brown adipocytes (Ahlabo and Barnard, 1971). Subse- above, the peroxisomal acyl-DHAP pathway of lipid synthesis quent studies showed that a hydrogen peroxide-producing produces ether phospholipids. Activities of various enzymes in oxidase was present in these particles together with catalase, this pathway increase during adipogenesis (Hajra et al., 2000). consistent with their identification as bona fide peroxisomes Knockdown of PexRAP, the terminal enzyme in this pathway (Pavelka et al., 1976). Pex genes involved in peroxisomal biogen- (Figure 4), impairs PPARg activation and adipogenesis in esis are significantly increased during differentiation of brown cultured cells. Antisense oligonucleotide-mediated knockdown adipocytes in culture and in BAT of mice exposed to cold temper- of PexRAP in mice decreases expression of PPARgdependent atures in a manner dependent on PGC1a (Bagattin et al., 2010). genes, reduces fat mass, increases leanness, and improves Peroxisomal metabolism may be related to heat production in insulin sensitivity (Lodhi et al., 2012). BAT. Acetyl CoAs generated by peroxisomal fatty acid oxidation We identified alkyl ether phospholipids as potential endoge- may enter the tricarboxylic acid cycle in brown fat mitochondria nous ligands of PPARg in an unbiased screen, and there is to fuel thermogenesis mediated by UCP1, a mitochondrial un- precedent for PPARg activation by this class of lipids. Azelaoyl coupling protein that allows protons to return to the mitochon- phosphatidylcholine (1-O-hexadecyl-2-O-(9-carboxyoctanoyl)- drial matrix without synthesizing ATP. Alternatively, because sn-glyceryl-3-phosphocholine), an oxidatively shortened peroxisomal b-oxidation itself generates heat instead of ATP phospholipid in LDL, was reported to be a high affinity ligand due to the lack of a peroxisomal electron transport system, per- (equipotent to rosiglitazone) for PPARg (Davies et al., 2001). An oxisomes may be involved in adaptive thermogenesis indepen- alkyl ether analog of LPA, 1-O-alkyl glycerol 3-phosphate dent of UCP1. Together, these studies indicate that peroxisomes (AGP), a direct product of the PexRAP-catalyzed reaction (see affect white and brown adipocyte differentiation and function. Figure 4), has been shown to be a high affinity partial agonist Peroxisome-Derived Phospholipids in PPARg Signaling for PPARg (McIntyre et al., 2003; Tsukahara et al., 2006; Zhang and Adipocyte Differentiation et al., 2004). Since PPARg can be activated by a variety of lipids, The nuclear receptor PPARg is required for adipocyte differenti- it is possible that endogenous ligands of this nuclear receptor in ation and function. Studies in mice and cultured cells have estab- general exhibit partial agonism, thus permitting selective recruit- lished PPARg as both necessary and sufficient for adipocyte ment of coactivators to regulate different subsets of genes. differentiation. Moreover, PPARg regulates whole-body lipid A direct requirement for peroxisome-derived lipids in adipose metabolism and insulin sensitivity through its transcriptional con- tissue development was suggested by studies in Pex7 knockout trol of various adipocyte activities, including fat storage, lipogen- mice that lack plasmalogens (Brites et al., 2009, 2011). These esis, adipokine production, and thermogenesis (Tontonoz and mice have severely reduced body fat. This decreased adiposity Spiegelman, 2008). Genome-wide mapping of PPARg regulatory was not due to a significant difference in food intake and could elements using chromatin immunoprecipitation (ChIP) assays be rescued by feeding a diet enriched in alkyl-glycerol, which combined with microarrays revealed several thousand distinct restored plasmalogen levels (Brites et al., 2011). Exogenously PPARg binding sites in adipocytes (Lefterova et al., 2008). How added alkyl-glycerol enters the ether lipid synthetic pathway does PPARg regulate such a large number of genes in adipo- downstream of the peroxisomal steps. Because endogenous cytes? One possibility is that PPARg is activated by multiple ether phospholipids are present at low abundance in adipose endogenous ligands that in turn recruit different transcriptional tissue (our unpublished observation), it is possible that ether coactivators controlling unique subsets of genes. PPARg has a phospholipids have a signaling function. Whether adipose tissue large ligand binding pocket capable of engaging a variety of deficiency in Pex7 knockout mice is due to defective PPARg agonists (Xu and Li, 2008). Originally identified as the target of signaling remains to be determined. Recently, an unbiased the diabetes drugs thiazolidinediones, PPARg binds several mass-spectrometry-based metabolomics screen showed that different lipids, with variable effects on transcriptional activity endogenous synthesis of alkyl-glycerol ether lipids increases (Schupp and Lazar, 2010). Identification of specific endogenous early during 3T3-L1 adipogenesis and that treatment with these ligands of PPARg has been difficult. A study using a PPARg- lipids promotes adipocyte differentiation (Homan et al., 2011). dependent reporter assay suggested that endogenous ligands for PPARg could be produced during early phase adipogenesis, Peroxisomal Disorders but these ligands remain unidentified (Tzameli et al., 2004). Some degree of peroxisome function is required for health in Our previous studies suggested that de novo lipogenesis humans since there are several devastating genetic disorders mediated by FAS is involved in generating an endogenous ligand caused by impaired peroxisomal activity or lack of peroxisomes for PPARa in liver (Chakravarthy et al., 2009). To determine if due to defective peroxisomal biogenesis (Aubourg and Wan- FAS is also involved in endogenous activation of PPARg in adi- ders, 2013). Peroxisomal disorders are clinically heterogeneous. pocytes, we generated mice with adipose-specific deletion of However, they are consistently associated with impaired perox- FAS. Studies with these mice and embryonic fibroblasts derived isomal lipid metabolism, resulting in the accumulation of VLCFAs from these animals suggested that FAS is part of a lipogenic and phytanic acid, and defective synthesis of ether lipids and bile pathway that promotes PPARg activation in adipose tissue. Us- acids. ing a mass-spectrometry-based approach, we identified several Peroxisomal Biogenesis Disorders FAS-dependent alkyl ether-linked phosphatidylcholine species Diseases caused by defects in peroxisomal biogenesis include that were associated with PPARg in a rosiglitazone-displaceable Zellweger spectrum disorders and rhizomelic chondrodysplasia

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punctata type 1. Zellweger spectrum disorders affect 1 in 30,000 Future Directions infants and include cerebrohepatorenal Zellweger syndrome, As an organelle, peroxisomes get little respect. This may change neonatal adrenoleukodystrophy, and infantile Refsum disease. given increasing appreciation for the complexity of their interac- These diseases result from mutations in one of the dozen Pex tions with lipid droplets, the manner in which they complement genes involved in peroxisomal biogenesis. Mutations in Pex3, metabolic functions of mitochondria, and the critical part they Pex16, and Pex19, which result in the complete absence of per- play in the generation of ether lipids. Several aspects of peroxi- oxisomes, cause the most severe phenotypes. Mutations in somal biology are ripe for novel translational pathways to human other Pex genes result in ghost peroxisomes. Features of Zell- disease. weger syndrome, which is the most severe end of the clinical Ether lipids have been known for decades to be increased in spectrum, include craniofacial dysmorphism, hepatomegaly, a variety of cancers, although the precise implications of this and neurological abnormalities such as disruption of normal finding are poorly understood. FAS is linked to ether lipid pro- development, hypotonia, seizures, glaucoma, retinal degenera- duction, FAS expression is known to be increased in certain can- tion, and deafness. Most Zellweger infants do not survive past cers, and ether lipids appear to be increased in particularly 1 year of age due to respiratory compromise, gastrointestinal aggressive cancers (Benjamin et al., 2013). It is possible that bleeding, and liver failure. They seldom reach appropriate devel- targeted inhibition of a peroxisomal lipogenic pathway leading opmental milestones. The features of neonatal adrenoleukodys- to ether lipid production specifically in cancer cells could treat trophy and infantile Refsum disease are similar to those of malignancies without inducing developmental abnormalities Zellweger syndrome, but these disorders progress more slowly. seen with congenital loss of ether lipid synthesis. Mitochondrial Children with neonatal adrenoleukodystrophy usually die be- function is altered in insulin-resistant states such as obesity tween the age of 2 and 3 years. Patients with infantile Refsum and diabetes. Based on the relationships between peroxisomes disease can live into early adulthood (Aubourg and Wanders, and mitochondria, modulating ether lipid synthesis in the heart (a 2013). site commonly affected in diabetes), white blood cells (which Rhizomelic chondrodysplasia punctata type 1 (RCDP1) is may propagate diabetes complications through effects on clinically and genetically distinct from Zellweger spectrum disor- inflammation), nerves (neuropathy is a common and inade- ders. It is characterized by distinctive facial features, including a quately treated complication of diabetes), and other tissues prominent forehead, hypertelorism (widely set eyes), and ante- could affect mitochondrial endpoints potentially beneficial in verted nares (nostrils that face anteriorly due to an upturned obesity-related diabetes. Defects in ether lipid synthesis cause nose). These patients also suffer from growth failure, develop- protean neurologic disorders, and elevations of certain ether mental delay, seizures, and congenital cataracts. Most die lipids are found in neurodegenerative disorders such as Alz- in early childhood. RCDP1 is caused by mutations in Pex7, a heimer’s disease (Pettegrew et al., 2001). Dietary interventions chaperone for PTS2-containing peroxisomal matrix proteins, to alter peroxisomal activities could impact the clinical course including ADHAPS. A mouse model of RCDP1 has been gener- of neurologic diseases. ated by knocking out the Pex7 gene. Feeding these mice a diet Peroxisomes are still mysterious. Uncovering their signaling enriched in alkyl-glycerol partially rescues their phenotype, mechanisms and how they leverage relationships with lipid drop- implicating defective ether lipid synthesis in the pathophysiology lets and mitochondria might lead to new approaches to disease (Brites et al., 2011). and prompt us to view peroxisomes as intracellular patricians Disorders Caused by an Impaired Peroxisomal Function instead of plebians. Defects in a single peroxisomal protein in the setting of intact peroxisomal structure result in adult Refsum disease (ARD), ACKNOWLEDGMENTS X-linked adrenoleukodystrophy (X-ALD), RCDP2, and RCDP3. ARD is characterized biochemically by accumulation of phy- This work was supported by NIH grants DK076729, DK088083, T32 tanic acid, which is found in dairy products. Phytanic acid, DK007120, DK20579, DK56341, and K99 DK094874. a branched chain fatty acid, must first undergo a-oxidation in peroxisomes prior to b-oxidation in mitochondria. 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