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Fatty acid transport proteins in disease: New insights from invertebrate models
Fatty acid transport proteins in disease: new insights from invertebrate models Pierre Dourlen, Alyson Sujkowski, Robert Wessells, Bertrand Mollereau PII: DOI: Reference:
S0163-7827(15)30006-0 doi: 10.1016/j.plipres.2015.08.001 JPLR 885
To appear in: Received date: Accepted date:
27 July 2015 18 August 2015
Please cite this article as: Dourlen Pierre, Sujkowski Alyson, Wessells Robert, Mollereau Bertrand, Fatty acid transport proteins in disease: new insights from invertebrate models, (2015), doi: 10.1016/j.plipres.2015.08.001
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ACCEPTED MANUSCRIPT Fatty acid transport proteins in disease: new insights from invertebrate models
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Pierre Dourlen1, Alyson Sujkowski2, Robert Wessells2, Bertrand Mollereau1
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1) Laboratory of Molecular Biology of the Cell, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, UMS 3444 Biosciences Lyon Gerland, Université de Lyon, Lyon, France
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2) Department of Physiology, Wayne State University School of Medecine, Detroit, MI, USA
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Corresponding authors: [email protected] and [email protected]
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ACCEPTED MANUSCRIPT Abstract The dysregulation of lipid metabolism has been implicated in various diseases, including diabetes, cardiopathies, dermopathies, retinal and neurodegenerative diseases. Mouse
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models have provided insights into lipid metabolism. However, progress in the understanding
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of these pathologies is hampered by the multiplicity of essential cellular processes and genes that modulate lipid metabolism. Drosophila and C. elegans have emerged as simple genetic
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models to improve our understanding of these metabolic diseases. Recent studies have
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characterized fatty acid transport protein (fatp) mutants in Drosophila and C. elegans, establishing new models of cardiomyopathy, retinal degeneration, fat storage disease and
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dermopathies. These models have generated novel insights into the physiological role of the Fatp protein family in vivo in multicellular organisms, and are likely to contribute substantially to progress in understanding the etiology of various metabolic disorders. Here, we describe
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and discuss the mechanisms underlying invertebrate fatp mutant models in the light of the
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current knowledge relating to FATPs and lipid disorders in vertebrates.
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ACCEPTED MANUSCRIPT Box1: Gene and protein nomenclature fatp, Drosophila gene Fatp, Drosophila protein
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Fatp1, mammalian gene
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FATP1, mammalian protein
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FATP and FATP, the general family of genes and proteins without consideration of species
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Box2: List of abbreviations: ACS: acyl-CoA synthetase
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ACSVL: very long-chain acyl-CoA synthetase AMD: age-related macular degeneration AMPK: AMP-activated protein kinase
CoA: Coenzyme A
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AMP, ATP: adenosine mono-phosphate, adenosine tri-phosphate
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DGAT2: diacylglycerol acyltransferase-2 DILPs: Drosophila insulin-like peptides ER: endoplasmic reticulum FA: fatty acid
LCFA: long-chain fatty acid
VLCFA: very long-chain fatty acid FABPpm: plasma membrane FA-binding protein FACS: FA-coA synthase FAT: fatty acid translocase FATP: fatty acid transport protein HFD: high-fat diet IPS: Ichthyosis Prematurity Syndrome LD: lipid droplet LRAT: lecithin retinol acetyl transferase
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ACCEPTED MANUSCRIPT N-ret-PE: N-retinylidene-phosphatidylethanolamine PDH: pigment-cell-enriched dehydrogenase PG: pheromone gland
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PR: photoreceptor neuron
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RDH: retinol dehydrogenase Rh1: Rhodopsin1
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RPE: Retinal pigment epithelium
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SLC27: solute carrier family 27 SNP: single nucleotide polymorphism
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TAG: triacylglycerol – triglycerides
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ACCEPTED MANUSCRIPT Introduction
Fatty acids (FA) and in particular long chain FA (LCFA) are major sources of energy in the
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cell. FA are transported into cells and activated by linking to a Coenzyme A (CoA) to
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generate FA-CoA, a necessary intermediate for FA elongation, triacylglycerol / triglyceride (TAG) synthesis and FA β-oxidation. Dysregulation of FA transport and activation can
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provoke a dysregulation of lipid homeostasis, which plays a major role in many medical
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conditions, including obesity, diabetes, cardiopathies, skin syndromes and several diseases of the central nervous system, including diseases of the retina [1–3].
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Key actors of FA transport and activation are the FATP family of proteins officially named solute carrier family 27 (SLC27), herein referred to as FATP for simplicity [4,5]. The
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mammalian FATP family includes six members that have distinct expression patterns. They
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exhibit varying degrees of specialization in FA transport or activation, and are associated with lipid disorders in humans. For example, several single nucleotide polymorphisms
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(SNPs) within Fatp1 are associated with increased plasma triglyceride levels [6,7]. A polymorphism in the Fatp4 gene is associated with insulin resistance [8]. Missense and nonsense mutations in Fatp4 have been identified in patients with ichthyosis prematurity syndrome (IPS) [9,10]. Finally, a polymorphism in Fatp5 is associated with hepatic injury, insulin resistance, and dyslipidemia [11]. The variety of FATP mutations that are associated with lipid disorders suggests that FATP proteins could be useful therapeutic targets to maintain lipid homeostasis. Our understanding of the pathological mechanisms associated with disruption of lipid homeostasis is hampered by gene redundancy between FATP family members and by redundancy with other FA transport proteins, such as the plasma membrane fatty acid translocase (FAT/CD36) and the plasma membrane fatty acid binding protein (FABPpm), as well as Acyl-CoA synthetase (ACS). In addition, due to the variety of mechanisms controlled by lipids, which include cell signaling, membrane composition and energy production, it is often difficult to pinpoint the causes and consequences of the deregulations [12].
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ACCEPTED MANUSCRIPT A way to address this issue is to investigate the fundamental mechanisms in simpler genetic model organisms in which pathways can be more clearly defined. For example, the molecular activity of FATPs has been successfully examined in the genetically tractable
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yeast model, Saccharomyces cerevisiae, as described below. For physiological studies in
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multicellular organisms, the classical genetic models are the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans.
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For many years, Drosophila and C. elegans were not considered to be suitable models for
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the study of lipid metabolism, because they provide little material for biochemistry compared with rodent models (Table 1). However, recent advances in high mass resolution shotgun
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lipidomics have made detailed lipidomic analyses possible for small organisms [13–15]. For example, it is now possible to quantify 250 lipid species from 14 major classes with only ½ of a gut or 5 brains from third instar Drosophila larvae [15]. Consequently, research in lipid
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metabolism can now benefit from the unquestioned genetic power of these model organisms. Not only the ease of knocking down or overexpressing any genes anywhere at any time, but
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the low gene redundancy in these organisms, allow the rapid dissection of key enzymatic steps [16]. With the emergence of these models for studying dysfunctions of lipid metabolism [1,17,18], significant improvements in our understanding of the role of lipid metabolism in living animals have been made possible. Here, in addition to vertebrate FATPs, we review recent analyses of fatp mutations in Drosophila and C.elegans and their use for the establishment of genetic models of lipid disorder-associated diseases, especially cardiac, retinal, and fat storage diseases, as well as dermopathies.
The FATP family throughout evolution. The FATP family is conserved from yeast to human, with one gene in S.cerevisiae (FAT1), two genes in C.elegans (acs-20 and acs-22), three genes in D.melanogaster (fatp, CG30194 and CG3394) and six genes in mammals (Fatp1 to Fatp6). In phylogenetic analyses, worm and fly genes cluster with vertebrate Fatp1 and Fatp4, whereas Fatp2, Fatp3, Fatp5 and Fatp6 constitute a group only present in vertebrates [19]. Fatp1 and Fatp4 are likely to have
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ACCEPTED MANUSCRIPT the evolutionarily conserved functions of the family, and therefore studies of their orthologs in worms and flies are informative. In vertebrates and invertebrates, the expression of FATP family members is tissue specific
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(Table 2). In mammals, each FATP family member has a defined expression profile and
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functions in specific tissues with differences among species [4,20,21] . Some Fatp genes, such as Fatp1 and Fatp4, are expressed in multiple, overlapping tissues and organs. In the
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mouse, Fatp1 and Fatp4 are both expressed in the adipose, colon, heart, kidney, liver, lung,
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ovary, small intestine, testis and placenta, with higher levels of Fatp1 in the adipose and higher levels of Fatp4 in the small intestine [20]. In contrast, other Fatp genes have a more
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restricted profile and are only found in a few dedicated organs. For example, in the mouse, Fatp2 is mainly expressed in the liver, kidney and small intestine while Fatp5 is mainly expressed in the liver [20].
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In invertebrates, FATP expression is also tissue-specific (http://www.flyatlas.org/ and
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http://www.wormbase.org/, Table2). In adult Drosophila, fatp is thought to be the major
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member of the family as it is very strongly expressed in the adult eye, hindgut, fat body, heart and carcass, whereas fatp paralogs, CG3394 and CG30194, are only moderately expressed in some of the tissues of the adult fly (Table 2). These latter are more expressed in the larval stage.
Molecular activity of FATP proteins FATP proteins are characterized by two subdomains, the ATP-AMP motif, common to all members of the adenylate-forming superfamily of enzymes, and the FATP-ACSVL motif, restricted to the FATP family [22]. The first member of the FATP family was named Fatty acid transport protein 1 (FATP1) due to its ability to increase the uptake of fluorescent FA by 3T3L1 adipocytes [23]. It fulfilled the criteria of a fatty acid transporter, as expression and localization to the plasma membrane of adipocytes caused specific and saturable uptake of LCFAs [23]. Sequence analysis of FATP1 also revealed motifs in common with ACSs,
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ACCEPTED MANUSCRIPT including the ATP-AMP forming motif. Accordingly, it was shown that FATP1 displays verylong-chain acyl-CoA synthetase (ACSVL) activity in cell culture [24]. These results raised controversies concerning the true nature of FATP proteins and their
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molecular activities: are they ACSs, FA transporters or both? In addition, is the ACS activity
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involved in FA import or independent? This issue was strongly debated for FATP4 in particular, due to a disagreement on whether it localized to the plasma membrane, as
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required for a membrane FA transporter, or instead in intracellular organelle membranes
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[25–28]. Studies using the yeast FATP ortholog gene, FAT1, which encodes Fat1p, have helped to clarify the issues. Using several assays, it has been shown that Fat1p is a
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bifunctional protein performing both FA transport and activation (Fig. 1), although with different FA specificities. Deletion of FAT1 impairs uptake of LCFA and impairs growth under conditions where yeast are auxotrophic for LCFA [29]. By contrast, activation of VLCFA in
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FAT1∆ strain is reduced by 70% without affecting LCFA activation [30–32]. Furthermore, specific amino acid substitutions can eliminate one of these activities while leaving the other
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intact, indicating that transport and activation functions of Fat1p are completely separable in yeast cells [33]. However, Fat1p does also contribute to the activation of LCFA by forming a physical complex with the fatty acyl-CoA synthetases (FACS), Faa1p and Faa4p, to perform FA import by vectorial acylation [34]. This process consists in the coupling of exogenous FA transport through the plasma membrane with the activation of the FA by esterification with a CoA [22,35]. Thus, import of LCFA is coupled to esterification, but not by the ACS activity of Fat1p itself. The yeast model system has also been used to dissect the properties of mammalian FATPs. All six murine FATP isoforms have been expressed and their activities compared in a yeast strain (fat1∆ faa1∆) deficient for both transport of LCFA and activation of VLCFA [36]. This model provides a well-defined heterologous system to distinguish transport and activation functions. In this model, all FATP isoforms except FATP5 exhibit ACSVL activities, while only FATP1, -2, and -4 restore LCFA transport function [36]. The ACS activity of FATP1, 2, 3 and 6 each appears to be specific of VLCFA substrates, while FATP4 can also target LCFA [36].
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ACCEPTED MANUSCRIPT A naturally occurring variant of FATP2, FATP2b, has no ACSVL activity, but nevertheless enhances exogenous FA import when expressed in fat1∆ faa1∆ yeast. This is consistent with distinct transport and activation functions of FATP2 [37]. FATP5, also known as Bile acyl-coA
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synthetase, is the only murine protein with no ACSVL activity in yeast cells. In mammals, this
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protein is found in liver, where it has the unique function among FATPs of conjugating bile acid to CoA, a necessary step for recycling of secreted bile [38,39]. The inability of FATP6 to
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complement the transport defect of the faa1∆ fat1∆ strain is more surprising as it has been
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described as a heart-specific FA transport protein [40]. This may be due to an inability of the murine FATP6 to interact with endogenous yeast proteins with ACS activity, such as Faa4p.
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Nevertheless, the potential role of FATP6 in cardiac FA transport requires further study. Mammalian FATPs have been proposed to cooperate with additional ACS proteins for vectorial acylation of FA. This idea is supported by the fact that none of the murine FATP
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isoforms are able to complement the ACS-deficient faa1∆ faa4∆ strain [36], indicating a requirement for endogenous proteins with ACS activity. Also, in 3T3 L1 adipocytes, FATP1
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and ACSL1 form a complex at the plasma membrane and synergistically increase LCFA uptake in fibroblasts [41,42], demonstrating a functional association in a key mammalian cell type. Of note, overexpression of Acsl1 in mouse and faa1 in yeast also increases fatty acid transport [23,36].
Plasma membrane localization of FATP1, FATP2 and FATP4 may not be required for increasing FA uptake. In this case, intracellular esterification of FA with CoA decreases the concentration of free FA within the cell and drives FA uptake by a partially undefined mechanism [26,43–45]. C. elegans and Drosophila FATP proteins exhibit clear sequence conservation with yeast Fat1p and mammalian FATPs, suggesting that they possess both ACSVL and fatty acid transport activities. The ACSVL activity has been demonstrated for the C.elegans FATP family proteins ACS-20 and ACS-22, as the incorporation of C26:0 fatty acids into sphingomyelin is reduced in acs-20;acs-22 mutant worms [46]. ACS-20 is localized at the
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ACCEPTED MANUSCRIPT endoplasmic reticulum, and has not been detected at the plasma membrane, suggesting it may not be involved in direct transport [46]. Transport and activation functions of Drosophila Fatp have not yet been experimentally
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verified. However, Fatp shows a strong conservation in the AMP-ATP and FATP-ACSVL
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motifs, and in the residues required for LCFA transport and ACSVL activities as defined by mutagenesis in yeast (Fig. 2) [33]. Drosophila Fatp is also more similar to FATP1 and FATP4
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than to FATP6 (respectively 66%, 68% and 43% similarities) in an additional region of
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FATP1 and FATP4 that appears to be important for fatty acid transport activity when expressed in yeast (Fig. 2) [47].
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Transport and activation functions of insect Fatp have been verified in the pheromone gland (PG) of the silkworm Bombyx mori [48]. PGs of female moths treated with BmFATP dsRNA accumulate noticeably smaller lipid droplets (LDs) with less dietary C18:2 and C18:3 FA in
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their TAG than control. It is associated with decreased import of fluorescent FA BODIPY and reduced uptake of extracellular radiolabeled LCFAs. The mechanism of transport could be
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similar to vectorial acylation. However, no interaction with any ACSL has been tested, although ACSL are conserved in insects. Such experiments in insects would be an opportunity to study complexes between FATPs and ACSLs in vivo in a multicellular organism. In addition, knockdown of BmFATP reduces the activation of C16:0 and C18:1 in PG homogenates [48] and the expression of Fatp from the moth Eilema japonica increases C18:0 and C20:0 uptake in Escherichia coli [49].
The role of FATP in cardiac function In vertebrates, cardiac tissue is highly dependent on FA utilization to meet the high energy needs of a continuously active tissue [50]. Therefore, cardiac function is critically dependent on the correct regulation of FA uptake and utilization. FA are thought to enter the cardiomyocyte in two forms: 1) as components of lipoproteins [51,52] and 2) as free FAs [53]. Free FA entry is partly dependent on the membrane localization and activity of FATP family proteins [40]. Studies in both intact mice and cardiac myocytes have shown that imbalances
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ACCEPTED MANUSCRIPT in FA transport and use, due to changes in Fatp expression, lead to the progression of various cardiopathies [54–56]. The relationship between FATP levels and lipotoxic cardiomyopathy is complex, as both
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underexpression and overexpression have been shown to reduce cardiac function. Protein
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levels of FAT/CD36, the plasma membrane FA binding protein (FABPpm), FATP1 and 6, were all found to be low in a rat model of myocardial infarction, causing a shift in metabolism
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away from FA oxidation. In this study, the degree of myocardial dysfunction was directly
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proportional to the decrease in FA oxidation [57]. In a second study, FATP1 was overproduced in mouse myocardial tissue. This cardiac overproduction of FATP1 resulted in
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higher levels of myocardial free FA uptake, and of cardiac FA accumulation and metabolism, leading to left ventricular dysfunction [58].
Cell culture studies have implicated hormonal signaling in the regulation of FATP trafficking
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to the plasma membrane, activating FA transport into the cell. The cellular distribution of FATPs seems to be critical for the transport of FAs to various metabolic pools, for storage
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and/or ATP production. Insulin induces the translocation of FATP1 to the plasma membrane, which is accompanied by an increase in LCFA uptake in cultured adipocytes [27]. Likewise, obesity has been observed to induce a relocalization of FABPpm and FAT/CD36 to the membranes of cardiomyocytes [59]. Interestingly, AMP-activated protein kinase (AMPK) overexpression can rescue insulin resistance in lipodystrophic cardiomyocytes, by mimicking contraction-stimulated glucose uptake without preventing lipid accumulation [60], providing a potential molecular link between exercise and the rescue of lipodystrophic cardiomyopathy. Studies of mutants in the fly model collectively suggest that the genetic regulation of cardiac development, contractility and metabolism in the fly heart is similar to vertebrates [61]. These similarities are such that the fly heart has been developed as a model for the identification of genetic factors linking high-fat or high-sugar diets to cardiac lipotoxicity in humans [62–64]. For example, flies on a high-fat diet accumulate lipid droplets in the myocardium and develop conduction blocks and cardiomyopathies. These phenotypes can be rescued by genetically reducing TOR Kinase pathway activity or by increasing expression of lipases in cardiac
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ACCEPTED MANUSCRIPT tissue [62]. The fly heart was further used to discover that the PGC1-α homolog, spargel, is regulated by TOR Kinase and by the ATGL homolog brummer in the presence of high-fat diet, and that the deleterious effects of a high-fat diet can be rescued by overexpression of
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spargel [65]. When flies are fed on high-sugar, they also accumulate lipid droplets, and
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develop fibrosis-like phenotypes. These phenotypes could be rescued by decreasing the activity of the hexosamine biosynthetic pathway [64]. Additionally, the fly heart has been
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used as a model system to detail the effects of isocalorically altering nutrient composition on
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cardiac performance [66]. Even the effects of time-restricted feeding on physiology have been examined using the Drosophila heart as a model [67].
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Decreasing Drosophila fatp expression, either by genomic mutation or by heart-specific knockdown, leads to increases in myocardial lipid accumulation and autophagy that are
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correlated with cardiac dysfunction [68]. Flies with low levels of fatp expression have a higher
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cardiac frequency and lower fractional shortening, and are more sensitive to pacing-induced stress. Interestingly, mice overexpressing Fatp1 in cardiac tissue also have an altered QT
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interval, indicating a disruption of electrical activity [58]. Defects in electrical conductance have also been observed in other organs, including the retinas in mutant flies and mice [69,70].
The cardiac phenotypes of flies with low levels of fatp expression are entirely attributable to the higher levels of lipid storage in the myocardium, as treatments rescuing this lipid accumulation phenotype, either through lipase overexpression (Wessells, unpublished observation) or endurance exercise [68], also rescue cardiac function. It remains unclear whether AMPK is required for exercise to rescue lipodystrophic phenotypes in the fly model; this could be addressed in the near future by exercising AMPK knockdown flies fed a HFD. Although mice and especially rats have established, effective exercise protocols available, the advantage of the flies for exercise lies in the availability of the many genetic tools available, while flies are exercising. The heart phenotypes of fatp mutant flies could also serve as a perfect readout for modifier screens.
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ACCEPTED MANUSCRIPT C. elegans have no heart. However, their pharynx has already been used as a heart model to study heart amyloidosis [71]. Indeed, pharynx muscle cells have autonomous contractile activity, reminiscent of cardiac myocytes. Nevertheless, the role of the C.elegans FATP
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family proteins ACS-20 and ACS-22 have not been studied in these cells yet.
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The role of FATP in retina
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FATP and the lipid content of retina
Mammals and Drosophila eyes are composed of equivalent cell types despite a different
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organization. C elegans, on the other hand, does not have eyes, per se, but some lightsensitive neurons exist and are essential for negative phototaxism [72]. Contrary to the single-chambered mammalian eye, Drosophila has a faceted/compound eye composed of
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800 units called ommatidia [73]. However, mammalian retina and each Drosophila ommatidium both contain light-sensitive photoreceptor neurons (PR), mammalian rod and
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cone PRs being functionally equivalent to the Drosophila R1-R6 outer PRs and R7-R8 inner PRs, respectively. The mammalian retina also contains epithelium cells in the retinal pigment epithelium (RPE). This epithelium makes the link between the neuroretina and blood circulation and has vital functions in nutrition, protection and maintenance of the retina [74]. The equivalent cell types in the fly retina are the interommatidial cells (IOCs). Both in mammalian and fly PRs, components of the phototransduction cascade are inserted in membranes, organized as thousands of discs in the outer segment of mammalian PR and as thousands of microvilli in the rhabdomere of fly PRs. The outer segment of mammalian PRs are phagocytosed daily by the RPE and concomitantly renewed by membrane biogenesis at their bases. Therefore these membranous structures are very dynamic. Maintenance of these structures requires high lipid transport and metabolism in the retina and a unique lipid composition of the retina. The outer segments of PR display the highest content of docosahexaenoic acid (DHA, C22:6), a polyunsaturated FA contributing to membrane fluidity, in the human body [75]. In contrast, Drosophila has very few FA longer
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ACCEPTED MANUSCRIPT than C18. Instead, Drosophila has high levels of unsaturated C16 (C16:1) and C18 (C18:1, C18:2 and C18:3) FA. Physical properties of PR membranes are important as structural parameters, but they are also important for signaling. In Drosophila, upon light exposure, the
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cleavage of the minor lipid membrane phosphatidylinositol 4,5-bisphosphate (PIP2) by the
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NORPA phospholipase C (PLC) leads to a contraction of the rhabdomere, which is able to activate mechanosensitive channels [76]. Lipid composition of PR membrane is therefore
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essential to anchor and regulate the activity of proteins involved in phototransduction.
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In the Drosophila retina, Fatp is detected mainly in IOCs and in PRs, which could fit with a role of fatp in the supply of FA to the retina and the PRs [69]. Drosophila fatp expression
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begins during pupal stage, i.e. at late stage of PR differentiation, period during which PR morphogenesis takes place and at the end of which, rhodopsin (Rh), the light-sensitive protein, starts to be expressed [69,77]. fatp expression has been shown to be
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developmentally regulated by Inositol-requiring enzyme 1 (Ire1), an essential gene of the
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unfolded protein response (UPR) [78]. fatp mRNA is a specific target of regulated Ire1-
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dependent decay (RIDD). This regulation of fatp expression by ER stress and UPR pathways could be very relevant in mammals in neurological and hepatic disorders in which both dysregulation of lipid metabolism and ER stress have been observed [79,80]. In Ire1 mutant retina, Fatp levels are elevated and this is associated with higher levels of phosphatidic acid, suggesting a role of Fatp in phosphatidic acid synthesis, potentially due to an increased cellular import of FA [78]. Phosphatidic acid is involved in the regeneration cycle of PIP2, a central component of the phototransduction cascade. Interestingly, fatp-/- retina exhibits higher amplitude of the depolarization upon light stimulation [69]. This result could be explained by an impact of fatp on the PIP2 cycle. Thus, Fatp could regulate the FA composition of the retina with functional consequences. Analysis of the lipid composition of fatp-/- retina would be very informative. In mammals, two FATP members, Fatp1 and Fatp4, have been shown to be expressed in retina [70,81,82]. These two members are also the predominant Fatp members expressed in the microvessel endothelial cells of the Blood Brain Barrier where they selectively facilitate
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ACCEPTED MANUSCRIPT the transport of long-chain fatty acids, such as oleate (C18:1) and linoleic acid (C18:2) [83,84]. Surprisingly loss of Fatp1 does not affect the composition of total FA in the retina [70]. This may be due to a redundancy with Fatp4 but the loss of Fatp1 is not associated with
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a compensatory increase of Fatp4. It remains possible that FA composition in the different
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lipid species of the Fatp1-/- retina is altered rather than the composition of total FA. Future analysis of the FA and lipid compositions of Fatp4-/- and double knock-out Fatp1-/- Fatp4-/-
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retina will help to better understand the role of FATP1 and FATP4 in the transport and
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activation of FA in the retina.
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FATP and retinal degeneration
The loss of fatp results in retinal degeneration and was first observed in a genetic screen for Drosophila PR degeneration [85]. The characterization of retinal degeneration by live
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imaging revealed that fatp-/- PRs were gradually lost during adulthood [69,86]. From these
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results, the Drosophila fatp mutant retina was proposed as a new model of progressive late-
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onset PR degeneration [69]. PR degeneration in fatp mutant is due to a defect in the trafficking or degradation of Rh1 [69]. As a result, toxic Rh1-Arrestin2 complexes accumulate leading to PR death. The mechanisms by which fatp regulates Rh1 levels are not clear but this could be due to the regulation of phosphatidic acid or ceramide, which are important for Rh1 trafficking and/or degradation [87–89]. The regulation of Rh1 trafficking by fatp was confirmed in late pupal stage, in which fatp is regulated by RIDD and inhibits Rh1 delivery to the rhabdomere of the PR [78]. Thus, fatp is an essential gene for proper regulation of Rh1 and PR viability during terminal differentiation of PR and adulthood. Interestingly, a vital role has recently been observed for Fatp1 and Fatp4 in mouse retina models [70,82]. Fatp1 and Fatp4 are the two members of the FATP family expressed in the mouse retina, and they are the two members that share the closest homology with Drosophila fatp gene. Young Fatp1-/- mice have no evident ocular defect, but older Fatp1-/mice show signs of Aged-related Macular Degeneration (AMD) with PR outer segment disorganization, laminar thickening of the brush membrane and modified choroidal vessels
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ACCEPTED MANUSCRIPT [70]. Although Fatp4-/- mice are embryonic lethal, study of Fatp4-/- retina has been performed in Fatp4-/- mice rescued by a keratinocyte-specific Fatp4 expression [82]. The keratinocyterescued Fatp4-/- mouse also exhibits retinal defects with a high light sensitivity. Light
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exposure for 1.5 h at 10,000 lux induces severe retinal degeneration with a decrease in the
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thickness of the upper outer nuclear layer and a decrease in Rhodopsin and M- and S-opsin levels due to the loss of rods and cones [82]. In the Fatp4-/- mouse model, light–induced
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degeneration correlates with the accumulation of the toxic all-trans retinal, the chromophore
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of Rhodopsin, released after Rhodopsin activation by light [82]. This results from the release of the inhibitory activity of FATP4 on the visual cycle (see the section below on the visual
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cycle for details). All together, these results indicate that the Fatp1 and Fatp4 genes are required for correct retinal function and survival. Construction of a double Fatp1-/- Fatp4-/- KO mouse will be necessary to determine a possible redundancy of the two proteins.
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The loss of FATP is associated with retinal degeneration in both the Drosophila and mouse
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evolution.
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models, indicating that the role of FATP in retinal physiology has been conserved throughout
Role of FATP proteins in the visual cycle The visual cycle regenerates the chromophore of Rhodopsin, the light-sensing part of the protein, after its activation (Fig. 3). Studies in mammals have revealed that FATP proteins play an important role in the RPE, in interactions with the key enzymes of the visual cycle (Fig. 3). Mouse FATP1 was initially identified as an RPE65-interacting protein in a yeast two-hybrid screen [81]. RPE65 is a key enzyme that isomerizes all-trans retinyl esters (atRE) to 11-cisretinol (11cROL) [90–92], which constitutes the rate-limiting step of the visual cycle [93]. FATP1 also physically interacts with the lecithin retinol acetyl transferase (LRAT), an enzyme catalyzing the production of all-trans retinyl ester in the RPE. Moreover, the authors of this study showed that FATP1 interacted functionally with RPE65 and LRAT, thereby inhibiting the production of 11-cis-retinol in vitro, which suggests that FATP1 is an inhibitor of the visual
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ACCEPTED MANUSCRIPT cycle [81]. However, no defect in chromophore regeneration could be detected in vivo in Fatp1-/- mice, although these mice display early signs of AMD with aging [70]. As with retinal survival (see above), the absence of a visual cycle phenotype in vivo may reflect functional
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redundancy of FATP1 and FATP4 for chromophore regeneration. Consistent with this
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possibility, FATP4 has also been identified as a negative regulator of RPE65 [82]. In vitro enzymatic analyses showed that FATP4 inhibits RPE65 activity by competing for the same
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substrate and the product of FATP4 competes with atRE for the hydrophobic pocket of
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RPE65. In vivo, the FATP4-deficient RPE has significantly higher levels of isomerase activity, the key activity of the visual cycle catalyzed by RPE65. Moreover, FATP4-deficient mice
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display particularly high levels of the cytotoxic all-trans retinal, and this is probably responsible for the light-induced PR degeneration seen in these mice. Thus, both FATP1 and FATP4 are negative regulators of the visual cycle in mammals. Interestingly, LRAT and
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RPE65 catalyze two steps of the visual cycle during which the chromophore is bound to a FA, suggesting that the ACS or transporter activity of FATP1/4 may be involved.
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By contrast, the role of Drosophila Fatp in the visual cycle remains to be explored. The visual cycle was discovered recently in Drosophila, with the identification of two retinol dehydrogenases:
the
pigment-cell-enriched
dehydrogenase
(PDH)
and
retinol
dehydrogenase B (RDHB) (Fig. 3) [94,95]. Studying the role of Fatp in the visual cycle in Drosophila would undoubtedly be informative. If Drosophila Fatp behaves as a negative regulator of the visual cycle, as in mammals, then chromophore availability should be higher in the fatp-/- retina and promote Rh1 synthesis as previously described [96]. This would account for the reported accumulation of Rh1 in the degenerating retina in flies [69]. The fly retina would then serve as a powerful model for future studies uncovering further genetic interactions with fatp in this process.
Role of FATP in fat storage TAG storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs) in specialized cells like the adipocytes in mammals, cells of the fat body in Drosophila
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ACCEPTED MANUSCRIPT and intestinal cells of nematodes (Table1). TAG may also be stored, to a lesser extent, in other cell types such as the heart; however, accumulation of lipid droplets in the myocardium can become pathologic. TAG storage depends on cellular FA uptake, TAG synthesis and
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processes directly or indirectly in species from worm to human.
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TAG catabolism, and as presented in detail below, FATP proteins are involved in all three
The role of FATP in TAG storage as a result of the regulation of cellular FA uptake is well
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shown for FATP1 in mammals. FATP1 is expressed in white and brown adipose tissue, and
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in skeletal and cardiac muscle and increases fatty acid uptake. It responds to insulin by translocating from an intracellular perinuclear compartment to the plasma membrane and
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mediates insulin-stimulated FA cellular uptake [27,97]. Loss of Fatp1 in mice reduces coldinduced FA uptake and the size of the LDs in brown adipose tissue, which results in defects in non-shivering thermogenesis [98]. Loss of Fatp1 also protects mice from diet-induced
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obesity and insulin desensitization, by reducing insulin-stimulated uptake of fatty acids into muscle tissue and adipocytes, suggesting that molecules of the Fatp family could be effective
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anti-diabetic targets [99]. In mice knocked out for Fatp5, specifically expressed in the basal membranes of hepatocytes , long-chain fatty acid uptake is reduced in hepatocytes, but not in muscle or adipose tissue [100]. These mice also fail to gain weight on a HFD because of both decreased food intake and increased energy expenditure [101]. Excitingly, induced knockdown of Fatp5 in the liver can reverse already established diet-induced hepatic steatosis in mice [100]. Thus, in mammals FATP1 and FATP5 promote TAG storage by increasing FA uptake and therefore are promising therapeutic candidates for obesity, diabetes and non-alcoholic fatty liver disease. In C.elegans, ACS-22 is part of a TAG synthesis complex that facilitates LD expansion. It was identified in a genetic suppressor screen of LD expansion in a daf-22 mutant background; in this background FA β-oxidation is disrupted in the peroxisome resulting in LD expansion [102]. ACS-22 on the endoplasmic reticulum (ER) membrane interacts physically and functionally with the diacylglycerol acyltransferase-2 (DGAT-2) on LD, an enzyme that catalyzes the conjugation of a fatty acyl-CoA to diacyl glycerol and generates TAG. This
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ACCEPTED MANUSCRIPT interaction is conserved in mammalian cells, where FATP1 interacts with DGAT-2 [102]. Thus, thanks to the C.elegans model, it has been shown that in addition to increasing FA uptake, FATP1 promotes TAG storage as a component of the complex that enables ER–LD
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interaction and couples the synthesis of TAG and its deposition into LDs.
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In Drosophila, loss of one of the two copies of fatp results in increased TAG storage [68], which is in apparent contradiction with the role of FATP1, FATP5 and ACS-22 described
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above. In support of the Drosophila result, Fatp4A-/- mice, with adipocyte-specific inactivation
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of the Fatp4 gene, are obese when fed a high-fat diet whereas controls with identical food consumption are not [103]. In addition, increased incorporation of FA into TAG has also been
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reported in Fatp4 mutant fibroblasts in vitro [104]; decreased incorporation of FA into acylCoA but not into TAG has been described in Fatp4 mutant adipocytes in vitro [25]; and enterocytes from a Fatp4-null mouse with a rescued skin phenotype and fed a Western diet
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exhibit increased TAG content [105].
In all these situations, what could be the origin of the increased TAG storage? An increase in
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FA uptake, an excessive trafficking of FA toward TAG, or a decrease in TAG lipolysis? It has been shown in the Fatp4A-/- mice that the higher TAG content in adipocytes does not result from a deregulation of FA uptake [103]. In the thicker subcutaneous adipose tissue, lipolysis enzymes are not affected but the lipid composition is: the abundance of complex lipids, such as phospholipids, sphingolipids and cholesterol ester, is reduced, suggesting that in the absence of FATP4 ACS activity at the ER, FA are not incorporated into these complex lipids but are channeled to other compartments in the ER to be incorporated into TAG [103]. This altered FA trafficking would be the reason of the increased TAG storage in the subcutaneous adipose tissue. In the visceral adipose tissue, the role of FATP4 seems different because the expression of the lipolysis enzymes is downregulated and the composition of complex lipids is not affected. In these cells, FATP4 may re-esterify FA originating from basal lipolysis [25]. In the absence of FATP4, the expression of lipolysis enzymes may be reduced such that potentially harmful nonesterified FA does not accumulate [103]. Reduction of lipolysis would result in increased
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ACCEPTED MANUSCRIPT TAG storage. In support of this hypothesis, in Drosophila, fatp belongs to a group of genes involved in lipid catabolism and beta-oxidation that are expressed under starvation under the control of the conserved transcription factor dHNF4 [106]. It would be interesting to
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determine if mammalian FATPs also belong to such a gene network.
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The high TAG content in heterozygous fatp mutant flies is accompanied by a low feeding rate [68]. This is reminiscent of the reduced food intake by Fatp5 mutant mice on a HFD and
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suggests conservation of dietary behavior between Drosophila and mammals [101].
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Heterozygous fatp mutant flies also exhibit increased lifespan, independent of dietary nutrient content with no reduction in spontaneous activity or capacity for enforced exercise [68].
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Interestingly, knockdown of fatp expression in the fly fat body, which plays roles in the fly analogous to both vertebrate adipocytes and vertebrate liver is also sufficient to increase TAG storage, extend lifespan/stress resistance and reduce the feeding rate. This
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combination of phenotypes is reminiscent of hepatocyte-specific knockdown of Fatp5 in the mouse liver [107], suggesting a conserved link between Fatp family activity in liver-type
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tissues and feeding rate/TAG storage. In summary, loss of fatp in Drosophila results in physiological phenotypes also observed in mammalian FATP knockdowns.
Role of FATP in the mammal skin and worm cuticle
The Ichthyosis Prematurity Syndrome (IPS) is a rare disease characterized by a defective barrier function of the skin and belongs to a heterogeneous group of keratinization disorders, called autosomal-recessive congenital ichthyosis (ARCI) [108,109]. In humans, the Fatp4 gene maps in the locus associated with IPS on chromosomes 9q33.3-q34.13. Nonsense and missense mutations in the human Fatp4 genes have been identified in patients with IPS, mostly in Scandinavian populations [9,10]. This is the first known example of a human inherited disease caused by deficiency of a FATP family member. Similarly to the human syndrome, mice invalidated for Fatp4 are affected by a skin disease. Spontaneous Fatp4 mutant mice, the wrinkle-free mice and the pigskin mice, and targeted
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ACCEPTED MANUSCRIPT Fatp4-/- knockout mice all display a very tight, thick, wrinkle-free, shiny and smooth skin, with reduced numbers of hair follicles and a defective permeability barrier that no longer prevents loss of water from within [110–112]. They die neonatally due to dehydration and restricted
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movement. Fatp4-/- mice are rescued by the overexpression of Fatp4 specifically in epidermal
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keratinocytes, indicating a tissue-specific requirement for Fatp4 in the epidermis [113]. Transgene-rescued Fatp4 null mice exhibit abnormal development of sebaceous glands and
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abnormal sebum composition, implicating FATP4 in the formation of the sebaceous gland
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and sebum [114].
The lipid composition of Fatp4 mutant skin is highly abnormal with only low concentrations of
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ceramides containing VLCFA, which are crucial for normal barrier integrity [115], although the total amount of ceramide is higher than normal [111,113]. This paucity of VLCFAcontaining ceramides is possibly a direct consequence of the absence of the ACS activity of mutant
skin
are
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Fatp4
also
characterized
by
a
defect
in
the
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FATP4.
proliferation/differentiation of the epidermis, with an abnormal thickness of the skin, due to an
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increased number of proliferating cells in the suprabasal layer of the epidermis [112,116], with an altered expression profile of keratins [110,112] and with an abnormal activation of the EGF receptor and the downstream STAT3 signaling pathways [116]. In C. elegans, loss of acs-20 alone or loss of acs-20 and acs-22 results in a mild or severe disruption of the barrier function of the cuticle [46]. The structure of the acs-20;acs-22 mutant worm cuticle is abnormal. Tree longitudinal ridges on the cuticule on the lateral sides of the body, the alae, are incompletely extended, with a collagen-like matrix accumulation underneath and the accumulation of an unidentified matrix in the cuticle layers [46]. This is consistent with the phenotypes of Fatp4 mutant mice. Such a similar phenotype between mouse and worm is surprising as the structure of the worm cuticle is very different from that of the mammalian skin. Furthermore, acs-20 and acs-20;acs-22 mutant worms can be rescued by expressing a human Fatp4 transgene [46], suggesting that the role of FATP4 in the epidermis is fundamental and conserved from worm to humans. A detailed comparison
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ACCEPTED MANUSCRIPT between the common features of worm cuticule and mammalian skin would be fruitful to better understand the role of FATP4 in the skin. Consistent with Fatp4 homologs having a role in the synthesis of lipids essential for the
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surface barrier, the incorporation of VLCFA into sphingomyelin is reduced in acs-20;acs-22
be
detected
at
the
level
of
epidermal
cells
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mutant animals [46]. However, no obvious defects in terms of cell fate or cell integrity could [46],
contrasting
with
the
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proliferation/differentiation defects in Fatp4 mutant mouse skin. This may be due to the
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different organization of nematode and mammalian epidermis, or it may reflect differences in the role of Fatp4 in epidermal proliferation/differentiation between species. Alternatively, the
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hyperproliferation of the mouse epidermis in Fatp4 mutant may be simply a response to loss of the permeability barrier function. In support of this hypothesis, the abnormal accumulation of matrix in acs-20 mutant worms may similarly compensate for the impaired lipid barrier [46].
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In Drosophila, loss of fatp is lethal at the transition between the first and second larval instar [69]. The cause of lethality has not been investigated and may involve cuticle defects, or
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other defects affecting, for example, lipid absorption or lipid metabolism. This lethal phenotype is an ideal model to perform a mutagenesis suppressor screen and identify functional partners of fatp.
Conclusions and future outlook
In conclusion, development of invertebrate models to study FATP functions has revealed unexpected and underestimated parallels between vertebrate and invertebrate FATPs (Table3), the most surprising being at the level of the feeding rate behavior and at the level of the skin/cuticle as detailed above. In addition, it has enabled the characterization of new roles of FATPs in retinal cell survival and function, and in the synthesis and deposition of TAG in LDs. Some properties identified in invertebrate models, such as the regulation of fatp mRNA by ER stress, still need to be studied in vertebrate models.
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ACCEPTED MANUSCRIPT The establishment of invertebrate models to study FATP now allows the use of the powerful genetics tools available in these models. In particular, unbiased genetic screening with genome-wide collections of mutants and knockdown lines and epistasis analyses will
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facilitate understanding the specific molecular interactions, the complex metabolic networks
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and the tissue-specific functions of FATPs in an in vivo, physiologically relevant context. A clear illustration is the identification of the FATP1-DGAT2 complex and its role in LD
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formation in C. elegans as described above. Lastly, these simple organisms are amenable to
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chemical testing and screening, and should be a powerful tool for inexpensive, in vivo testing of drugs for the treatment of lipid metabolic diseases. Altogether, the use of the invertebrate
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models should contribute to a broad understanding of the FATP family.
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ACCEPTED MANUSCRIPT Figure legends
Fig. 1: Transport and ACS activities of FATP proteins. These proteins promote the uptake of
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free FA by the cell and catalyse their esterification with CoA to generate acyl-CoA. In yeast, Fat1p makes a complex with the ACSL Faa1p and Faa4p to couple FA transport with
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activation, a process called vectorial acylation. FATP proteins are localized in the plasma
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membrane and in the membrane of internal organel such as the endoplasmic reticulum. FATP1 and FATP4 proteins translocate from intracellular compartment to the plasma
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membrane upon insulin-induced stimulation of the cell or muscle contraction.
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Fig. 2: Protein sequence conservation between Drosophila Fatp, human FATP1, 4 and 6, worm ACS-20 and ACS-22, and yeast Fat1p. The alignment was done with the MultAlin tool
D
[117] based on sequence similarity. Residues in red and blue correspond to residues for
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which there is a strong consensus (>90%) and a weak consensus (>50%), respectively. For the consensus sequence: ! is used for a I or V residue, $ for a L or M residue, % for a F or Y
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residue and # for a N, D, Q or E residue. The magenta bars indicate the five consensus sequences for human acyl-coenzyme A synthetase (ACS) motifs, motif I and II correspond respectively to the ATP-AMP and FATP-ACSVL motifs [19]. Although originally proposed as a fatty acid-binding “signature motif” promoting acyl chain length specificity [118], examination of the crystal structure of Thermus thermophilus long-chain ACS suggests that Motif II may not be involved in chain length recognition [119,120]. Motif IV comprises the first five residues of the gate motif of Thermus thermophilus ACS (226-VPMFHVNAW-234), which controls the access of the fatty acid substrate to the catalytic site [119]. In the gate motif, the indole ring of W234 acts as the gate. This residue is not conserved in human ACS. Instead, a Y or F residue upstream of the motif IV has been proposed to play the role in ACSL6 [121,122]. Of note, in FATPs, a conserved aromatic residue (Y or F) is located 3 amino acid upstream of the motif IV. The blue stars denote the site required for yeast Fat1p activity [33]. The light blue stars label residues at which mutations distinguish between the
24
ACCEPTED MANUSCRIPT LCFA transport activity and the VLCFA ACS activity of Fat1p. The green bar indicates the position of the 73-amino acid segment of Fatp1 and 4 which is also important for their fatty
Fig. 3: Role of FATP in mouse and Drosophila retina
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acid transport activity in fat1∆ faa1∆ yeast [47].
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In mouse (A), the visual cycle recycles the chromophore, 11-cis retinal, of Rh, the light-
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sensitive protein of PRs [for a review, see [123][124]]. Enzymes of this cycle are distributed over PRs and RPE. FATP1 has been shown to interact with and to inhibit two of these
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enzymes, LRAT and RPE65 [81]. FATP4 has been shown to inhibit the activity of RPE65 [82]. RPE65 is the isomerase and the rate-limiting enzyme of the cycle.
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In Drosophila (B), Fatp is also expressed in PRs and pigment cells and is required for PR
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survival through Rh1 regulation [69]. The chromophore of Rh1 is a molecule of 3-hydroxyretinal (OHRal). The visual cycle in flies is not well documented: only two enzymes, PDH and
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RDHB, involved in this cycle have been described and not all the retinoid intermediates have been identified [94,95]. 11c-Ral: 11-cis retinal, at-Ral: all-trans retinal, at-Rol: all-trans retinol, PE: phosphatidyl ethanolamine, N-ret-PE: N-retinylidene-PE, RDH: retinol dehydrogenase, LRAT: lecithin-retinol acetyl transferase
25
ACCEPTED MANUSCRIPT
Acknowledgments BM’s research was supported by grants from the Fondation pour la Recherche Médicale, the
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CNRS (ATIP) and the ANR-12-BSV1-0019-01. PD was supported by the Retina France
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Association and the Ecole Normale Supérieure of Lyon (France). AS and RW are supported by grants from the MPOB and the Physiology Department of the Wayne State University
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School of Medicine.
Competing interest disclosure
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The authors have no financial or competing interests to declare.
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ACCEPTED MANUSCRIPT [111] Herrmann T, van der Hoeven F, Grone H-J, Stewart AF, Langbein L, Kaiser I, et al. Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy. J Cell Biol 2003;161:1105–15.
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ACCEPTED MANUSCRIPT [126] Gutierrez E, Wiggins D, Fielding B, Gould AP. Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature 2007;445:275–80. [127] Grönke S, Beller M, Fellert S, Ramakrishnan H, Jäckle H, Kühnlein RP. Control of fat storage by a Drosophila PAT domain protein. Curr Biol 2003;13:603–6.
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Figure 1
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Figure 2
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Figure 3
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ACCEPTED MANUSCRIPT Table 1: Conservation of cells and organs involved in lipid metabolism in mammals, nematodes and insects. Nematodes (C.elegans) 4µg
Oenocytes and cells of the fat body fulfill functions of liver hepatocytes including fat storage in lipid droplets and lipid mobilization [1,126,127].
Neutral lipids are stored in LRO and LD, localized in intestinal cells* [128].
Beta cells in the pancreas, secrete insulin which regulates lipid metabolism [2]
Eight Drosophila insulinlike peptides (DILPs) regulate lipid metabolism. DILPs synthesized by cells analogous to beta cells located in the brain and other tissues [129– 131].
No orthologous structure but insulin signaling regulates lipid mass in response to fasting and growth
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Liver (hepatocytes) Adipose tissue Fat storage in lipid droplets and lipid mobilization [125]
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Cells regulating carbohydrates and lipid metabolism
Insects (D. melanogaster) 0,5mg
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Animal approx. weight Organs dedicated to fat storage and metabolism
Mammals (M. musculus) 30g
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* There is still some debate about the nature of fatty acid storage organelles in C. elegans. They have been called gut granules, lysosome-related organelles (LRO) [132], vesicles distinct from LRO [133] and lipid droplets [134,135]. Using the selective staining properties of Nile Red (LRO only) and BODIPY (LRO and LD), Mak and col. were able to separate LD from LRO by centrifugation [128] and study the mechanisms regulating LD expansion [102,128]. These studies indicate that LD are fat storage structures conserved through evolution; they can be studied by applying the powerful genetic techniques available in C. elegans and Drosophila [136,137].
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ACCEPTED MANUSCRIPT Table 2: Nomenclature and tissue expression of FATP genes in mouse, Drosophila and C. elegans. For the mouse genes, the nomenclature corresponds to the FATP nomenclature, the official nomenclature and the ACSVL nomenclature. FATP1 and FATP4 are in bold
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Superscript symbols show analogous organ between species.
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Fatp3/Slc27a3/Acsvl3 Fatp4/Slc27a4/Acsvl5
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Mouse
Fatp2/Slc27a2/Acsvl1
Fatp5/Slc27a5/Acsvl6
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Fatp6/Slc27a6/Acsvl2
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D.melanogaster
Tissue expression mainly in: heart !, muscle#, retina§, adipose tissue$, and in: brain%, kidney, liver, lung, colon¤, placenta, ovary, testis, pancreas, small intestine¤ mainly in: liver, kidney, small intestine¤ and in: lung, colon¤, placenta, ovary, testis, Kidney, lung, ovary, testis, adrenal gland, mainly in: heart !, muscle#, retina§, small intestine¤, skin, and in: brain%, kidney, liver, lung, colon¤, placenta, ovary, testis, adipose tissue$ Mainly in: liver and in: kidney, lung mainly in: lung, testis, placenta and in: liver, ovary,heart ! Adult -very strongly expressed in: retina§, hindgut¤, fat body$, heart !, carcass -highly expressed in: head, crop, midgut¤, male accessory gland -moderately expressed in: brain%, malpighian tubules, salivary gland, virgin female spermatheca, inseminated female spermatheca Larva: -highly expressed in: midgut¤ -moderately expressed in: central nervous system%, hindgut¤, malpighian tubules, fat body$, salivary gland, trachea, carcass Adult -moderately expressed in: midgut¤, malpighian tubules, fat body$, virgin female spermatheca, inseminated female spermatheca, ovary, testis Larva: -very strongly expressed in: malpighian tubules, fat body$ -highly expressed in: midgut¤ -moderately expressed in: hindgut¤
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Gene name Fatp1/Slc27a1/Acsvl4
fatp
CG3394
CG30194
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because they are more closely related to the Drosophila and C.elegans orthologs.
moderately expressed in: testis
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head neuron% intestine¤ pharynx! hypodermis
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acs-22
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Intestine¤ hyp7 syncytium hypodermis* reproductive system pharynx! seam cell anal sphincter muscle# anal depressor muscle# vulval muscle# head
C.elegans
acs-20
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Table3 : Main processes and associated human diseases in which FATP genes are involved in mammals, Drosophila and C.elegans. “X” means required in the process, “0” not required in the process or in the organ, “?” means that the role of FATP is unknown. Processes Associated human Mammals Drosophila C.elegans diseases fat storage Obesity, nonX X ? alcoholic fatty liver disease LD expansion X ? X Feeding rate X X ? Heart lipotoxicity cardiomyopathy X X 0 Electrophysiological X X ? activity Retinal Macular X X 0 degeneration degenerations Epidermis Ichthyosis X ? X development and Prematurity maintenance syndrome thermogenesis X 0 0 Resistance to ? X ? stress/longevity
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S0163-7827(15)30006-0 doi: 10.1016/j.plipres.2015.08.001 JPLR 885
To appear in: Received date: Accepted date:
27 July 2015 18 August 2015
Please cite this article as: Dourlen Pierre, Sujkowski Alyson, Wessells Robert, Mollereau Bertrand, Fatty acid transport proteins in disease: new insights from invertebrate models, (2015), doi: 10.1016/j.plipres.2015.08.001
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ACCEPTED MANUSCRIPT Fatty acid transport proteins in disease: new insights from invertebrate models
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Pierre Dourlen1, Alyson Sujkowski2, Robert Wessells2, Bertrand Mollereau1
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1) Laboratory of Molecular Biology of the Cell, UMR5239 CNRS/Ecole Normale Supérieure de Lyon, UMS 3444 Biosciences Lyon Gerland, Université de Lyon, Lyon, France
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2) Department of Physiology, Wayne State University School of Medecine, Detroit, MI, USA
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Corresponding authors: [email protected] and [email protected]
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ACCEPTED MANUSCRIPT Abstract The dysregulation of lipid metabolism has been implicated in various diseases, including diabetes, cardiopathies, dermopathies, retinal and neurodegenerative diseases. Mouse
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models have provided insights into lipid metabolism. However, progress in the understanding
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of these pathologies is hampered by the multiplicity of essential cellular processes and genes that modulate lipid metabolism. Drosophila and C. elegans have emerged as simple genetic
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models to improve our understanding of these metabolic diseases. Recent studies have
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characterized fatty acid transport protein (fatp) mutants in Drosophila and C. elegans, establishing new models of cardiomyopathy, retinal degeneration, fat storage disease and
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dermopathies. These models have generated novel insights into the physiological role of the Fatp protein family in vivo in multicellular organisms, and are likely to contribute substantially to progress in understanding the etiology of various metabolic disorders. Here, we describe
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and discuss the mechanisms underlying invertebrate fatp mutant models in the light of the
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current knowledge relating to FATPs and lipid disorders in vertebrates.
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ACCEPTED MANUSCRIPT Box1: Gene and protein nomenclature fatp, Drosophila gene Fatp, Drosophila protein
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Fatp1, mammalian gene
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FATP1, mammalian protein
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FATP and FATP, the general family of genes and proteins without consideration of species
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Box2: List of abbreviations: ACS: acyl-CoA synthetase
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ACSVL: very long-chain acyl-CoA synthetase AMD: age-related macular degeneration AMPK: AMP-activated protein kinase
CoA: Coenzyme A
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AMP, ATP: adenosine mono-phosphate, adenosine tri-phosphate
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DGAT2: diacylglycerol acyltransferase-2 DILPs: Drosophila insulin-like peptides ER: endoplasmic reticulum FA: fatty acid
LCFA: long-chain fatty acid
VLCFA: very long-chain fatty acid FABPpm: plasma membrane FA-binding protein FACS: FA-coA synthase FAT: fatty acid translocase FATP: fatty acid transport protein HFD: high-fat diet IPS: Ichthyosis Prematurity Syndrome LD: lipid droplet LRAT: lecithin retinol acetyl transferase
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ACCEPTED MANUSCRIPT N-ret-PE: N-retinylidene-phosphatidylethanolamine PDH: pigment-cell-enriched dehydrogenase PG: pheromone gland
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PR: photoreceptor neuron
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RDH: retinol dehydrogenase Rh1: Rhodopsin1
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RPE: Retinal pigment epithelium
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SLC27: solute carrier family 27 SNP: single nucleotide polymorphism
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TAG: triacylglycerol – triglycerides
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ACCEPTED MANUSCRIPT Introduction
Fatty acids (FA) and in particular long chain FA (LCFA) are major sources of energy in the
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cell. FA are transported into cells and activated by linking to a Coenzyme A (CoA) to
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generate FA-CoA, a necessary intermediate for FA elongation, triacylglycerol / triglyceride (TAG) synthesis and FA β-oxidation. Dysregulation of FA transport and activation can
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provoke a dysregulation of lipid homeostasis, which plays a major role in many medical
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conditions, including obesity, diabetes, cardiopathies, skin syndromes and several diseases of the central nervous system, including diseases of the retina [1–3].
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Key actors of FA transport and activation are the FATP family of proteins officially named solute carrier family 27 (SLC27), herein referred to as FATP for simplicity [4,5]. The
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mammalian FATP family includes six members that have distinct expression patterns. They
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exhibit varying degrees of specialization in FA transport or activation, and are associated with lipid disorders in humans. For example, several single nucleotide polymorphisms
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(SNPs) within Fatp1 are associated with increased plasma triglyceride levels [6,7]. A polymorphism in the Fatp4 gene is associated with insulin resistance [8]. Missense and nonsense mutations in Fatp4 have been identified in patients with ichthyosis prematurity syndrome (IPS) [9,10]. Finally, a polymorphism in Fatp5 is associated with hepatic injury, insulin resistance, and dyslipidemia [11]. The variety of FATP mutations that are associated with lipid disorders suggests that FATP proteins could be useful therapeutic targets to maintain lipid homeostasis. Our understanding of the pathological mechanisms associated with disruption of lipid homeostasis is hampered by gene redundancy between FATP family members and by redundancy with other FA transport proteins, such as the plasma membrane fatty acid translocase (FAT/CD36) and the plasma membrane fatty acid binding protein (FABPpm), as well as Acyl-CoA synthetase (ACS). In addition, due to the variety of mechanisms controlled by lipids, which include cell signaling, membrane composition and energy production, it is often difficult to pinpoint the causes and consequences of the deregulations [12].
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ACCEPTED MANUSCRIPT A way to address this issue is to investigate the fundamental mechanisms in simpler genetic model organisms in which pathways can be more clearly defined. For example, the molecular activity of FATPs has been successfully examined in the genetically tractable
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yeast model, Saccharomyces cerevisiae, as described below. For physiological studies in
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multicellular organisms, the classical genetic models are the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans.
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For many years, Drosophila and C. elegans were not considered to be suitable models for
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the study of lipid metabolism, because they provide little material for biochemistry compared with rodent models (Table 1). However, recent advances in high mass resolution shotgun
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lipidomics have made detailed lipidomic analyses possible for small organisms [13–15]. For example, it is now possible to quantify 250 lipid species from 14 major classes with only ½ of a gut or 5 brains from third instar Drosophila larvae [15]. Consequently, research in lipid
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metabolism can now benefit from the unquestioned genetic power of these model organisms. Not only the ease of knocking down or overexpressing any genes anywhere at any time, but
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the low gene redundancy in these organisms, allow the rapid dissection of key enzymatic steps [16]. With the emergence of these models for studying dysfunctions of lipid metabolism [1,17,18], significant improvements in our understanding of the role of lipid metabolism in living animals have been made possible. Here, in addition to vertebrate FATPs, we review recent analyses of fatp mutations in Drosophila and C.elegans and their use for the establishment of genetic models of lipid disorder-associated diseases, especially cardiac, retinal, and fat storage diseases, as well as dermopathies.
The FATP family throughout evolution. The FATP family is conserved from yeast to human, with one gene in S.cerevisiae (FAT1), two genes in C.elegans (acs-20 and acs-22), three genes in D.melanogaster (fatp, CG30194 and CG3394) and six genes in mammals (Fatp1 to Fatp6). In phylogenetic analyses, worm and fly genes cluster with vertebrate Fatp1 and Fatp4, whereas Fatp2, Fatp3, Fatp5 and Fatp6 constitute a group only present in vertebrates [19]. Fatp1 and Fatp4 are likely to have
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ACCEPTED MANUSCRIPT the evolutionarily conserved functions of the family, and therefore studies of their orthologs in worms and flies are informative. In vertebrates and invertebrates, the expression of FATP family members is tissue specific
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(Table 2). In mammals, each FATP family member has a defined expression profile and
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functions in specific tissues with differences among species [4,20,21] . Some Fatp genes, such as Fatp1 and Fatp4, are expressed in multiple, overlapping tissues and organs. In the
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mouse, Fatp1 and Fatp4 are both expressed in the adipose, colon, heart, kidney, liver, lung,
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ovary, small intestine, testis and placenta, with higher levels of Fatp1 in the adipose and higher levels of Fatp4 in the small intestine [20]. In contrast, other Fatp genes have a more
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restricted profile and are only found in a few dedicated organs. For example, in the mouse, Fatp2 is mainly expressed in the liver, kidney and small intestine while Fatp5 is mainly expressed in the liver [20].
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In invertebrates, FATP expression is also tissue-specific (http://www.flyatlas.org/ and
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http://www.wormbase.org/, Table2). In adult Drosophila, fatp is thought to be the major
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member of the family as it is very strongly expressed in the adult eye, hindgut, fat body, heart and carcass, whereas fatp paralogs, CG3394 and CG30194, are only moderately expressed in some of the tissues of the adult fly (Table 2). These latter are more expressed in the larval stage.
Molecular activity of FATP proteins FATP proteins are characterized by two subdomains, the ATP-AMP motif, common to all members of the adenylate-forming superfamily of enzymes, and the FATP-ACSVL motif, restricted to the FATP family [22]. The first member of the FATP family was named Fatty acid transport protein 1 (FATP1) due to its ability to increase the uptake of fluorescent FA by 3T3L1 adipocytes [23]. It fulfilled the criteria of a fatty acid transporter, as expression and localization to the plasma membrane of adipocytes caused specific and saturable uptake of LCFAs [23]. Sequence analysis of FATP1 also revealed motifs in common with ACSs,
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ACCEPTED MANUSCRIPT including the ATP-AMP forming motif. Accordingly, it was shown that FATP1 displays verylong-chain acyl-CoA synthetase (ACSVL) activity in cell culture [24]. These results raised controversies concerning the true nature of FATP proteins and their
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molecular activities: are they ACSs, FA transporters or both? In addition, is the ACS activity
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involved in FA import or independent? This issue was strongly debated for FATP4 in particular, due to a disagreement on whether it localized to the plasma membrane, as
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required for a membrane FA transporter, or instead in intracellular organelle membranes
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[25–28]. Studies using the yeast FATP ortholog gene, FAT1, which encodes Fat1p, have helped to clarify the issues. Using several assays, it has been shown that Fat1p is a
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bifunctional protein performing both FA transport and activation (Fig. 1), although with different FA specificities. Deletion of FAT1 impairs uptake of LCFA and impairs growth under conditions where yeast are auxotrophic for LCFA [29]. By contrast, activation of VLCFA in
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FAT1∆ strain is reduced by 70% without affecting LCFA activation [30–32]. Furthermore, specific amino acid substitutions can eliminate one of these activities while leaving the other
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intact, indicating that transport and activation functions of Fat1p are completely separable in yeast cells [33]. However, Fat1p does also contribute to the activation of LCFA by forming a physical complex with the fatty acyl-CoA synthetases (FACS), Faa1p and Faa4p, to perform FA import by vectorial acylation [34]. This process consists in the coupling of exogenous FA transport through the plasma membrane with the activation of the FA by esterification with a CoA [22,35]. Thus, import of LCFA is coupled to esterification, but not by the ACS activity of Fat1p itself. The yeast model system has also been used to dissect the properties of mammalian FATPs. All six murine FATP isoforms have been expressed and their activities compared in a yeast strain (fat1∆ faa1∆) deficient for both transport of LCFA and activation of VLCFA [36]. This model provides a well-defined heterologous system to distinguish transport and activation functions. In this model, all FATP isoforms except FATP5 exhibit ACSVL activities, while only FATP1, -2, and -4 restore LCFA transport function [36]. The ACS activity of FATP1, 2, 3 and 6 each appears to be specific of VLCFA substrates, while FATP4 can also target LCFA [36].
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ACCEPTED MANUSCRIPT A naturally occurring variant of FATP2, FATP2b, has no ACSVL activity, but nevertheless enhances exogenous FA import when expressed in fat1∆ faa1∆ yeast. This is consistent with distinct transport and activation functions of FATP2 [37]. FATP5, also known as Bile acyl-coA
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synthetase, is the only murine protein with no ACSVL activity in yeast cells. In mammals, this
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protein is found in liver, where it has the unique function among FATPs of conjugating bile acid to CoA, a necessary step for recycling of secreted bile [38,39]. The inability of FATP6 to
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complement the transport defect of the faa1∆ fat1∆ strain is more surprising as it has been
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described as a heart-specific FA transport protein [40]. This may be due to an inability of the murine FATP6 to interact with endogenous yeast proteins with ACS activity, such as Faa4p.
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Nevertheless, the potential role of FATP6 in cardiac FA transport requires further study. Mammalian FATPs have been proposed to cooperate with additional ACS proteins for vectorial acylation of FA. This idea is supported by the fact that none of the murine FATP
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isoforms are able to complement the ACS-deficient faa1∆ faa4∆ strain [36], indicating a requirement for endogenous proteins with ACS activity. Also, in 3T3 L1 adipocytes, FATP1
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and ACSL1 form a complex at the plasma membrane and synergistically increase LCFA uptake in fibroblasts [41,42], demonstrating a functional association in a key mammalian cell type. Of note, overexpression of Acsl1 in mouse and faa1 in yeast also increases fatty acid transport [23,36].
Plasma membrane localization of FATP1, FATP2 and FATP4 may not be required for increasing FA uptake. In this case, intracellular esterification of FA with CoA decreases the concentration of free FA within the cell and drives FA uptake by a partially undefined mechanism [26,43–45]. C. elegans and Drosophila FATP proteins exhibit clear sequence conservation with yeast Fat1p and mammalian FATPs, suggesting that they possess both ACSVL and fatty acid transport activities. The ACSVL activity has been demonstrated for the C.elegans FATP family proteins ACS-20 and ACS-22, as the incorporation of C26:0 fatty acids into sphingomyelin is reduced in acs-20;acs-22 mutant worms [46]. ACS-20 is localized at the
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ACCEPTED MANUSCRIPT endoplasmic reticulum, and has not been detected at the plasma membrane, suggesting it may not be involved in direct transport [46]. Transport and activation functions of Drosophila Fatp have not yet been experimentally
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verified. However, Fatp shows a strong conservation in the AMP-ATP and FATP-ACSVL
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motifs, and in the residues required for LCFA transport and ACSVL activities as defined by mutagenesis in yeast (Fig. 2) [33]. Drosophila Fatp is also more similar to FATP1 and FATP4
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than to FATP6 (respectively 66%, 68% and 43% similarities) in an additional region of
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FATP1 and FATP4 that appears to be important for fatty acid transport activity when expressed in yeast (Fig. 2) [47].
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Transport and activation functions of insect Fatp have been verified in the pheromone gland (PG) of the silkworm Bombyx mori [48]. PGs of female moths treated with BmFATP dsRNA accumulate noticeably smaller lipid droplets (LDs) with less dietary C18:2 and C18:3 FA in
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their TAG than control. It is associated with decreased import of fluorescent FA BODIPY and reduced uptake of extracellular radiolabeled LCFAs. The mechanism of transport could be
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similar to vectorial acylation. However, no interaction with any ACSL has been tested, although ACSL are conserved in insects. Such experiments in insects would be an opportunity to study complexes between FATPs and ACSLs in vivo in a multicellular organism. In addition, knockdown of BmFATP reduces the activation of C16:0 and C18:1 in PG homogenates [48] and the expression of Fatp from the moth Eilema japonica increases C18:0 and C20:0 uptake in Escherichia coli [49].
The role of FATP in cardiac function In vertebrates, cardiac tissue is highly dependent on FA utilization to meet the high energy needs of a continuously active tissue [50]. Therefore, cardiac function is critically dependent on the correct regulation of FA uptake and utilization. FA are thought to enter the cardiomyocyte in two forms: 1) as components of lipoproteins [51,52] and 2) as free FAs [53]. Free FA entry is partly dependent on the membrane localization and activity of FATP family proteins [40]. Studies in both intact mice and cardiac myocytes have shown that imbalances
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ACCEPTED MANUSCRIPT in FA transport and use, due to changes in Fatp expression, lead to the progression of various cardiopathies [54–56]. The relationship between FATP levels and lipotoxic cardiomyopathy is complex, as both
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underexpression and overexpression have been shown to reduce cardiac function. Protein
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levels of FAT/CD36, the plasma membrane FA binding protein (FABPpm), FATP1 and 6, were all found to be low in a rat model of myocardial infarction, causing a shift in metabolism
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away from FA oxidation. In this study, the degree of myocardial dysfunction was directly
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proportional to the decrease in FA oxidation [57]. In a second study, FATP1 was overproduced in mouse myocardial tissue. This cardiac overproduction of FATP1 resulted in
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higher levels of myocardial free FA uptake, and of cardiac FA accumulation and metabolism, leading to left ventricular dysfunction [58].
Cell culture studies have implicated hormonal signaling in the regulation of FATP trafficking
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to the plasma membrane, activating FA transport into the cell. The cellular distribution of FATPs seems to be critical for the transport of FAs to various metabolic pools, for storage
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and/or ATP production. Insulin induces the translocation of FATP1 to the plasma membrane, which is accompanied by an increase in LCFA uptake in cultured adipocytes [27]. Likewise, obesity has been observed to induce a relocalization of FABPpm and FAT/CD36 to the membranes of cardiomyocytes [59]. Interestingly, AMP-activated protein kinase (AMPK) overexpression can rescue insulin resistance in lipodystrophic cardiomyocytes, by mimicking contraction-stimulated glucose uptake without preventing lipid accumulation [60], providing a potential molecular link between exercise and the rescue of lipodystrophic cardiomyopathy. Studies of mutants in the fly model collectively suggest that the genetic regulation of cardiac development, contractility and metabolism in the fly heart is similar to vertebrates [61]. These similarities are such that the fly heart has been developed as a model for the identification of genetic factors linking high-fat or high-sugar diets to cardiac lipotoxicity in humans [62–64]. For example, flies on a high-fat diet accumulate lipid droplets in the myocardium and develop conduction blocks and cardiomyopathies. These phenotypes can be rescued by genetically reducing TOR Kinase pathway activity or by increasing expression of lipases in cardiac
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ACCEPTED MANUSCRIPT tissue [62]. The fly heart was further used to discover that the PGC1-α homolog, spargel, is regulated by TOR Kinase and by the ATGL homolog brummer in the presence of high-fat diet, and that the deleterious effects of a high-fat diet can be rescued by overexpression of
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spargel [65]. When flies are fed on high-sugar, they also accumulate lipid droplets, and
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develop fibrosis-like phenotypes. These phenotypes could be rescued by decreasing the activity of the hexosamine biosynthetic pathway [64]. Additionally, the fly heart has been
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used as a model system to detail the effects of isocalorically altering nutrient composition on
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cardiac performance [66]. Even the effects of time-restricted feeding on physiology have been examined using the Drosophila heart as a model [67].
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Decreasing Drosophila fatp expression, either by genomic mutation or by heart-specific knockdown, leads to increases in myocardial lipid accumulation and autophagy that are
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correlated with cardiac dysfunction [68]. Flies with low levels of fatp expression have a higher
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cardiac frequency and lower fractional shortening, and are more sensitive to pacing-induced stress. Interestingly, mice overexpressing Fatp1 in cardiac tissue also have an altered QT
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interval, indicating a disruption of electrical activity [58]. Defects in electrical conductance have also been observed in other organs, including the retinas in mutant flies and mice [69,70].
The cardiac phenotypes of flies with low levels of fatp expression are entirely attributable to the higher levels of lipid storage in the myocardium, as treatments rescuing this lipid accumulation phenotype, either through lipase overexpression (Wessells, unpublished observation) or endurance exercise [68], also rescue cardiac function. It remains unclear whether AMPK is required for exercise to rescue lipodystrophic phenotypes in the fly model; this could be addressed in the near future by exercising AMPK knockdown flies fed a HFD. Although mice and especially rats have established, effective exercise protocols available, the advantage of the flies for exercise lies in the availability of the many genetic tools available, while flies are exercising. The heart phenotypes of fatp mutant flies could also serve as a perfect readout for modifier screens.
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ACCEPTED MANUSCRIPT C. elegans have no heart. However, their pharynx has already been used as a heart model to study heart amyloidosis [71]. Indeed, pharynx muscle cells have autonomous contractile activity, reminiscent of cardiac myocytes. Nevertheless, the role of the C.elegans FATP
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family proteins ACS-20 and ACS-22 have not been studied in these cells yet.
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The role of FATP in retina
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FATP and the lipid content of retina
Mammals and Drosophila eyes are composed of equivalent cell types despite a different
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organization. C elegans, on the other hand, does not have eyes, per se, but some lightsensitive neurons exist and are essential for negative phototaxism [72]. Contrary to the single-chambered mammalian eye, Drosophila has a faceted/compound eye composed of
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800 units called ommatidia [73]. However, mammalian retina and each Drosophila ommatidium both contain light-sensitive photoreceptor neurons (PR), mammalian rod and
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cone PRs being functionally equivalent to the Drosophila R1-R6 outer PRs and R7-R8 inner PRs, respectively. The mammalian retina also contains epithelium cells in the retinal pigment epithelium (RPE). This epithelium makes the link between the neuroretina and blood circulation and has vital functions in nutrition, protection and maintenance of the retina [74]. The equivalent cell types in the fly retina are the interommatidial cells (IOCs). Both in mammalian and fly PRs, components of the phototransduction cascade are inserted in membranes, organized as thousands of discs in the outer segment of mammalian PR and as thousands of microvilli in the rhabdomere of fly PRs. The outer segment of mammalian PRs are phagocytosed daily by the RPE and concomitantly renewed by membrane biogenesis at their bases. Therefore these membranous structures are very dynamic. Maintenance of these structures requires high lipid transport and metabolism in the retina and a unique lipid composition of the retina. The outer segments of PR display the highest content of docosahexaenoic acid (DHA, C22:6), a polyunsaturated FA contributing to membrane fluidity, in the human body [75]. In contrast, Drosophila has very few FA longer
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ACCEPTED MANUSCRIPT than C18. Instead, Drosophila has high levels of unsaturated C16 (C16:1) and C18 (C18:1, C18:2 and C18:3) FA. Physical properties of PR membranes are important as structural parameters, but they are also important for signaling. In Drosophila, upon light exposure, the
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cleavage of the minor lipid membrane phosphatidylinositol 4,5-bisphosphate (PIP2) by the
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NORPA phospholipase C (PLC) leads to a contraction of the rhabdomere, which is able to activate mechanosensitive channels [76]. Lipid composition of PR membrane is therefore
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essential to anchor and regulate the activity of proteins involved in phototransduction.
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In the Drosophila retina, Fatp is detected mainly in IOCs and in PRs, which could fit with a role of fatp in the supply of FA to the retina and the PRs [69]. Drosophila fatp expression
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begins during pupal stage, i.e. at late stage of PR differentiation, period during which PR morphogenesis takes place and at the end of which, rhodopsin (Rh), the light-sensitive protein, starts to be expressed [69,77]. fatp expression has been shown to be
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developmentally regulated by Inositol-requiring enzyme 1 (Ire1), an essential gene of the
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unfolded protein response (UPR) [78]. fatp mRNA is a specific target of regulated Ire1-
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dependent decay (RIDD). This regulation of fatp expression by ER stress and UPR pathways could be very relevant in mammals in neurological and hepatic disorders in which both dysregulation of lipid metabolism and ER stress have been observed [79,80]. In Ire1 mutant retina, Fatp levels are elevated and this is associated with higher levels of phosphatidic acid, suggesting a role of Fatp in phosphatidic acid synthesis, potentially due to an increased cellular import of FA [78]. Phosphatidic acid is involved in the regeneration cycle of PIP2, a central component of the phototransduction cascade. Interestingly, fatp-/- retina exhibits higher amplitude of the depolarization upon light stimulation [69]. This result could be explained by an impact of fatp on the PIP2 cycle. Thus, Fatp could regulate the FA composition of the retina with functional consequences. Analysis of the lipid composition of fatp-/- retina would be very informative. In mammals, two FATP members, Fatp1 and Fatp4, have been shown to be expressed in retina [70,81,82]. These two members are also the predominant Fatp members expressed in the microvessel endothelial cells of the Blood Brain Barrier where they selectively facilitate
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ACCEPTED MANUSCRIPT the transport of long-chain fatty acids, such as oleate (C18:1) and linoleic acid (C18:2) [83,84]. Surprisingly loss of Fatp1 does not affect the composition of total FA in the retina [70]. This may be due to a redundancy with Fatp4 but the loss of Fatp1 is not associated with
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a compensatory increase of Fatp4. It remains possible that FA composition in the different
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lipid species of the Fatp1-/- retina is altered rather than the composition of total FA. Future analysis of the FA and lipid compositions of Fatp4-/- and double knock-out Fatp1-/- Fatp4-/-
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retina will help to better understand the role of FATP1 and FATP4 in the transport and
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activation of FA in the retina.
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FATP and retinal degeneration
The loss of fatp results in retinal degeneration and was first observed in a genetic screen for Drosophila PR degeneration [85]. The characterization of retinal degeneration by live
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imaging revealed that fatp-/- PRs were gradually lost during adulthood [69,86]. From these
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results, the Drosophila fatp mutant retina was proposed as a new model of progressive late-
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onset PR degeneration [69]. PR degeneration in fatp mutant is due to a defect in the trafficking or degradation of Rh1 [69]. As a result, toxic Rh1-Arrestin2 complexes accumulate leading to PR death. The mechanisms by which fatp regulates Rh1 levels are not clear but this could be due to the regulation of phosphatidic acid or ceramide, which are important for Rh1 trafficking and/or degradation [87–89]. The regulation of Rh1 trafficking by fatp was confirmed in late pupal stage, in which fatp is regulated by RIDD and inhibits Rh1 delivery to the rhabdomere of the PR [78]. Thus, fatp is an essential gene for proper regulation of Rh1 and PR viability during terminal differentiation of PR and adulthood. Interestingly, a vital role has recently been observed for Fatp1 and Fatp4 in mouse retina models [70,82]. Fatp1 and Fatp4 are the two members of the FATP family expressed in the mouse retina, and they are the two members that share the closest homology with Drosophila fatp gene. Young Fatp1-/- mice have no evident ocular defect, but older Fatp1-/mice show signs of Aged-related Macular Degeneration (AMD) with PR outer segment disorganization, laminar thickening of the brush membrane and modified choroidal vessels
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ACCEPTED MANUSCRIPT [70]. Although Fatp4-/- mice are embryonic lethal, study of Fatp4-/- retina has been performed in Fatp4-/- mice rescued by a keratinocyte-specific Fatp4 expression [82]. The keratinocyterescued Fatp4-/- mouse also exhibits retinal defects with a high light sensitivity. Light
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exposure for 1.5 h at 10,000 lux induces severe retinal degeneration with a decrease in the
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thickness of the upper outer nuclear layer and a decrease in Rhodopsin and M- and S-opsin levels due to the loss of rods and cones [82]. In the Fatp4-/- mouse model, light–induced
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degeneration correlates with the accumulation of the toxic all-trans retinal, the chromophore
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of Rhodopsin, released after Rhodopsin activation by light [82]. This results from the release of the inhibitory activity of FATP4 on the visual cycle (see the section below on the visual
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cycle for details). All together, these results indicate that the Fatp1 and Fatp4 genes are required for correct retinal function and survival. Construction of a double Fatp1-/- Fatp4-/- KO mouse will be necessary to determine a possible redundancy of the two proteins.
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The loss of FATP is associated with retinal degeneration in both the Drosophila and mouse
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evolution.
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models, indicating that the role of FATP in retinal physiology has been conserved throughout
Role of FATP proteins in the visual cycle The visual cycle regenerates the chromophore of Rhodopsin, the light-sensing part of the protein, after its activation (Fig. 3). Studies in mammals have revealed that FATP proteins play an important role in the RPE, in interactions with the key enzymes of the visual cycle (Fig. 3). Mouse FATP1 was initially identified as an RPE65-interacting protein in a yeast two-hybrid screen [81]. RPE65 is a key enzyme that isomerizes all-trans retinyl esters (atRE) to 11-cisretinol (11cROL) [90–92], which constitutes the rate-limiting step of the visual cycle [93]. FATP1 also physically interacts with the lecithin retinol acetyl transferase (LRAT), an enzyme catalyzing the production of all-trans retinyl ester in the RPE. Moreover, the authors of this study showed that FATP1 interacted functionally with RPE65 and LRAT, thereby inhibiting the production of 11-cis-retinol in vitro, which suggests that FATP1 is an inhibitor of the visual
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ACCEPTED MANUSCRIPT cycle [81]. However, no defect in chromophore regeneration could be detected in vivo in Fatp1-/- mice, although these mice display early signs of AMD with aging [70]. As with retinal survival (see above), the absence of a visual cycle phenotype in vivo may reflect functional
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redundancy of FATP1 and FATP4 for chromophore regeneration. Consistent with this
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possibility, FATP4 has also been identified as a negative regulator of RPE65 [82]. In vitro enzymatic analyses showed that FATP4 inhibits RPE65 activity by competing for the same
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substrate and the product of FATP4 competes with atRE for the hydrophobic pocket of
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RPE65. In vivo, the FATP4-deficient RPE has significantly higher levels of isomerase activity, the key activity of the visual cycle catalyzed by RPE65. Moreover, FATP4-deficient mice
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display particularly high levels of the cytotoxic all-trans retinal, and this is probably responsible for the light-induced PR degeneration seen in these mice. Thus, both FATP1 and FATP4 are negative regulators of the visual cycle in mammals. Interestingly, LRAT and
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RPE65 catalyze two steps of the visual cycle during which the chromophore is bound to a FA, suggesting that the ACS or transporter activity of FATP1/4 may be involved.
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By contrast, the role of Drosophila Fatp in the visual cycle remains to be explored. The visual cycle was discovered recently in Drosophila, with the identification of two retinol dehydrogenases:
the
pigment-cell-enriched
dehydrogenase
(PDH)
and
retinol
dehydrogenase B (RDHB) (Fig. 3) [94,95]. Studying the role of Fatp in the visual cycle in Drosophila would undoubtedly be informative. If Drosophila Fatp behaves as a negative regulator of the visual cycle, as in mammals, then chromophore availability should be higher in the fatp-/- retina and promote Rh1 synthesis as previously described [96]. This would account for the reported accumulation of Rh1 in the degenerating retina in flies [69]. The fly retina would then serve as a powerful model for future studies uncovering further genetic interactions with fatp in this process.
Role of FATP in fat storage TAG storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs) in specialized cells like the adipocytes in mammals, cells of the fat body in Drosophila
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ACCEPTED MANUSCRIPT and intestinal cells of nematodes (Table1). TAG may also be stored, to a lesser extent, in other cell types such as the heart; however, accumulation of lipid droplets in the myocardium can become pathologic. TAG storage depends on cellular FA uptake, TAG synthesis and
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processes directly or indirectly in species from worm to human.
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TAG catabolism, and as presented in detail below, FATP proteins are involved in all three
The role of FATP in TAG storage as a result of the regulation of cellular FA uptake is well
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shown for FATP1 in mammals. FATP1 is expressed in white and brown adipose tissue, and
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in skeletal and cardiac muscle and increases fatty acid uptake. It responds to insulin by translocating from an intracellular perinuclear compartment to the plasma membrane and
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mediates insulin-stimulated FA cellular uptake [27,97]. Loss of Fatp1 in mice reduces coldinduced FA uptake and the size of the LDs in brown adipose tissue, which results in defects in non-shivering thermogenesis [98]. Loss of Fatp1 also protects mice from diet-induced
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obesity and insulin desensitization, by reducing insulin-stimulated uptake of fatty acids into muscle tissue and adipocytes, suggesting that molecules of the Fatp family could be effective
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anti-diabetic targets [99]. In mice knocked out for Fatp5, specifically expressed in the basal membranes of hepatocytes , long-chain fatty acid uptake is reduced in hepatocytes, but not in muscle or adipose tissue [100]. These mice also fail to gain weight on a HFD because of both decreased food intake and increased energy expenditure [101]. Excitingly, induced knockdown of Fatp5 in the liver can reverse already established diet-induced hepatic steatosis in mice [100]. Thus, in mammals FATP1 and FATP5 promote TAG storage by increasing FA uptake and therefore are promising therapeutic candidates for obesity, diabetes and non-alcoholic fatty liver disease. In C.elegans, ACS-22 is part of a TAG synthesis complex that facilitates LD expansion. It was identified in a genetic suppressor screen of LD expansion in a daf-22 mutant background; in this background FA β-oxidation is disrupted in the peroxisome resulting in LD expansion [102]. ACS-22 on the endoplasmic reticulum (ER) membrane interacts physically and functionally with the diacylglycerol acyltransferase-2 (DGAT-2) on LD, an enzyme that catalyzes the conjugation of a fatty acyl-CoA to diacyl glycerol and generates TAG. This
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ACCEPTED MANUSCRIPT interaction is conserved in mammalian cells, where FATP1 interacts with DGAT-2 [102]. Thus, thanks to the C.elegans model, it has been shown that in addition to increasing FA uptake, FATP1 promotes TAG storage as a component of the complex that enables ER–LD
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interaction and couples the synthesis of TAG and its deposition into LDs.
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In Drosophila, loss of one of the two copies of fatp results in increased TAG storage [68], which is in apparent contradiction with the role of FATP1, FATP5 and ACS-22 described
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above. In support of the Drosophila result, Fatp4A-/- mice, with adipocyte-specific inactivation
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of the Fatp4 gene, are obese when fed a high-fat diet whereas controls with identical food consumption are not [103]. In addition, increased incorporation of FA into TAG has also been
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reported in Fatp4 mutant fibroblasts in vitro [104]; decreased incorporation of FA into acylCoA but not into TAG has been described in Fatp4 mutant adipocytes in vitro [25]; and enterocytes from a Fatp4-null mouse with a rescued skin phenotype and fed a Western diet
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exhibit increased TAG content [105].
In all these situations, what could be the origin of the increased TAG storage? An increase in
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FA uptake, an excessive trafficking of FA toward TAG, or a decrease in TAG lipolysis? It has been shown in the Fatp4A-/- mice that the higher TAG content in adipocytes does not result from a deregulation of FA uptake [103]. In the thicker subcutaneous adipose tissue, lipolysis enzymes are not affected but the lipid composition is: the abundance of complex lipids, such as phospholipids, sphingolipids and cholesterol ester, is reduced, suggesting that in the absence of FATP4 ACS activity at the ER, FA are not incorporated into these complex lipids but are channeled to other compartments in the ER to be incorporated into TAG [103]. This altered FA trafficking would be the reason of the increased TAG storage in the subcutaneous adipose tissue. In the visceral adipose tissue, the role of FATP4 seems different because the expression of the lipolysis enzymes is downregulated and the composition of complex lipids is not affected. In these cells, FATP4 may re-esterify FA originating from basal lipolysis [25]. In the absence of FATP4, the expression of lipolysis enzymes may be reduced such that potentially harmful nonesterified FA does not accumulate [103]. Reduction of lipolysis would result in increased
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ACCEPTED MANUSCRIPT TAG storage. In support of this hypothesis, in Drosophila, fatp belongs to a group of genes involved in lipid catabolism and beta-oxidation that are expressed under starvation under the control of the conserved transcription factor dHNF4 [106]. It would be interesting to
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determine if mammalian FATPs also belong to such a gene network.
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The high TAG content in heterozygous fatp mutant flies is accompanied by a low feeding rate [68]. This is reminiscent of the reduced food intake by Fatp5 mutant mice on a HFD and
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suggests conservation of dietary behavior between Drosophila and mammals [101].
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Heterozygous fatp mutant flies also exhibit increased lifespan, independent of dietary nutrient content with no reduction in spontaneous activity or capacity for enforced exercise [68].
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Interestingly, knockdown of fatp expression in the fly fat body, which plays roles in the fly analogous to both vertebrate adipocytes and vertebrate liver is also sufficient to increase TAG storage, extend lifespan/stress resistance and reduce the feeding rate. This
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combination of phenotypes is reminiscent of hepatocyte-specific knockdown of Fatp5 in the mouse liver [107], suggesting a conserved link between Fatp family activity in liver-type
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tissues and feeding rate/TAG storage. In summary, loss of fatp in Drosophila results in physiological phenotypes also observed in mammalian FATP knockdowns.
Role of FATP in the mammal skin and worm cuticle
The Ichthyosis Prematurity Syndrome (IPS) is a rare disease characterized by a defective barrier function of the skin and belongs to a heterogeneous group of keratinization disorders, called autosomal-recessive congenital ichthyosis (ARCI) [108,109]. In humans, the Fatp4 gene maps in the locus associated with IPS on chromosomes 9q33.3-q34.13. Nonsense and missense mutations in the human Fatp4 genes have been identified in patients with IPS, mostly in Scandinavian populations [9,10]. This is the first known example of a human inherited disease caused by deficiency of a FATP family member. Similarly to the human syndrome, mice invalidated for Fatp4 are affected by a skin disease. Spontaneous Fatp4 mutant mice, the wrinkle-free mice and the pigskin mice, and targeted
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ACCEPTED MANUSCRIPT Fatp4-/- knockout mice all display a very tight, thick, wrinkle-free, shiny and smooth skin, with reduced numbers of hair follicles and a defective permeability barrier that no longer prevents loss of water from within [110–112]. They die neonatally due to dehydration and restricted
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movement. Fatp4-/- mice are rescued by the overexpression of Fatp4 specifically in epidermal
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keratinocytes, indicating a tissue-specific requirement for Fatp4 in the epidermis [113]. Transgene-rescued Fatp4 null mice exhibit abnormal development of sebaceous glands and
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abnormal sebum composition, implicating FATP4 in the formation of the sebaceous gland
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and sebum [114].
The lipid composition of Fatp4 mutant skin is highly abnormal with only low concentrations of
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ceramides containing VLCFA, which are crucial for normal barrier integrity [115], although the total amount of ceramide is higher than normal [111,113]. This paucity of VLCFAcontaining ceramides is possibly a direct consequence of the absence of the ACS activity of mutant
skin
are
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Fatp4
also
characterized
by
a
defect
in
the
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FATP4.
proliferation/differentiation of the epidermis, with an abnormal thickness of the skin, due to an
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increased number of proliferating cells in the suprabasal layer of the epidermis [112,116], with an altered expression profile of keratins [110,112] and with an abnormal activation of the EGF receptor and the downstream STAT3 signaling pathways [116]. In C. elegans, loss of acs-20 alone or loss of acs-20 and acs-22 results in a mild or severe disruption of the barrier function of the cuticle [46]. The structure of the acs-20;acs-22 mutant worm cuticle is abnormal. Tree longitudinal ridges on the cuticule on the lateral sides of the body, the alae, are incompletely extended, with a collagen-like matrix accumulation underneath and the accumulation of an unidentified matrix in the cuticle layers [46]. This is consistent with the phenotypes of Fatp4 mutant mice. Such a similar phenotype between mouse and worm is surprising as the structure of the worm cuticle is very different from that of the mammalian skin. Furthermore, acs-20 and acs-20;acs-22 mutant worms can be rescued by expressing a human Fatp4 transgene [46], suggesting that the role of FATP4 in the epidermis is fundamental and conserved from worm to humans. A detailed comparison
21
ACCEPTED MANUSCRIPT between the common features of worm cuticule and mammalian skin would be fruitful to better understand the role of FATP4 in the skin. Consistent with Fatp4 homologs having a role in the synthesis of lipids essential for the
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surface barrier, the incorporation of VLCFA into sphingomyelin is reduced in acs-20;acs-22
be
detected
at
the
level
of
epidermal
cells
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mutant animals [46]. However, no obvious defects in terms of cell fate or cell integrity could [46],
contrasting
with
the
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proliferation/differentiation defects in Fatp4 mutant mouse skin. This may be due to the
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different organization of nematode and mammalian epidermis, or it may reflect differences in the role of Fatp4 in epidermal proliferation/differentiation between species. Alternatively, the
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hyperproliferation of the mouse epidermis in Fatp4 mutant may be simply a response to loss of the permeability barrier function. In support of this hypothesis, the abnormal accumulation of matrix in acs-20 mutant worms may similarly compensate for the impaired lipid barrier [46].
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In Drosophila, loss of fatp is lethal at the transition between the first and second larval instar [69]. The cause of lethality has not been investigated and may involve cuticle defects, or
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other defects affecting, for example, lipid absorption or lipid metabolism. This lethal phenotype is an ideal model to perform a mutagenesis suppressor screen and identify functional partners of fatp.
Conclusions and future outlook
In conclusion, development of invertebrate models to study FATP functions has revealed unexpected and underestimated parallels between vertebrate and invertebrate FATPs (Table3), the most surprising being at the level of the feeding rate behavior and at the level of the skin/cuticle as detailed above. In addition, it has enabled the characterization of new roles of FATPs in retinal cell survival and function, and in the synthesis and deposition of TAG in LDs. Some properties identified in invertebrate models, such as the regulation of fatp mRNA by ER stress, still need to be studied in vertebrate models.
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ACCEPTED MANUSCRIPT The establishment of invertebrate models to study FATP now allows the use of the powerful genetics tools available in these models. In particular, unbiased genetic screening with genome-wide collections of mutants and knockdown lines and epistasis analyses will
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facilitate understanding the specific molecular interactions, the complex metabolic networks
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and the tissue-specific functions of FATPs in an in vivo, physiologically relevant context. A clear illustration is the identification of the FATP1-DGAT2 complex and its role in LD
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formation in C. elegans as described above. Lastly, these simple organisms are amenable to
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chemical testing and screening, and should be a powerful tool for inexpensive, in vivo testing of drugs for the treatment of lipid metabolic diseases. Altogether, the use of the invertebrate
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models should contribute to a broad understanding of the FATP family.
23
ACCEPTED MANUSCRIPT Figure legends
Fig. 1: Transport and ACS activities of FATP proteins. These proteins promote the uptake of
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free FA by the cell and catalyse their esterification with CoA to generate acyl-CoA. In yeast, Fat1p makes a complex with the ACSL Faa1p and Faa4p to couple FA transport with
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activation, a process called vectorial acylation. FATP proteins are localized in the plasma
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membrane and in the membrane of internal organel such as the endoplasmic reticulum. FATP1 and FATP4 proteins translocate from intracellular compartment to the plasma
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membrane upon insulin-induced stimulation of the cell or muscle contraction.
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Fig. 2: Protein sequence conservation between Drosophila Fatp, human FATP1, 4 and 6, worm ACS-20 and ACS-22, and yeast Fat1p. The alignment was done with the MultAlin tool
D
[117] based on sequence similarity. Residues in red and blue correspond to residues for
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which there is a strong consensus (>90%) and a weak consensus (>50%), respectively. For the consensus sequence: ! is used for a I or V residue, $ for a L or M residue, % for a F or Y
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residue and # for a N, D, Q or E residue. The magenta bars indicate the five consensus sequences for human acyl-coenzyme A synthetase (ACS) motifs, motif I and II correspond respectively to the ATP-AMP and FATP-ACSVL motifs [19]. Although originally proposed as a fatty acid-binding “signature motif” promoting acyl chain length specificity [118], examination of the crystal structure of Thermus thermophilus long-chain ACS suggests that Motif II may not be involved in chain length recognition [119,120]. Motif IV comprises the first five residues of the gate motif of Thermus thermophilus ACS (226-VPMFHVNAW-234), which controls the access of the fatty acid substrate to the catalytic site [119]. In the gate motif, the indole ring of W234 acts as the gate. This residue is not conserved in human ACS. Instead, a Y or F residue upstream of the motif IV has been proposed to play the role in ACSL6 [121,122]. Of note, in FATPs, a conserved aromatic residue (Y or F) is located 3 amino acid upstream of the motif IV. The blue stars denote the site required for yeast Fat1p activity [33]. The light blue stars label residues at which mutations distinguish between the
24
ACCEPTED MANUSCRIPT LCFA transport activity and the VLCFA ACS activity of Fat1p. The green bar indicates the position of the 73-amino acid segment of Fatp1 and 4 which is also important for their fatty
Fig. 3: Role of FATP in mouse and Drosophila retina
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acid transport activity in fat1∆ faa1∆ yeast [47].
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In mouse (A), the visual cycle recycles the chromophore, 11-cis retinal, of Rh, the light-
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sensitive protein of PRs [for a review, see [123][124]]. Enzymes of this cycle are distributed over PRs and RPE. FATP1 has been shown to interact with and to inhibit two of these
MA
enzymes, LRAT and RPE65 [81]. FATP4 has been shown to inhibit the activity of RPE65 [82]. RPE65 is the isomerase and the rate-limiting enzyme of the cycle.
D
In Drosophila (B), Fatp is also expressed in PRs and pigment cells and is required for PR
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survival through Rh1 regulation [69]. The chromophore of Rh1 is a molecule of 3-hydroxyretinal (OHRal). The visual cycle in flies is not well documented: only two enzymes, PDH and
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RDHB, involved in this cycle have been described and not all the retinoid intermediates have been identified [94,95]. 11c-Ral: 11-cis retinal, at-Ral: all-trans retinal, at-Rol: all-trans retinol, PE: phosphatidyl ethanolamine, N-ret-PE: N-retinylidene-PE, RDH: retinol dehydrogenase, LRAT: lecithin-retinol acetyl transferase
25
ACCEPTED MANUSCRIPT
Acknowledgments BM’s research was supported by grants from the Fondation pour la Recherche Médicale, the
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CNRS (ATIP) and the ANR-12-BSV1-0019-01. PD was supported by the Retina France
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Association and the Ecole Normale Supérieure of Lyon (France). AS and RW are supported by grants from the MPOB and the Physiology Department of the Wayne State University
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School of Medicine.
Competing interest disclosure
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The authors have no financial or competing interests to declare.
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ACCEPTED MANUSCRIPT [111] Herrmann T, van der Hoeven F, Grone H-J, Stewart AF, Langbein L, Kaiser I, et al. Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy. J Cell Biol 2003;161:1105–15.
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ACCEPTED MANUSCRIPT [126] Gutierrez E, Wiggins D, Fielding B, Gould AP. Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature 2007;445:275–80. [127] Grönke S, Beller M, Fellert S, Ramakrishnan H, Jäckle H, Kühnlein RP. Control of fat storage by a Drosophila PAT domain protein. Curr Biol 2003;13:603–6.
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ACCEPTED MANUSCRIPT Table 1: Conservation of cells and organs involved in lipid metabolism in mammals, nematodes and insects. Nematodes (C.elegans) 4µg
Oenocytes and cells of the fat body fulfill functions of liver hepatocytes including fat storage in lipid droplets and lipid mobilization [1,126,127].
Neutral lipids are stored in LRO and LD, localized in intestinal cells* [128].
Beta cells in the pancreas, secrete insulin which regulates lipid metabolism [2]
Eight Drosophila insulinlike peptides (DILPs) regulate lipid metabolism. DILPs synthesized by cells analogous to beta cells located in the brain and other tissues [129– 131].
No orthologous structure but insulin signaling regulates lipid mass in response to fasting and growth
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Liver (hepatocytes) Adipose tissue Fat storage in lipid droplets and lipid mobilization [125]
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Cells regulating carbohydrates and lipid metabolism
Insects (D. melanogaster) 0,5mg
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Animal approx. weight Organs dedicated to fat storage and metabolism
Mammals (M. musculus) 30g
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* There is still some debate about the nature of fatty acid storage organelles in C. elegans. They have been called gut granules, lysosome-related organelles (LRO) [132], vesicles distinct from LRO [133] and lipid droplets [134,135]. Using the selective staining properties of Nile Red (LRO only) and BODIPY (LRO and LD), Mak and col. were able to separate LD from LRO by centrifugation [128] and study the mechanisms regulating LD expansion [102,128]. These studies indicate that LD are fat storage structures conserved through evolution; they can be studied by applying the powerful genetic techniques available in C. elegans and Drosophila [136,137].
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ACCEPTED MANUSCRIPT Table 2: Nomenclature and tissue expression of FATP genes in mouse, Drosophila and C. elegans. For the mouse genes, the nomenclature corresponds to the FATP nomenclature, the official nomenclature and the ACSVL nomenclature. FATP1 and FATP4 are in bold
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Superscript symbols show analogous organ between species.
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Fatp3/Slc27a3/Acsvl3 Fatp4/Slc27a4/Acsvl5
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Mouse
Fatp2/Slc27a2/Acsvl1
Fatp5/Slc27a5/Acsvl6
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Fatp6/Slc27a6/Acsvl2
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Tissue expression mainly in: heart !, muscle#, retina§, adipose tissue$, and in: brain%, kidney, liver, lung, colon¤, placenta, ovary, testis, pancreas, small intestine¤ mainly in: liver, kidney, small intestine¤ and in: lung, colon¤, placenta, ovary, testis, Kidney, lung, ovary, testis, adrenal gland, mainly in: heart !, muscle#, retina§, small intestine¤, skin, and in: brain%, kidney, liver, lung, colon¤, placenta, ovary, testis, adipose tissue$ Mainly in: liver and in: kidney, lung mainly in: lung, testis, placenta and in: liver, ovary,heart ! Adult -very strongly expressed in: retina§, hindgut¤, fat body$, heart !, carcass -highly expressed in: head, crop, midgut¤, male accessory gland -moderately expressed in: brain%, malpighian tubules, salivary gland, virgin female spermatheca, inseminated female spermatheca Larva: -highly expressed in: midgut¤ -moderately expressed in: central nervous system%, hindgut¤, malpighian tubules, fat body$, salivary gland, trachea, carcass Adult -moderately expressed in: midgut¤, malpighian tubules, fat body$, virgin female spermatheca, inseminated female spermatheca, ovary, testis Larva: -very strongly expressed in: malpighian tubules, fat body$ -highly expressed in: midgut¤ -moderately expressed in: hindgut¤
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Gene name Fatp1/Slc27a1/Acsvl4
fatp
CG3394
CG30194
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because they are more closely related to the Drosophila and C.elegans orthologs.
moderately expressed in: testis
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head neuron% intestine¤ pharynx! hypodermis
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acs-22
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Intestine¤ hyp7 syncytium hypodermis* reproductive system pharynx! seam cell anal sphincter muscle# anal depressor muscle# vulval muscle# head
C.elegans
acs-20
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Table3 : Main processes and associated human diseases in which FATP genes are involved in mammals, Drosophila and C.elegans. “X” means required in the process, “0” not required in the process or in the organ, “?” means that the role of FATP is unknown. Processes Associated human Mammals Drosophila C.elegans diseases fat storage Obesity, nonX X ? alcoholic fatty liver disease LD expansion X ? X Feeding rate X X ? Heart lipotoxicity cardiomyopathy X X 0 Electrophysiological X X ? activity Retinal Macular X X 0 degeneration degenerations Epidermis Ichthyosis X ? X development and Prematurity maintenance syndrome thermogenesis X 0 0 Resistance to ? X ? stress/longevity
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