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The Lipid Droplet as a Potential Therapeutic Target in NAFLD David L. Silver, PhD1

1 Signature Research Program in Cardiovascular & Metabolic Disorders,

Duke-NUS Graduate Medical School, Singapore Semin Liver Dis 2013;33:312–320.

Abstract

Keywords

► nonalcoholic fatty liver disease ► lipid droplets ► triglyceride

Address for correspondence David L. Silver, PhD, Signature Research Program in Cardiovascular & Metabolic Disorders, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857 (e-mail: [email protected]).

Nonalcoholic fatty liver disease (NAFLD) is a growing problem worldwide. Nonalcoholic fatty liver disease is characterized by an abnormal accumulation of triglyceride-rich lipid droplets (LDs) in the liver, which can lead to liver inflammation and metabolic disturbances. Lipid droplets are dynamic organelles that have recently gained considerable scientific interest. Their formation and growth are regulated processes requiring the participation of many endoplasmic reticulum- (ER-) and LD-associated proteins, which may serve as potential therapeutic targets for NAFLD. Protein families such as fatinducing transmembrane proteins 1 and 2 (FITM1/FIT1 and FITM2/FIT2), the CIDE family of proteins, and the perilipin family, play important roles in LD biology. In this review, the authors discuss current views on LD formation and growth, and how various proteins may affect LD metabolism and lipoprotein assembly in the pathogenesis of NAFLD.

NAFLD and Triglyceride Lipid Droplets Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world.1 Nonalcoholic fatty liver disease is characterized by an excess accumulation of triglyceride (TG) within the hepatocyte, in organelles called lipid droplets (LDs). Nonalcoholic fatty liver disease is not a singly defined disease, but can manifest as a spectrum of disorders ranging from hepatic steatosis to nonalcoholic steatohepatitis (NASH). Accordingly, the severity of NAFLD varies widely from patient to patient, often incidentally detected in some asymptomatic individuals, while leading to liver inflammation, cirrhosis, and even hepatocellular carcinoma in others.2 A recent data analysis of the literature revealed the strongest risk factors for NAFLD to be central obesity, type 2 diabetes, dyslipidemia, and hypertension.3 Despite its widespread occurrence, current treatments for NAFLD remain limited. A recently published meta-analysis of randomized trials found that weight loss and thiazolidinediones improved liver histology and cardiometabolic profiles, although weight gain for the latter intervention was common.4 Other current and novel therapeutics for NAFLD have been previously discussed4–9 and are beyond the scope of this review. With the

Issue Theme Lipids and the Liver; Guest Editor, David E. Cohen, MD, PhD

prevalence of NAFLD on the rise,10 it is no surprise that the disease has garnered immense interest in the scientific community. Several comprehensive reviews have been published on the topic in recent years.2,3,11–17 In this review, we will present a LD-centric perspective on the pathogenesis of NAFLD. Because excessive deposition of lipid within hepatocyte cytosolic LDs characterizes NAFLD, we will take an indepth look at the mechanisms of LD biogenesis and growth.

Lipid Droplet Composition: More than a Droplet of Fat Once perceived as relatively simple storage particles for TG, LDs are now widely recognized as dynamic organelles. Lipid droplets are found in the cytoplasm, and are made up of a hydrophobic core of neutral and minimally charged lipids surrounded by a phospholipid monolayer. The mammalian LD phospholipid monolayer consists mainly of phosphatidylcholine (PC), followed in descending order of abundance by phosphatidylethanolamine (PE), phosphatidylinositol (PI), lyso-PC, and lyso-PE.18 Peripherally associated with or embedded within the phospholipid monolayer is an ecosystem of proteins that play important roles in LD biology. Lipid

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DOI http://dx.doi.org/ 10.1055/s-0033-1358521. ISSN 0272-8087.

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Vera J. Goh, BSc1

droplets are phylogenetically conserved organelles found in all eukaryotes. Lipid droplet composition has been examined using lipidomic and proteomic approaches in diverse eukaryotes including yeast, worm, fly, and mammals.18–28 Among the LD-associated proteins in mammals, the perilipin (Plin) family stands out as the most well characterized to date. There are five members of the Plin family, namely Plin1/ perilipin, Plin2/adipophilin, Plin3/tail-interacting protein of 47 kDa (TIP47), Plin4/S3–12, and Plin5/OXPAT. Plin proteins differ in terms of tissue expression: Plin2 and Plin3 are expressed ubiquitously; Plin1, Plin4, and Plin5 are expressed in a tissuerestricted manner. Plin1 and Plin2 are primarily found on the LD surface, whereas Plin3, Plin4, and Plin5 are stable in the cytoplasm and target to the LD surface upon fatty acid loading of cells, a stimulus that induces rapid LD accumulation.29–31 Plin proteins have been shown to distribute preferentially to LDs of different sizes in adipocytes,32 although it is unclear what mechanisms govern Plin targeting to LDs, or whether Plins have distinct functional roles on specific populations of LDs. For example, Plin3 coats very small-sized LDs in steatotic hepatocytes, whereas Plin1 and Plin2 associate with larger LDs.33 In general, Plin proteins are known to play important roles in stabilizing LDs and regulating lipolysis in the cell. The few and small LDs found in normal human and mouse livers contain Plin2 and Plin3, whereas Plin1 is additionally found only in steatotic livers.33 The expression of Plin1 in steatotic livers may be due to transcriptional transactivation by PPARgamma,34,35 a master regulator of adipogenesis that directly regulates the expression of Plin1 in adipose tissue. Indeed, induction of other PPARgamma targets has been seen in steatotic livers of rodents and humans.36–44 Plin1 and Plin2, but not Plin3, correlate positively with the amount of hepatocytic LDs in the steatotic liver.33 In a hepatoma cell model fed with oleic acid, de novo synthesized LDs stained for Plin2 and Plin3, but not Plin1, indicating that expression of these proteins preceded that of Plin1 during steatogenesis.33 Interestingly, like normal hepatocytes, enterocytes only express Plin2 and Plin3,45 suggesting a role for these proteins in lipoprotein assembly. Further differentiation between Plin functions was discovered by coherent anti-Stokes Raman scattering (CARS) microscopy, when Plin3 was found to coat cytosolic LDs in enterocytes after an acute fat challenge, whereas Plin2 was found to coat cytosolic LDs only after chronic fat challenge. The functional significance that Plin2 and Plin3 impart to hepatocytic LDs under acute or chronic fat challenge is unknown, but it is likely that each has multiple functions in regulating LD metabolism. Similar to its postulated function in enterocytes, Plin3 also seems to play a role in LD accumulation in the hepatocyte. Plin3 antisense oligonucleotide treatment in mice reduced histological features of hepatic steatosis. At the same time, reduction in Plin3 levels significantly blunted hepatic TG secretion, suggesting a role in hepatic lipoprotein metabolism.46

Lipid Droplets in Lipoprotein Assembly Lipoprotein assembly begins with the cotranslational lipidation of apoB during its translocation into the ER lumen. Very

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low-density lipoprotein (VLDL) is the primary lipoprotein synthesized and secreted by the liver. Its assembly is believed to occur via a two-step lipidation pathway whereby apoB-100 is first cotranslationally lipidated to form a dense pre-VLDL particle, then secondarily lipidated when the VLDL precursors combine with ER intraluminal TG-rich LDs to form maturing VLDL particles.47,48 Microsomal triglyceride transfer protein (MTTP), a heterodimer predominantly expressed in hepatocytes and enterocytes, lipidates the elongating apoB-100 polypeptide chain.49 In addition to apoB-100 lipidation in the liver, the 97kDa lipid-binding and transfer subunit of MTTP is also essential for the lipidation of the intestinespecific truncated form of apoB called apoB-48 during chylomicron synthesis.50 The lipidation of apoB stabilizes the polypeptide chain and prevents it from being degraded via the ER-associated degradation (ERAD) pathway. Apo B can also be degraded even when apoB lipidation is normal by a process called post-ER presecretory proteolysis.51 Humans with inactivating mutations in the MTTP gene suffer from a condition called familial abetalipoproteinemia. Patients with this disease have almost undetectable plasma apoB levels and develop hepatic steatosis. Human apoB mRNA can be translated into the full-length apoB-100 polypeptide chain in hepatocytes, or edited to translate apoB-48 containing only the N-terminal 48% of the polypeptide. Both truncating and nontruncating mutations in the human APOB gene cause familial hypobetalipoproteinemia (FHBL). Familial hypobetalipoproteinemia patients have defective apoB secretion and hepatic steatosis,52,53 and in some cases, intestinal fat malabsorption.53,54 Because MTTP and apoB represent two protein factors essential for the assembly and secretion of neutral lipid-containing VLDL, disrupting the function of either protein leads to hepatocyte TG retention and fatty liver. An important LD-associated protein affecting VLDL assembly and secretion is CIDEB. CIDEB is part of the cell deathinducing DNA fragmentation factor 45-like effector (CIDE) family, which also includes CIDEA and CIDEC/Fsp27. CIDEB is detected in many tissues, but is most highly expressed in the liver where it exists as both an ER- and LD-associated protein. CIDEB interacts with apoB and is involved in VLDL lipidation and maturation in mice.55 Peng Li and colleagues found that CIDEB null mice displayed reduced VLDL secretion with a concomitant increase in hepatic TG content as compared with control littermates. They proposed a model in which CIDEB is localized to the cytosolic side of the ER and LDs, and upon binding to apoB in VLDL precursors, facilitates the transfer of TG from cytosolic LDs or de novo synthesized TG within the ER bilayer onto the pre-VLDL. It remains to be tested if nascent LDs that remain attached to the ER are the source of apoB lipidation and sites of apoB-CIDEB interactions. In the subsequent steps of VLDL assembly, maturing VLDL particles travel from the ER to the Golgi via coat complex II(COPII-) coated vesicles.56 There, they acquire more phospholipid and neutral lipids to form mature VLDL particles.56,57 Formation of the COPII-coated vesicles, termed VLDL transport vesicles (VTVs), has been suggested to occur via CIDEB interaction with COPII components, Sar1 and Sec24.58 In a recent publication by Siddiqi et al, proteomic and Western Seminars in Liver Disease

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Lipid Droplet as a Potential Therapeutic Target in NAFLD

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blotting data show the presence of CIDEB in VTVs, but no other ER-derived vesicles. Furthermore, blocking CIDEB in an in vitro ER budding assay inhibited VTV budding.58 These combined studies indicate that CIDEB is important in both assembly of nascent VLDL and export of nascent VLDL to the Golgi. It is still unclear precisely how cytosolic LD metabolism is coordinated with lipoprotein synthesis and secretion, but some evidence exists that suggests a tug of war between the distribution of TG to cytosolic LDs versus distribution to the ER lumen. In support of this idea, it has recently been shown that CIDEB and Plin2 were found to play opposing roles in VLDL lipidation.59 CIDEB null mice were observed to have increased Plin2 levels, suggesting that ablation of CIDEB decreased VLDL lipidation, shunting neutral lipids synthesized in the ER toward the formation of cytosolic LDs. In further evidence for the opposing effects of Plin2 and CIDEB on VLDL synthesis, liver-specific knockdown of Plin2 in CIDEB null mice reduced the hepatic TG content and increased VLDL-TG secretion to wild-type levels.59 Accordingly, Plin2 knockout mice displayed reduced hepatic TG content and resistance to HFD-induced hepatic steatosis, possibly due to increased TG secretion from the liver.60 Chang and colleagues postulated that Plin2 could play a role in regulating LD biogenesis, whereby ablation of the protein would lead to accumulation of TG within its site of synthesis in the ER, inducing TG to be preferentially transferred toward ER intraluminal VLDL assembly. Although this idea has not been directly shown, it is supported by the increase in MTTP levels and hepatocyte microsomal TG content in Plin2 knockout mice.60 However, reduction of hepatic Plin2 levels using antisense oligonucleotide injections in two other mouse models of hepatic steatosis61 did not corroborate the previous observation in CIDEB / Plin2 knockdown mice of increased TG export as VLDL. Instead, expression of lipogenic genes was decreased along with VLDL secretion, suggesting that the protective effects of Plin2 deficiency against liver steatosis in these models were due to reduced lipogenesis rather than increased VLDL secretion. In a hepatoma cell line, knocking down Plin2 reduced the formation of LDs while increasing VLDL secretion.62 Increasing Plin2 levels in this cell modelinduced storage of TG in cytosolic LDs while reducing its secretion as VLDL. In view of contradicting results from different experimental approaches, it is more likely that LDs can both facilitate VLDL production as well as limit it by sequestering fatty substrates away from the ER. This underscores a recent finding that some LDs remain attached to the ER, while others do not, indicating the existence of distinct populations of LDs within a given cell. Such distinct populations could have opposing effects on VLDL assembly. Clearly, more work needs to be done to fully understand the role of the Plin proteins in modulating LD formation and VLDL secretion from the liver.

as Saccharomyces cerevisiae. Their conservation throughout evolution likely points to a conserved function in storing TG. Stored TG is used for phospholipid biosynthesis during logarithmic phase growth in yeast, or for storing excess energy in higher eukaryotes. Indeed, LDs provide the largest storage depot for energy. They store energy in the form of esterified fatty acids that can be mobilized to peripheral tissues for ATP production. Lipid droplets also help to maintain cellular lipid homeostasis in several ways. First, they function as reservoirs for sterols and fatty acids, which are important building blocks for the synthesis of cell membranes. Second, LDs are thought to compartmentalize lipids to prevent cellular damage caused by their excess. In particular, unesterified fatty acids may serve as bioactive signaling molecules, detergents, or may drive ceramide synthesis within cells, leading to effects often referred to as lipotoxicity.63–66 Acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) is an enzyme that catalyzes the final, committed step in the TGsynthesizing pathway preceding LD formation. As Farese and coworkers discovered, human DGAT1 deficiency results in a congenital diarrheal disorder,67 which in turn is postulated to be due to the lipotoxic effects of accumulating DGAT1 substrates, DAG, and fatty acids, on enterocyte function. DGAT1 is one of two unrelated genes in mammals. The other DGAT in mammals is DGAT2 that mediates the same enzymatic reaction of fatty acid esterification with DAG to form TG. Interestingly, only DGAT1 and not DGAT2 is expressed in human intestine. Conversely, murine intestine contains both DGATs, and loss of murine DGAT1 alone does not lead to the diarrheal phenotype seen in humans.68,69 The gastrointestinal phenotype of loss of both DGATs in mice has yet to be reported. Another function of LDs is their purported role in regulating protein metabolism. In leukocytes, LDs are formed in response to infectious and inflammatory stimuli, and thought to serve as niduses for the biosynthesis of eicosanoids such as prostaglandins and leukotrienes.70–73 Lipid droplets may also serve as parking spots for hydrophobic proteins, as many proteins have previously been observed to localize to LDs.20 Another function of LDs is their possible role in the ERAD of misfolded ER membrane proteins via the ubiquitin-proteasome pathway. Cytosolic LDs harbor some proteins involved in ERAD, such as the ubiquitin-like (UBX) domain-containing protein Ubx2/Ubxd8 and the ancient ubiquitous protein 1 (AUP1),74–76 and may play a role in ER quality control. Furthermore, cytosolic LD-associated apoB has been shown to represent a lipidated apoB pool targeted for ERAD,77,78 possibly via an ER-to-cytoplasm escape hatch mechanism,79 which is separate from the poorly lipidated apoB degradation at the translocon.80 This suggests that LDs may help to regulate the amount and quality of secreted apoB by participating in protein degradation. However, there is also evidence suggesting that LDs are dispensable for the ERAD of proteins in yeast.81 Finally, LDs participate in some pathogen-host interactions, such as when they serve as assembly platforms for virion synthesis during hepatitis C virus (HCV) replication.82 In HCV-infected hepatocytes, the HCV core is thought to promote virus-induced steatosis by decreasing LD turnover.83

Lipid Droplet Function Lipid droplets are found ubiquitously in almost all eukaryotic cells including the most ancient extant species of yeast such Seminars in Liver Disease

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Lipid Droplet Formation Lipid droplets are formed at the ER where neutral lipid synthesis enzymes like acyl-CoA:cholesterol acyltransferases (ACATs) and DGATs reside, catalyzing the formation of sterol esters and TG, respectively. There have been several proposed models of LD formation, although one particular model has gained considerable popularity. This model posits that LDs form from a “lens” in the cytoplasmic leaflet of the ER membrane that pinches out from the ER, taking with it the newly synthesized TG that accumulates within the ER bilayer. In a recent publication, such nascent LDs may have been visualized during fatty acid loading of drosophila S2 cells,84 but are not commonly reported. This paucity in evidence for visualizing nascent LD structures may be due to technical challenges or the short-lived nature of these structures. It would likely be energetically unfavorable for TG to cluster at one location between the ER membrane leaflets, and TG should instead travel freely within the bilayer to reduce overall stress on the membrane.85 Based on the “lens” hypothesis of LD formation, it is therefore logical to hypothesize the role of a TG-binding protein localized to the ER membrane that could sequester TG at one point and physically facilitate the partitioning of TG into a LD. Fatty acids composing PC and lyso-PC in LDs are uniquely more unsaturated than those found in the ER,19 suggesting that these rare species of phospholipids either partition onto LDs after being synthesized in the ER, or are synthesized through desaturation and phospholipase action on the LDs themselves. It is still unknown what factors participate in physically partitioning TG into a forming LD, and how phospholipids move from the ER membrane to form a monolayer delineating the budding particle. A promising candidate protein family postulated to play a role in de novo LD biogenesis is the fat-inducing transmembrane (FIT) protein family, comprised of FITM1/FIT1 and FITM2/FIT2. Fat-inducing transmembrane proteins were first identified from a genetic screen using PPARalpha-agonist treated mouse livers. PPARalpha activation induced FIT1 and FIT2 gene expression in murine liver but not heart. Fatinducing transmembrane proteins are localized to the ER and have six transmembrane domains with both N- and C-termini facing the cytosol.86 Fat-inducing transmembrane proteins were found to be important in the partitioning of neutral lipid into cytosolic LDs, but do not participate in neutral lipid synthesis.87 Furthermore, overexpression of FIT2 protein in human embryonic kidney cells, murine liver,87 and muscle88 induced the formation of LDs, while shRNA-mediated FIT2 knockdown in cultured adipocytes drastically reduced the number of LDs.87 Using purified FIT proteins in detergent micelles, it was shown that both proteins bind directly to TG and DAG, with FIT2 having a higher affinity and capacity to bind these neutral lipids than FIT1. It is perhaps not coincidental that overexpression of FIT2 in cells produces larger LDs than overexpression of FIT1, as size of the LDs directly correlates with TG binding affinity to FIT. Binding preference for both FIT proteins was higher for TG and lower for DAG. Mutagenesis studies identified a gain-of-function mutant of FIT2 that generates larger LDs than wild-type FIT2, and a

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partial loss-of-function mutant that produces smaller LDs than wild-type FIT2. Importantly, the gain-of-function mutant exhibited a significant increase in TG and DAG binding, while the partial loss-of-function mutant showed reduced binding.89 These findings are consistent with the conclusion that TG binding by FIT proteins is important in FIT-mediated LD formation. One possible model of FIT function is that FIT proteins play a role in the initial local accumulation of TG within the leaflets of the ER and thus act as regulatory proteins for determining where and when a nascent LD will form. Although the ability to bind TG is essential to FIT protein function in generating LDs, it is unknown whether this TG-binding property alone is sufficient for FIT to induce LD formation. It is also possible that either FIT proteins bind to other LD-stabilizing proteins that promote LD formation or they harbor more complex activity in partitioning TG or phospholipids onto a growing LD. That said, evidence for direct binding of phospholipids to FIT is lacking.89 Lipoprotein-secreting liver and intestine can form lipid-containing particles that bud into the cytosol or the ER lumen.49 That FIT proteins bind directly to TG and are ER transmembrane proteins raises the possibility that they could mediate both types of budding (►Fig. 1). Fat-inducing transmembrane proteins have distinct tissue distributions, with FIT1 being expressed primarily in muscle and to a lesser extent in heart. Although FIT1 mRNA is expressed in mouse liver, the tissue from which it was initially discovered, surprisingly FIT1 protein has not been found in liver. FIT2 is ubiquitously expressed in mouse and human tissues, with the highest levels in adipose tissue.87 The high level of FIT2 in both white and brown adipose tissue of mouse is likely due to its direct regulation by PPARgamma.90,91 Indeed, FIT2 expression is induced during adipogenesis of 3T3-L1 cells.87 The tissue distribution of FIT proteins suggests unique functions in regulating LD formation. The small and large LDs formed by FIT1 and FIT2 overexpression, respectively, are reminiscent of the characteristic LDs found in muscle and adipocytes. These observations suggest that FIT proteins might be in part responsible for the size distribution of LDs in various tissues. Normal skeletal muscle contains small LDs adjacent to mitochondria; adipose tissue contains large or unilocular LDs. The FIT protein family is highly evolutionarily conserved, with FIT orthologs found in mammals, birds, amphibians, worms, insects, and yeast.87 In fact, Saccharomyces cerevisiae has two homologs of FIT2, named SCS3 and YFT2. Expression of yeast homologs of FIT2 in human embryonic kidney cells promotes LD formation, while expression of human FIT2 in yeast rescues the inositol auxotrophic phenotype of SCS3deleted yeast.92 This indicates that despite the passage of greater than 170 million years and crossing the species barrier, yeast orthologs and human FIT retain some common function. It has recently been shown using synthetic genetic interaction arrays that yeast strains deleted for either or both genes display shared and unique phenotypes involving phospholipid biosynthesis and lipid metabolism.92 However, deletion of SCS3 or YFT2 did not abrogate LD formation in yeast, indicating that FIT2 may in fact play a more fundamental role Seminars in Liver Disease

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Lipid Droplet as a Potential Therapeutic Target in NAFLD

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Fig. 1 Model of cytosolic lipid droplet (LD) and very low-density lipoprotein (VLDL) assembly in the liver. This model proposes that FIT2 binds triglyceride (TG) at the endoplasmic reticulum (ER) and generates a nascent LD by concentrating TG between the leaflets of the ER. The production of a nascent LD or a mature LD that remains attached to the ER might supply the TG for apoB lipidation. Thus, FIT2 might facilitate both extraluminal and intraluminal LD budding. During this process, other important LD-associated proteins like CIDEB and Plin2 could regulate the directional flux of LD formation either into the ER lumen or into the cytosol. MTTP, microsomal triglyceride transfer protein.

in lipid metabolism at the ER, which precedes the development of its function in LD formation.

Lipid Droplet Growth: Expansion and Fusion Lipid droplets are dynamic organelles that contract in volume through lipolysis of the TG core, and expand in volume when excess fatty acids are present in the cell. Two models for LD growth are strongly supported by experimental evidence—in situ TG synthesis at the LD84 and fusion of two LDs. With the intent of identifying genes involved in LD formation and growth, Guo et al used a genome-wide RNAi screen in drosophila S2 cells. This screen revealed that genes encoding enzymes of the phospholipid biosynthesis pathway determined LD size and number.93 Lipid droplet growth requires the addition of surface phospholipids, of which PC is the most abundant18 and acts like a surfactant to prevent LD coalescence.94 It is thought that when PC is limiting, LDs fuse to form a larger LD, decreasing the surface area to volume ratio of the LD. CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting enzyme of PC synthesis, which converts phosphocholine to CDP-choline. CDP-choline then condenses with DAG to form PC via the action of ER-membrane protein CDP-choline:1,2 diacylglycerol cholinephosphotransferase (CPT). Krahmer and colleagues have shown that the source of additional PC required during LD surface expansion is provided by the Kennedy pathway, which in turn is activated by the targeting of CCT to the expanding LD surface.94 Even so, LDs do not contain CPT activity that is Seminars in Liver Disease

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confined to the ER, and it is still unknown how synthesized PC in the ER membrane reaches the expanding LD surface. Alternatively, PC can also be synthesized by the PE-methyltransferase (PEMT) pathway or by fatty acid remodelling of existing phospholipids.95 In the PEMT pathway, which is highly expressed in the liver, PEMT on the ER and mitochondria catalyzes the conversion of PE to PC in close proximity to LDs.96 Again, it remains unknown whether the PC formed via this pathway is used for LD expansion, or how it would reach the expanding LD surface. Phosphatidylcholine formation from fatty acid remodeling on existing phospholipids takes place via the sequential actions of phospholipase A2 (PLA2) and lysophospholipid acyltransferases (LPCATs). This is supported by the discovery of robust LPCAT activity on LDs,97 and the observations that LD PC contains more monounsaturated fatty acids93 than the PC derived from the PEMT pathway,98 or the PC found on ER membranes.18,19,99 In a recent publication by Krahmer et al, just over 100 LD proteins were identified with high confidence using a methodology combining proteomics and correlative profiling on LDs from insect cells.21 Enzymes of lipid metabolism such as acyl CoA synthetases, lipases, and CTP:phosphocholine cytidyltransferases (CCTs) were identified on LDs. Importantly, LDs were found to contain all the enzymes required for de novo in situ synthesis of TG,21,84 supporting the hypothesis that LDs can grow while being completely detached from the ER. Wilfling et al reported that membrane bridges form between the ER and LD that help provide a conduit for the relocalization of TG-synthesizing enzymes from the ER to a

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differentiation and lipogenesis in adipocytes, while suppressing lipogenesis in nonadipose tissues.

Future Perspectives The discovery of the FIT proteins, Fsp27, and many other proteins involved in LD biology, as well as the localization of enzymes involved in TG and phospholipid biosynthesis to the LD, are important steps toward a deeper understanding of LD biogenesis and expansion. It will be important to determine a detailed biochemical mechanism for FIT protein function in LD biogenesis, and for the LD fusion process involving Fsp27. Whether FIT plays a role in regulating lipoprotein secretion in the liver and intestine must also be ascertained. Questions regarding the physiological contribution of FIT proteins to the generation of LDs in tissues must similarly be addressed using in vivo knockout models. Additionally, it is important to investigate the prevalence of de novo LD formation versus LD expansion/fusion under lipogenic conditions in the liver. An ever-expanding amount of research is being focused on LD biology. It is inevitable that these and other important questions regarding LD biogenesis, LD fusion and expansion, and LD protein targeting will be answered. It is hoped that multiple therapeutic targets to treat NAFLD and other lipid storage disorders will be identified and characterized from research on LD biology.

Acknowledgments This work was supported in part by grants from the Singapore Ministry of Health’s National Medical Research Council CBRG/0012/2012 (to D.L.S.), and the Agency for Science, Technology and Research (ASTAR) National Science Scholarship award (to V.J.G.).

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subset of forming LDs. This results in two different LD populations: one that contains TG synthesis enzymes and can expand via in situ synthesis of TG, and another that lacks these enzymes and remains small during fatty acid loading.84 CIDEA and CIDEC/Fsp27 of the CIDE family discussed above play essential roles in mediating LD fusion. CIDEA and CIDEC/ Fsp27 are direct targets of PPARgamma37,100,101 and are primarily expressed in adipose tissue where they promote the formation of large unilocular LDs.102,103 This function however is not restricted to adipocytes, and both proteins are also significantly upregulated in steatotic livers where they are thought to induce hepatic TG accumulation.37,104,105 A human mutation in Fsp27 resulting in the expression of a truncated protein has been reported to cause partial lipodystrophy and insulin-resistant diabetes in the afflicted individual.106 It has been shown that Fsp27 enhances LD clustering and then fusion107 by mediating the transfer of TG from smaller to larger LDs at LD contact sites (LDCS) where the proteins are highly enriched.108 The transfer of neutral lipid from the smaller to the larger LD was postulated to occur because of higher internal pressure within the smaller organelle. Live cell imaging of LD fusion events further revealed that after lipid transfer, the smaller LD would be absorbed by the larger one to form an even larger LD.108 It remains to be determined how the phospholipid monolayer of the smaller droplet fuses with the monolayer of the larger droplet. As suggested by Gong et al, enrichment of CIDEA and Fsp27 at LDCS could promote the formation of a passageway between the LDs that allows neutral lipids to be exchanged,108 but the functional significance of CIDEA localization to the LDCS remains to be determined. Indeed, in a recent publication by Sun et al, the CIDE-N domain of Fsp27 was found to form homodimers in a configuration important for Fsp27-mediated lipid transfer.109 In this model, Plin1 is suggested to play a role in Fsp27-mediated lipid transfer by interacting with Fsp27, changing its conformation to enhance its activity.109 Human mutations in the BSCL2 gene encoding the ER resident membrane protein seipin cause a disorder called congenital generalized lipodystrophy type 2 (CGL2).110 CGL2 manifests as a near complete loss of adipose tissue, insulin resistance, and hepatic steatosis. Seipin null mice develop a severe lipodystrophy syndrome with fatty liver resembling the human condition.111,112 It has been proposed that seipin functions in LD biogenesis. However, knockdown of seipin in mammalian fibroblast-like cells induces TG biosynthesis and proliferation of small clusters of LDs, while overexpression resulted in reduction in TG biosynthesis and LD accumulation.113 Other important studies indicate an important role of seipin in the regulation of lipogenesis and adipogenesis.114–117 As suggested by Yang and coworkers, seipin could play a more fundamental role in phospholipid metabolism such that in its absence, phosphatidic acid accumulation leads to the downstream effects of impaired adipogenesis, increased TG synthesis, and LD fusion.113,118,119 Indeed, knockdown of seipin in the fat body of drosophila is associated with increased phosphatidic acid (PA) accumulation rather than LD formation.120 Collectively, these results indicate that seipin’s role in lipid homeostasis is highly tissue-specific, promoting

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