InteractiveFly: GeneBrief

Lipid storage droplet-1 & Lipid storage droplet-2 : Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Lipid storage droplet-1 & Lipid storage droplet-2

Synonyms - PLIN1 & PLIN2

Cytological map positions - 95B1-95B1 and 13A8-9

Function - lipid storage protein

Keywords - fat storage, energy homeostasis, intracellular neutral lipid droplet storage protein, mesoderm

Symbol - Lsd-1 & Lsd-2

FlyBase ID: FBgn0039114 & FBgn0030608

Genetic map position - chr3R:19589579-19592388 & chrX:14965489-14970236

Classification - Perilipin family

Cellular location - intracellular



NCBI links for LSD-1 : Entrez Gene
NCBI links for LSD-2 : Entrez Gene
Recent literature
Men, T. T., Binh, T. D., Yamaguchi, M., Huy, N. T. and Kamei, K. (2016). Function of Lipid Storage Droplet 1 (Lsd1) in wing development of Drosophila melanogaster. Int J Mol Sci 17. PubMed ID: 27136547
Summary:
Perilipins are evolutionarily conserved from Drosophila to humans, the Lipid storage droplet 1 (Lsd1) is a Drosophila homolog of human perilipin 1. The function of Lsd1 as a regulator of lipolysis in Drosophila has been demonstrated, as the Lsd1 mutant causes an increase of lipid droplet size. However, the functions of this gene during development are still under investigation. In order to determine the function of Lsd1 during development, Lsd1 was knocked down in Drosophila using the GAL4-UAS system. Selective knockdown of Lsd1 in the dorsal wing disc caused an atrophied wing phenotype. The generation of reactive oxygen species in the wing pouch compartment of the Lsd1-knockdown flies was significantly higher than in the control. Immunostaining with caspase-3 antibody revealed a greater number of apoptotic cells in Lsd1-knockdown wing discs than in the control. Cell death by autophagy was also induced in the knockdown flies. Moreover, cells deprived of Lsd1 showed mitochondrial expansion and decreased ATP levels. These results strongly suggest that knockdown of Lsd1 induces mitochondrial stress and the production of reactive oxygen species that result in cell death, via apoptosis and the autophagy pathway. These results highlight the roles of Drosophila Lsd1 during wing development (Men, 2016).
BIOLOGICAL OVERVIEW

In Drosophila, the masses and sheets of adipose tissue that are distributed throughout the fly are collectively called the fat body. Like mammalian adipocytes, insect fat body cells provide the major energy reserve of the animal organism. Both cell types accumulate triacylglycerols (TAG) in intracellular lipid droplets; this finding suggests that the strategy of energy storage as well as the machinery and the control to achieve fat storage might be evolutionarily conserved. Studies addressing the control of lipid-based energy homeostasis of mammals identified proteins of the PAT domain family, such as Perilipin, which reside on lipid droplets. Perilipin knockout mice are lean and resistant to diet-induced obesity. Conversely, Perilipin expression in preadipocyte tissue culture increases lipid storage by reducing the rate of TAG hydrolysis. Factors that mediate corresponding processes in invertebrates are still unknown. The function of Lsd2, one of only two PAT domain-encoding genes in the Drosophila genome has been analyzed. Lsd2 acts in a Perilipin-like manner, suggesting that components regulating homeostasis of lipid-based energy storage at the lipid droplet membrane are evolutionarily conserved. Lsd2 is predominantly expressed in tissues engaged in high levels of lipid metabolism, the fat body and the germ line of females. Ultrastructural analysis in the germ line shows that Lsd2 localizes to the surface of lipid droplets. Mutant adults have a reduced level of neutral lipid content compared to wild type, showing that Lsd2 is required for normal lipid storage. Ovaries from Lsd2 mutant females exhibit an abnormal pattern of accumulation of neutral lipids from mid-oogenesis, which results in reduced deposition of lipids in the egg. Consistent with its expression in the female germ line, Lsd2 is shown to be a maternal effect gene that is required for normal embryogenesis (Gronke, 2003; Teixeira; 2003).

Lipids are a major form of energy storage in animals. They are stored in the intracellular neutral lipid droplets of specialized tissues such as adipose tissue in mammals and the fat body in insects. Although initially found in fat-related tissues, lipid droplets are organelles present in many, if not all, cell types. Hence, understanding the role of lipid metabolism at the level of the cell and whole organisms requires identification of the molecular mechanisms governing the biogenesis, trafficking and turnover of lipid droplets (Teixeira, 2003 and references therein).

Lipid droplets are formed by a unique monolayer of amphipatic phospholipids surrounding a central hydrophobic core of neutral lipids, mainly consisting of triacylglycerol (TAG) and sterol esters. Two mammalian proteins have been studied for their property to specifically localize at the surface of these organelles: Perilipin and ADRP (adipocyte differentiation-related protein also known as adipophilin) (Blanchette-Mackie, 1995; Brasaemle, 1997b; Greenberg, 1991). In addition to this particular property, ADRP and Perilipin also show sequence similarity, especially in their N-terminal region where they are ~40% identical (Lu, 2001). This N-terminal region, also present in another mammalian protein––TIP47, has been termed PAT domain (Lu, 2001). TIP47 was originally identified as a protein required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. Although initially controversial, the association of TIP47 to lipid droplets was recently verified (Miura, 2002). The presence of a PAT domain correlates with the ability of proteins to localize to lipid droplets, although it has been recently shown not to be absolutely required (Garcia, 2003: McManaman, 2003; Targett-Adams, 2003). Proteins with a PAT domain have been found in a wide variety of species, including Drosophila, and together form the PAT family (Lu, 2001; Teixeira, 2003 and references therein).

Perilipin and ADRP were originally identified as genes highly expressed in adipose tissue (Greenberg, 1991; Jiang, 1992). Further studies have shown that Perilipin expression is restricted to differentiated adipocytes and steroidogenic cells (Greenberg, 1993; Servetnick, 1995), whereas ADRP is more ubiquitously expressed (Brasaemle, 1997b; Heid, 1998). In cultured cells, it has been shown that the ectopic expression of Perilipin or ADRP increases the capacity of cells to take up long fatty acids from the medium and to accumulate neutral lipids (Brasaemle, 2000; Gao, 1999; Imamura, 2002; Souza, 2002). Reciprocally, the addition of fatty acids to the culture medium stimulates neutral lipid accumulation in cells and increases intracellular levels of Perilipin or ADRP (Brasaemle, 1997a: Gao, 2000; Souza, 2002). The reciprocal regulation of Perilipin/ADRP and neutral lipids suggests that these two proteins have a role in lipid metabolism regulation (Teixeira, 2003 and references therein).

The function of Perilipin in vivo has been analyzed in Perilipin-deficient mice (Martinez-Botas, 2000; Tansey, 2001). These mice are viable, fertile and have normal size and weight. However, they have a reduced adipose tissue mass and are more muscular than controls. They are resistant to induced obesity and have a higher metabolic rate. These phenotypes are explained by the observed increase in basal lipolysis activity. This analysis together with data from cell culture experiments ( [Brasaemle, 2000; Souza, 1998) led to the proposal that Perilipin has a protective role against lipases. The viability of Perilipin mutant mice could be explained by a partial compensation by other mammalian PAT-members. The identification of the in vivo role of the other members of this family awaits the generation of mutants (Teixeira, 2003).

Two Drosophila melanogaster members of the PAT family, Lsd1 and Lsd2, have been identified by BLAST search (Lu, 2001). Both are equally related to any of the three mammalian members of the PAT family (Miura, 2002). Ectopically expressed GFP-tagged Lsd1 or Lsd2 localize to lipid droplets, both in mammalian cell culture and in the fat body of Drosophila (Miura et, 2002). This shows that the targeting to lipid droplets is a feature conserved between Drosophila and mammalian PAT-family members (Teixeira, 2003).

Lsd2 is expressed during all stages of the Drosophila life cycle, and transcripts accumulate in specific spatiotemporal patterns. Northern blot analysis demonstrates a strong enrichment of the 2.4 kb Lsd2 mRNA in early embryos, and this enrichment reflects a maternal contribution, as also visualized by whole-mount in situ hybridization of syncytial blastoderm-staged embryos. Maternal Lsd2 mRNA becomes subsequently degraded, except in germline precursor cells, where the transcripts are enriched up until midembryonic stages. There is transient Lsd2 expression in the amnioserosa and continuous expression in the developing fat body and the anterior midgut, two tissues known to function in lipid storage and nutrient lipid resorption, respectively. During the first and second larval stages, Lsd2 is only moderately expressed. In third instar larvae, the gene is strongly expressed in the fat body, the major TAG storage tissue (Gronke, 2003).

In order to visualize the intracellular localization of LSD2 in vivo, expression of an LSD2-EGFP fusion protein was targeted to the third instar larval fat body by using the Gal4/UAS system in conjunction with a fat body-specific Gal4 driver (FB-Gal4). In living fat body cells of such individuals, LSD2-EGFP is associated with vesicular structures of various sizes. Purification of their fat bodies' intracellular vesicles by density gradient fractionation results in the detection of LSD2-EGFP on lipid droplet surfaces, as identified by Nile red staining. Moreover, Western blot analysis of density-fractionated fat body homogenates of third instar larva shows LSD2 enrichment in the lipid droplet fraction. The spatiotemporal expression patterns and the intracellular localization of LSD2 are therefore consistent with the proposal that the protein plays a regulatory role in global TAG storage by acting at the level of lipid droplets (Gronke, 2003).

Lsd2 mutant flies contain significantly less TAG levels than controls. For simplicity, those individuals are referred to as lean, and those exceeding the TAG levels of controls are referred to as obese. As compared to freshly hatched Lsd2revKG00149 male individuals that carry a functional Lsd2 allele, the TAG content of Lsd251, Lsd240, and Lsd2KG00149 mutants is reduced by 34.5%, 28%, and 37.2%, respectively. In order to unambiguously establish whether the TAG reduction is caused by the loss of LSD2 function in the fat body, a cDNA-based Lsd2 transgene (UAS-Lsd2) was expressed in response to the FB-Gal4 driver in Lsd2 mutant individuals. Expression of Lsd2 in the fat body reverts the leanness of Lsd2KG00149 mutant flies, indicating that loss of LSD2 activity is the cause of the mutant phenotype. Thus, LSD2 is an essential component in the regulation of lipid storage in the fly fat body (Gronke, 2003).

In order to test whether LSD2 activity is capable of modulating the TAG level of otherwise wild-type flies, Lsd2 was overexpressed in the fat body. Western blots with proteins extracted from freshly hatched male flies tested with α-LSD2-specific antiserum show gradually increased levels of UAS-Lsd2 transgene-dependent LSD2 activity in the fat body, and these increased levels result in increasingly severe obesity phenotypes. Flies moderately overexpressing LSD2 elevate organismal TAG storage by 28%, whereas strong Lsd2 overexpression causes a TAG storage increase by 48.5% (Adh-Gal4; lane 8) compared to control individuals bearing the noninduced UAS-Lsd2 transgene. These data demonstrate that modulation of LSD2 levels is sufficient to adjust TAG storage in ad libitum fed flies. The obese FB-Gal4:UAS-Lsd2 flies are more starvation resistant than control flies, whereas the lean Lsd240 mutant flies are starvation sensitive. Starvation resistance of Lsd2-overexpressing flies is accompanied by a delayed but complete premortal depletion of the TAG stores. Collectively, these results indicate that Lsd2 activity can adjust TAG storage at an organismal level at times when food is accessible to ensure extended survival when food supply is limiting (Gronke, 2003).

These results provide evidence that Lsd2 of Drosophila is an essential component of the genetic circuitry that controls energy homeostasis at the level of fat storage. The finding that varying the amount of LSD2 causes a dosage-dependent increase of TAG storage, whereas the lack of LSD2 results in lean flies, is reminiscent of results obtained with the vertebrate PAT domain protein Perilipin. This suggests that LSD2 operates in a Perilipin-like manner by modulating the rate of lipolysis. The results also suggest that PAT domain proteins, which are found in higher eukaryotes as diverse as human, fly, and the slime mold Dictyostelium, share an ancestral function in the organismal control of lipid storage homeostasis. The Drosophila flies with Lsd2 lack-of-function and gain-of-function genotypes therefore represent a genetically accessible model system to identify the components and mechanisms underlying the phenomenon of energy homeostasis in order to address questions concerning energy storage disorders (Gronke, 2003).

Opposite and redundant roles of the two Drosophila perilipins in lipid mobilization

Lipid droplets are the main lipid storage sites in cells. Lipid droplet homeostasis is regulated by the surface accessibility of lipases. Mammalian adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are two key lipases for basal and stimulated lipolysis, respectively. Perilipins, the best known lipid droplet surface proteins, can either recruit lipases or prevent the access of lipases to lipid droplets. Mammals have five perilipin proteins, which often exhibit redundant functions, precluding the analysis of the exact role of individual perilipins in vivo. Drosophila have only two perilipins, PLIN1/LSD-1 and PLIN2/LSD-2. Previous studies revealed that PLIN2 is important for protecting lipid droplets from lipolysis mediated by Brummer (BMM), the Drosophila homolog of ATGL. This study reports the functional analysis of (Lipid storage droplet-1) PLIN1 and Drosophila Hormone-sensitive lipase ortholog (HSL). Loss-of-function and overexpression studies reveal that unlike PLIN2, PLIN1 probably facilitates lipid mobilization. HSL is recruited from the cytosol to the surface of lipid droplets under starved conditions and PLIN1 is necessary for the starved induced lipid droplet localization of HSL. Moreover, phenotypic analysis of plin1;plin2 double mutants revealed that PLIN1 and PLIN2 might have redundant functions in protecting lipid droplets from lipolysis. Therefore, the two Drosophila perilipins have both opposite and redundant roles. Domain swapping and deletion analyses indicate that the C-terminal region of PLIN1 confers functional specificity to PLIN1. This study highlights the complex roles of Drosophila perilipin proteins and the evolutionarily conserved regulation of HSL translocation by perilipins (Bi, 2012).

The analysis of dHSL reveals several interesting points. Under fed conditions, both the TAG level and the size of lipid droplets are slightly increased in dHSL mutant larvae, indicating that dHSL may function under basal condition. In supporting that, dHSL mutation enhances the large lipid droplet phenotype of bmm mutants. The location of dHSL-EGFP to lipid droplets under starvation highlights that the mechanism by which HSL regulates stimulated lipolysis is likely conserved from Drosophila to mammals. This study took advantage of a dHSL-EGFP reporter to establish a strong connection between defective fat mobilization in plin1 mutants and the lipid droplet surface localization of dHSL (Bi, 2012).

The fact that plin1 mutant larvae have larger lipid droplets than bmm;dHSL double mutants can be explained by the proposed structural role of PLIN1 in lipid droplets (Beller, 2010). Since plin1 mutants have giant lipid droplets, it is possible that PLIN1 may be involved in lipid droplet fission or fusion. Several recent studies have revealed that phosphatidic acid (PA) is important for the formation of supersized lipid droplets in Seipin mutants. It remains to be determined whether PLIN1 affects the metabolism of fatty acids or phospholipids, such as PA. Moreover, these results also extend previous findings (Beller, 2010) by showing that PLIN1 has PLIN2-like function in protecting lipid droplets from lipolysis. Currently, it is not known how PLIN1 performs this protective role. It is possible that it acts by blocking the access of BMM. Previous finding (Beller, 2010) that more BMM localizes to lipid droplets in plin1 mutants is consistent with this possibility (Bi, 2012).

The dual role of Drosophila PLIN1 prompts comparison between Drosophila PLIN1 and mammalian Perilipin1. Both PLIN1 and Perilipin1 have two opposing functions in lipid droplets: preventing lipolysis and facilitating lipolysis. The two roles of Perilipin1 are regulated by phosphorylation. Unphosphorylated Perilipin1 protects lipid droplets from lipolysis by blocking the access of lipases, while phosphorylated Perilipin1 releases the ATGL activator CGI58, resulting in activation of ATGL, which promotes lipolysis (Zimmermann, 2004). Phosphorylated Perilipin1 can also elicit translocation of HSL from the cytosol to the lipid droplet surface. Similarly, studies using purified Drosophila PLIN1 implied that PKA phosphorylation of PLIN1 had a direct effect on lipase activity. Moreover, this study found that PLIN1 is important for dHSL lipid droplet location. Therefore, the regulation of HSL localization by Perilipins is likely highly conserved from Drosophila to mammals. It remains to be determined whether PLIN1 regulates the activity of BMM, the Drosophila ATGL. In contrast, plin1 differs from Perilipin1 in the following ways. First, plin1 mutants show different phenotypes under normal conditions to Perilipin1 mutants. Unlike Perilipin1 knockout mice, Drosophila plin1 mutants are not lean; indeed, a recent study showed that plin1 mutant animals develop adult-onset obesity (Beller, 2010). Second, overexpression of Perilipin1 results in aggregated lipid droplets (Marcinkiewicz, 2006), while overexpression of plin1 leads to small lipid droplets. Lastly, the partially redundant function of PLIN1 was revealed in the plin2 mutant background. It is not known whether Perilipin1 has other functions in the absence of other Perilipins in vivo (Bi, 2012).

The results suggest that PLIN2, together with PLIN1, may protect small lipid droplets at an early stage of lipid droplet biogenesis from BMM- and probably dHSL-mediated lipolysis, while PLIN1 facilitates dHSL-mediated lipolysis in large lipid droplets. Based on the phenotypic analysis, it is thought that the major function of PLIN1 is in facilitating fat mobilization. Because large lipid droplets have greater lipid content, lipolysis of large droplets may be an efficient way to support the cell’s energy needs and to balance lipid usage with lipid droplet biogenesis. Such fine regulation is important for maintaining lipid homeostasis. Moreover, the functional complexity of PLIN1 may reflect the evolution of ancient Perilipins from simple barriers that protect lipid droplets to more active regulators of lipid homeostasis. How are the dual functions of PLIN1 regulated? It is possible that PLIN1 may have different structures/states and binding partner(s) in lipid droplets of different sizes. Phosphorylated PLIN1 was found to affect the activity of lipase in in vitro assays (Arrese, 2008). Therefore, the phosphorylation state of PLIN1 may be different in small and large lipid droplets. Although the functional importance of PLIN1 phosphorylation remains to be determined in vivo, a recent study showed that the canonical PKA target sites are not important for PLIN1 function (Beller, 2010). Therefore, identification of the phosphorylation site of PLIN1 will lead to better understanding of the regulation of PLIN1 function. Since the C-terminal region of PLIN1 determines its functional specificity, regulation of the dual role may be a property of the C terminus. The N-terminal portion of PLIN1 may be sufficient for its function in protecting lipid droplets from lipolysis. The C-terminal region of PLIN1 is highly conserved among Drosophila species. Identifying protein partners of the C-terminal region could help to reveal the regulatory mechanisms involved. Similarly, compared to ADRP and TIP47, Perilipin1 has an extended C-terminal region. Phosphorylation of key residues in the C-terminal region of Perilipin1 is important for ATGL activation and lipid droplet dispersal. Frame-shift mutations at the C-terminal region of Perilipin1 result in dominant partial lipodystrophy in human, supporting the functional importance of the C-terminal region (Bi, 2012).

The study reveals the functions of the only two Perilipins in Drosophila. The fact that plin1;plin2 double mutants have small lipid droplets indicates that Perilipins are dispensable for the initial biogenesis of lipid droplets, but are required for the growth of lipid droplets. Together with a recent study on PLIN1 (Beller, 2010), these findings provide a better understanding of the exact function of Perilipins in vivo. plin1, plin2, and dHSL mutants can be used as models to further probe the homeostasis of lipid droplets. More functional studies of Drosophila lipid-related genes may facilitate a deeper understanding of diseases related to fat metabolism, such as obesity and diabetes (Bi, 2012).


REGULATION

Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. This study identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system. These regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets (Beller, 2008).

Lipid homeostasis is critical in health and disease, but remains poorly understood. Non-esterified free fatty acid (NEFA) is used for energy generation in beta-oxidation, membrane phospholipid synthesis, signaling, and in regulation of transcription factors such as the peroxisome proliferator-activated receptors (PPARs). Essentially all cells take up excess NEFA and convert it to energy-rich neutral lipids in the form of triglycerides (TG). TG is packaged into specialized organelles called lipid droplets. NEFA is regenerated from lipid droplet stores to meet metabolic and energy needs, and lipid droplets protect cells against lipotoxicity by sequestering excess NEFA. Lipid droplets are the main energy storage organelles and are thus central to the understanding of energy homeostasis. Despite their importance, little is known about the ontogeny and regulation of these organelles (Beller, 2008).

Lipid droplets are believed to form in the ER membrane by incorporating a growing TG core between the leaflets of the bilayer, and ultimately are released surrounded by a phospholipid monolayer. Cytosolic lipid droplets possess a protein coat and grow by synthesis of TG at the lipid droplet surface and by fusion with other lipid droplets. Formation of nascent droplets and aggregation of existing droplets is likely to require a dynamic exchange of lipids and proteins from and to the droplet. Indeed, the range of proteins identified in lipid droplet proteomic studies suggests extensive trafficking between lipid droplets and other cellular compartments, including the endoplasmic reticulum (ER). Additionally, lipid droplet-associated proteins translocate between the cytosol and lipid droplets. For example, tail interacting protein of 47 kDa (TIP47) associates with small, putative nascent, lipid droplets, but is not found on larger droplets, which are coated by other members of the perilipin, adipocyte differentiation related protein (ADRP), TIP47 (PAT) protein family. Intriguingly, TIP47 mediates mannose 6-phosphate receptor trafficking between the lysosome and Golgi, raising the possibility that trafficking is involved in lipid droplet ontogeny or fate. However, unlike the well-studied Golgi trafficking system, the routes to and from the lipid droplet are unknown (Beller, 2008).

Once lipid droplets are formed, stored TG is mobilized in a regulated manner. Triglyceride, diglyceride (DG), and monoglyceride lipases convert TG back into NEFA. Most of the knowledge concerning lipolysis is based on extensively studied adipocytes in which at least two lipolytic enzymes have been identified: adipocyte triglyceride lipase (ATGL; Drosophila brummer) and hormone sensitive lipase (HSL). Due to the hydrophobic properties of the lipid droplet TG core, lipases are likely to act at the surface of lipid droplets, where members of the PAT protein family regulate lipase access to the TG core. Mammalian genomes encode at least five PAT-proteins. Whereas perilipin is the dominant PAT protein in adipocytes, ADRP is the dominant PAT protein in nonadipose tissues in which it is tightly associated with the lipid droplet surface. PAT members appear to have a hierarchical affinity for the lipid droplet surface. In nonmammalian genomes, there are fewer PAT proteins. For example, two PAT proteins termed Lipid storage droplet 1 and 2 (LSD-1 and LSD-2) are found in Drosophila (Miura, 2002). The crucial role of PAT proteins is evolutionary conserved as the absence of perilipin in mice, or LSD-2 in flies results in lean animals. Overexpression of LSD-2 results in obese flies. These data indicate the conserved PAT proteins at the lipid droplet surface are important regulators of energy storage (Beller, 2008 and references therein).

It seems likely that PAT proteins protect lipid from lipolysis, but the role of PAT proteins may not be limited to passive steric hindrance of lipase access to the TG core, as illustrated by perilipin. Unphosphorylated perilipin protects the lipid droplet from lipase activity. Following stimulation by protein kinase A (PKA), however, phospho-perilipin acts as a docking site for HSL, which translocates from the cytosol to the droplet surface. Whereas phospho-perilipin promotes massive NEFA release from the droplet, this is not mediated exclusively by HSL, as mice lacking HSL function show a relatively mild phenotype marked by the accumulation of DG, thus demonstrating that HSL acts as a DG lipase in vivo. The TG lipase functioning in HSL null mice is ATGL. In the current view of adipocyte lipolysis, ATGL is responsible for the first step in TG hydrolysis, liberating DG and NEFA, whereas HSL acts as a DG lipase. Very little is known about how ATGL is targeted to the lipid droplet (Beller, 2008).

In contrast to the lean phenotype in animals lacking perilipin (mouse) or LSD-2 (fly), both mice and flies lacking ATGL are obese. In mice, the absence of ATGL results in excessive TG accumulation in liver and muscle. Similarly, human patients suffering from neutral lipid storage disease carry mutations resulting in truncated ATGL isoforms. ATGL function is evolutionary conserved, as flies lacking the Drosophila ATGL ortholog, Brummer, accumulate copious amounts of body fat. The lipid droplet-associated protein Comparative Gene Identification-58 (CGI-58) acts as an ATGL colipase (Lass, 2006). Mutations in the CGI-58 gene result in ectopic fat accumulation in patients suffering from Chanarin Dorfman Syndrome (CDS), supporting the idea that both ATGL and CGI-58 are required for mobilizing lipid stores in nonadipose tissue. Interestingly, CGI-58 physically interacts with perilipin as demonstrated by both coimmunoprecipitation and fluorescence resonance energy transfer (FRET) studies. In addition, there are other lipases and probably many more cofactors encoded in the genome. Understanding which ones act at the lipid droplet surface and how their localization is regulated will be important (Beller, 2008).

Drosophila is a powerful model for pathway discovery due to well-developed genetics. Additionally, greater than 60% of the genes associated with human disease have clear orthologs in Drosophila. Drosophila is highly relevant to lipid droplet study, as lipid droplets in Drosophila and mammals are associated with many of the same proteins. Finally, the emerging model of lipid storage and endocrine regulation are similar in humans and Drosophila, suggesting that Drosophila will be a good genetic model for lipid storage and lipid storage diseases in humans. This study therefore utilized genome-wide RNA interference (RNAi) screening in Drosophila tissue culture cells to identify and characterize novel regulators of lipid storage. The function of these regulators was tested in mouse lipid droplet regulation by directed RNAi studies. 318 Drosophila genes were identified that were required to limit lipid storage and 208 Drosophila genes were identified that were required to promote lipid storage. These genes encode known regulators of lipid storage as well as genes not previously associated with lipid storage regulation (Beller, 2008).

Positive regulation of lipolysis by the COPI retrograde-vesicle trafficking pathway was the most striking and unexpected result of the screen. Interference with COPI function, either by RNAi or compounds, in Drosophila Kc167 or S3 cells, or in mouse 3T3-L1 or AML12 cells, results in increased lipid storage. Furthermore, recent and parallel studies in yeast and Drosophila S2 cells (Guo, 2008) also suggested a role of COPI function in lipid droplet regulation. Interestingly, only the epsilon-subunit of the COPI complex failed to result in a lipid droplet deposition phenotype on knockdown. Although limited RNAi efficacy or increased protein stability cannot be ruled out, epsilonCOP was the only canonical COP subunit not resulting in a lipid storage phenotype in a parallel study using different cells and reagents (Guo, 2008), and targeting of epsilonCOP transcripts by RNAi in AML12 cells had a weak effect on lipid storage at best. Finally, epsilonCOP is the only dispensable subunit in a recent study identifying COPI activity coupled with fatty acid biosynthesis as a host factor important for Drosophila C virus replication (Cherry, 2006). This is especially interesting, since certain enveloped viruses, including Hepatitis C virus, assemble on lipid droplets. Taken together, these results indicate that six out of the seven wild-type COPI subunits mediate lipid storage by positively regulating lipolysis (Beller, 2008).

COPI could have a direct or indirect effect on lipid storage. The indirect mechanism is poorly defined, but if the Golgi is a 'sink' for phospholipids derived from TG stores, then decreased Golgi function could simply decrease demand for TG substrate. If non-esterified free fatty acid (from the media in fed cells, and from biosynthesis in unfed cells) conversion to TG continues, then increased lipid droplet volume would occur. It is also possible that canonical COPI function transporting lipids and proteins from the Golgi to the ER is ultimately responsible for lipid droplet utilization and protein composition at the lipid droplet surface. For example, COPI might be required for the particular phospholipid composition in hemimembranes formed on nascent droplets, which secondarily alter TIP47 and ATGL localization in mature lipid droplets (Beller, 2008).

However, evidence that Golgi function per se is not linked to lipid storage phenotypes, as well as direct association of COPI members and regulators with the lipid droplet or PAT proteins supports a more direct model. The COPI and COPII pathways have established roles as constitutive vesicle transport systems that cycle proteins as well as lipid from the Golgi to the ER (COPI), or vice versa (COPII). Interference with either of the COP trafficking systems results in disturbed ER and Golgi function. The lipid overstorage phenotype was seen only in the case of interference with COPI trafficking. This indicates that the lipid overstorage phenotype is not a simple consequence of ER and Golgi function. Finally, in an indirect model in which COPI shuttles only between the Golgi and the ER, COPI should not be lipid droplet associated. However, COPI subunits are directly associated with the lipid droplet surface as shown by proteomics. Additionally, Arf1 binds to ADRP, which is exclusively associated with the lipid droplet surface. Arf79F, the Drosophila homolog of mammalian Arf1, also localizes to lipid droplets in Drosophila S2 cells (Beller, 2008 and references therein).

It is proposed that COPI is likely to function directly at the lipid droplet surface and not indirectly through the Golgi. Perhaps COPI is a destination-specific transporter returning lipid droplet surface hemimembrane and Golgi membrane to the ER. The transport system that brings nascent lipid droplets from the ER to the lipid droplet has not been elucidated, but it is intriguing that the transport/PAT protein TIP47 is found preferentially on small lipid droplets. Small lipid droplets derived from the ER are thought to help build larger droplets by fusion. TIP47-coated droplets might form in the ER, and then COPI could return TIP47 to the ER after the lipid cargo is deposited. In this model, TIP47 becomes trapped at the lipid droplet surface in the absence of COPI (Beller, 2008).

Although increased TIP47 was observed on ADRP-positive droplets by both western blot and cell staining, the cell staining result was more dramatic. The current model might also explain why. The punctate staining of TIP47 in untreated cells could be due to TIP47 on nascent droplets that might also cofractionate with the larger ADRP-positive droplets in the western blots, leading to a less dramatic enrichment for TIP47 relative to ADRP in that experiment. However, other explanations cannot be ruled out, such as nonlinear detection of antigen concentration or epitope masking in the cell staining experiments (Beller, 2008).

COPI perturbation increases stored TG by decreasing the lipolysis rate indicating that the wild-type COPI complex promotes lipolysis. COPI directly or indirectly removes TIP47 from the lipid droplet surface and promotes ATGL localization to the droplet surface, where lipolysis occurs. ATGL has a key role in lipid droplet utilization, and ATGL association with the droplet is reduced by ADRP and Tip47 (Bell, 2008). The epistasis experiments combining siRNA-mediated ATGL knockdown and BFA or Exo1 compound treatment demonstrated that the decrease in lipolysis rate is due to loss of ATGL activity. COPI activity specifically alters lipid droplet surface composition by increasing the amount of TIP47 and reducing the amount of ATGL at ADRP-coated lipid droplets. It is suggested that COPI negatively regulates localization of TIP47. TIP47 in turn prevents ATGL localization. The rescue of the double-knockdown phenotype of TIP47 and ADRP by BFA or Exo1 suggests that COPI has an independent feed-forward effect on ATGL levels at the lipid droplet surface (Beller, 2008).

Although this study focused attention here on COPI, systematic and genome-wide exploration of gene functions required for lipid storage in Drosophila significantly increases experimental access to the complex molecular processes regulating lipid storage and utilization. Further, the use of multiple screens using different cell types and different organisms greatly increases confidence in the genes in the intersection. Given widespread concerns about RNAi screening efficacy and off-target effects, as well as the time and effort required for downstream analysis, systematic use of multiple species and libraries to address a single biological question might be cost effective in addition to resulting in more durable datasets. Primary screens in Drosophila cells followed by secondary screens in mouse cells are much less expensive than a similar genome-wide screen in mammalian cells. Additionally, the availability of mutants in most Drosophila genes, along with demonstrated translation to mammalian systems, provides a valuable entry point for in-depth analyses in both fly and mouse; and eventually for the selection of therapeutic targets for emerging problems associated with obesity and other metabolic disorders (Beller, 2008).

Membrane attachment and structure models of lipid storage droplet protein 1

Neutral lipid triglycerides, a main reserve for fat and energy, are stored in organelles called lipid droplets. The storage and release of triglycerides are actively regulated by several proteins specific to the droplet surface, one of which in insects is PLIN1. PLIN1 plays a key role in the activation of triglyceride hydrolysis upon phosphorylation. However, the structure of PLIN1 and its relation to functions remain elusive due to its insolubility and crystallization difficulty. This study reports the first solid-state NMR study on the Drosophila melanogaster PLIN1 in combination with molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a trans-model agreed with experimental spectra. In this model, lipids from the bottom leaflet were very close to the surface in the region enclosed by membrane attachment motif. This may imply that in real lipid droplet, triglyceride molecules might be brought close to the surface by the same mechanism, ready to leave the droplet in the event of lipolysis. Juxtaposition of triglyceride lipase structure to the trans-model suggested a possible interaction of a conserved segment with the lipase by electrostatic interactions, opening the lipase lid to expose the catalytic center (Lin, 2014).


DEVELOPMENTAL BIOLOGY

Embryonic

As a first approach towards the investigation of Drosophila Lsd2, its pattern of expression during embryo development was examined by in situ hybridization. A high level of uniformly distributed Lsd2 transcript was found in the early stages of embryogenesis. The mRNA present at these stages is provided maternally. Upon cellularization of the embryo at stage 5, Lsd2 mRNA disappears except in the pole cells, the germ-line precursors at the posterior pole of the embryo. Later, at stage 11, zygotic expression begins in the amnioserosa. At stage 14, Lsd2 is relatively broadly expressed with an enrichment in the fat body and the midgut. At the end of embryogenesis, these tissues, together with the hindgut, are the main sites of Lsd2 expression. The enrichment of Lsd2 in the fat body is particularly interesting because it is the main organ for lipid storage in insects (Teixeira, 2003).

The enrichment of Lsd2 mRNA in the fat body at the end of embryogenesis prompted a determination of whether Lsd2 is also expressed in larval fat body. The larval fat body is a site of active synthesis and storage of lipids while the larva rapidly grows and prepares for metamorphosis. Immunodetection of Lsd2 protein was detected in wild type and Lsd21 homozygous third instar larvae, using Lsd2 antiserum. At this stage, Lsd2 is mainly found in the fat body of wild-type larvae. Mutant larvae present no staining in this tissue, showing the specificity of the detection. The enrichment of Lsd2 protein in the larval fat body supports a role for this protein in lipid metabolism (Teixeira, 2003).

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. A close juxtaposition of TIP47 with the surfaces of lipid storage droplets was detected using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, related proteins from species as diverse as Drosophila and Dictyostelium are shown to also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization (Miura, 2002).

PAT family proteins are related to Peri and ADRP. Sequence analyses based upon neighbor-joining comparisons confirm separate vertebrate groupings for Peri, ADRP, and TIP47. A novel murine family member, PAT1, was also identified. Although the Drosophila and Dictyostelium proteins are more distantly related, they demonstrate clear PAT group association (Miura, 2002).

The subcellular localization of TIP47 has been controversial. TIP47 was originally identified in a screen for proteins that interact with the MPR/IGF-IIR. Subsequently, antibodies to TIP47 were shown to detect protein at lipid droplet surfaces, but because these antibodies cross-reacted with ADRP, it was not possible to assert unequivocally that TIP47 exhibits lipid droplet co-localization. The data indicate that GFP-TIP47 can associate closely with the lipid droplet surface, but they do not exclude an ability of TIP47 to interact with other subcellular components or structures. Affinity-purified antibody to human TIP47 was used to examine subcellular localization of endogenous TIP47 in HeLa cells. The small lipid droplets characteristic of HeLa cells were clearly evident by Nile Red fluorescence, and TIP47 was found predominantly in very close juxtaposition. The GFP-TIP47 fusion experiments in conjunction with other reports raise the possibility that TIP47 may be multifunctional, potentially trafficking proteins and/or lipids among several compartments. Perhaps different environmental parameters alter the relative distribution of TIP47 among various intracellular compartments. TIP47 would not be unique in its ability to associate with LSD and non-LSDs, depending upon the physiological state of the cell (Miura, 2002).

To determine whether lipid droplet localization is a general quality of the more diverged PAT protein members, the intracellular targeting of Drosophila LSD-1 and -2 and of Dictyostelium LSD1 in CHO cells were examined as fusions with GFP. All of the proteins showed selective localization of fluorescence to the lipid droplet surface that was exclusive of any other compartment. Fluorescent GFP rings were in close apposition with the neutral lipids. These data indicate that lipid droplet targeting is characteristic of PAT family proteins and that sites for their recruitment are highly conserved despite their effective separation through nearly one billion years of evolution (Miura, 2002).

The localization of Drosophila LSD-1 and -2 proteins were examined in their endogenous environment. Transgenic Drosophila were generated that expressed GFP-LSD-1 or -2 genes driven with a GAL4-inducible promoter. The GAL4 transcriptional regulatory protein was induced by heat shock response. The GFP-LSD-1 and GFP-LSD-2 proteins were clearly shown to associate with lipid droplets within cells of larval (first instar) fat bodies, as evidenced by the rings of GFP fluorescence surrounding the large lipid droplets in these cells. These data confirm that LSD-1 and -2 proteins can associate specifically with lipid droplets in a native environment, as well as in mammalian CHO cells (Miura, 2002).

In summary, the PAT protein family has ancient progenitors that define a novel protein targeting component for association with intracellular lipid storage droplets. A common sequence and/or structural element among these proteins is necessary and sufficient for this functional conservation even within exogenous cellular environments. Further, structure/function studies of Peri demonstrate its essential role in lipid storage droplet deposition and mobilization, strongly suggesting related roles for ADRP, TIP47, and other PAT proteins in lipid metabolism and trafficking. The PAT family has no apparent sequence relationship with the variety of other proteins capable of lipid droplet association in plants, yeasts, or mammalian cells, including the oleosins, caveolin, and synuclein. Nonetheless, the confirmation that the distantly related PAT proteins of Drosophila and Dictyostelium also possess the essential structural elements for LSD targeting emphasizes this functional characteristic. Directed and complementary studies using both mammalian and non-mammalian systems will be required to dissect the molecular mechanisms that are a fundamental property of this important protein family (Miura, 2002).

Specialized hepatocyte-like cells regulate Drosophila lipid metabolism

Lipid metabolism is essential for growth and generates much of the energy needed during periods of starvation. In Drosophila, fasting larvae release large quantities of lipid from the fat body but it is unclear how and where this is processed. This study identified the oenocyte as the principal cell type accumulating lipid droplets during starvation. Tissue-specific manipulations of the Slimfast amino-acid channel, the Lsd2 fat-storage regulator and the Brummer lipase indicate that oenocytes act downstream of the fat body. In turn, oenocytes are required for depleting stored lipid from the fat body during fasting. Hence, lipid-metabolic coupling between the fat body and oenocytes is bidirectional. When food is plentiful, oenocytes have critical roles in regulating growth, development and feeding behaviour. In addition, they specifically express many different lipid-metabolizing proteins, including Cyp4g1, an omega-hydroxylase regulating triacylglycerol composition. These findings provide evidence that some lipid-processing functions of the mammalian liver are performed in insects by oenocytes (Gutierrez, 2007).

In mammals, specialized cells of the adipose tissue and liver are critical for coordinating fat metabolism. This physiological axis regulates a complex set of lipid uptake, storage, synthesis, modification and degradation reactions essential for normal growth and development. Lipid metabolism also has a particularly critical role in providing energy during periods of starvation. Food deprivation (fasting) stimulates the lipolysis of triglycerides (also called triacylglycerol, TAG) stored in adipocyte fat droplets via increases in hormone-sensitive lipase activity and adipose triglyceride lipase (ATGL) expression (Zechner, 2005). A large proportion of the fatty acids and other lipids thereby released into the circulation are then captured, modified and broken down by the major cell type of the liver, the hepatocyte. An intriguing feature of the starvation response is that, in contrast to many other cell types, hepatocytes accumulate large numbers of fat droplets, resulting in hepatic steatosis. Fatty acids are released from hepatic lipid droplets during starvation and oxidized into shorter chain fatty acids and ultimately into soluble ketone bodies that can be discharged into the circulation for use as an energy source by many tissues. This fatty acid oxidation process involves chain shortening by α- and β-oxidation pathways active in peroxisomes and mitochondria. Although lipid catabolism predominates during starvation, in the postprandial state, hepatocytes are highly active in synthesizing fatty acids for incorporation into triglycerides. These can then be assembled into lipoprotein particles, delivered to adipocytes and stored in lipid droplets. One critical step for incorporating newly synthesized fatty acids into TAG is catalysed by stearoyl CoA-desaturase-1 (SCD-1), a hepatic enzyme converting palmitic (C16:0) and stearic (C18:0) acids into monounsaturated palmitoleic (C16:1) and oleic (C18:1) acids, respectively. The importance of maintaining an appropriate balance between hepatic fatty acid synthesis and oxidation is highlighted by human diseases arising from mutations in fatty acid oxidation enzymes, and also by widespread diet-influenced pathologies such as non-alcoholic fatty liver disease and metabolic syndrome (Gutierrez, 2007).

Invertebrate model organisms offer a powerful means to identify and functionally analyse lipid-metabolising genes. In Caenorhabditis elegans, fat is stored by intestinal epithelial cells and many regulators of this process have been identified using reverse genetic screens. In contrast, Drosophila and other insects store lipid in a specialized tissue that resembles the adipose tissue of mammals, the fat body. Diet-derived lipids, exported from the midgut as lipoproteins, are taken up from the haemolymph by the fat body via a mechanism involving Low-Density Lipoprotein (LDL) receptor-like molecules called Lipophorin receptors. These lipids accumulate in fat body cells in the form of intracellular droplets but, when larvae are food-deprived, there is a net efflux of lipid into the haemolymph. The mobilization process is regulated by TSC/TOR signalling and a nutrient sensor in the fat body that monitors amino-acid levels via the Slimfast (Slif) amino-acid channel. Fasting-induced fat release is accompanied by increased lipolysis, at least in part associated with upregulation of Brummer, an ATGL-related lipase localized to lipid droplets. Fat mobilization is also influenced by Lsd2, a lipid droplet protein related to a mammalian negative regulator of TAG hydrolysis called perilipin. In addition to its involvement in lipid storage and release, the fat body produces a humoral signal regulating larval tissue growth in response to food availability. Thus far, efforts to harness the power of Drosophila genetics to model human fat metabolism have been limited by the lack of information on how and where insect lipids are processed once they have been released from the fat body. For example, it is not known whether there is a specialized Drosophila tissue that synthesizes, modifies and oxidizes fatty acids in a similar way to the mammalian liver, nor is it clear to what degree the mammalian biochemical pathways metabolizing fatty acids are conserved in Drosophila. This study addresses both of these issues using a combination of bioinformatics, genetics and integrative physiology (Gutierrez, 2007).

How fat is redistributed throughout the larval body after food deprivation was studied. Using Oil Red O staining, three cell types in the third instar (L3) larva were found to contain numerous large (0.5-2.5 microm) lipid droplets under fed and/or fasting conditions: fat body cells, midgut epithelial cells and larval oenocytes. The fat body of L3 larvae has such a large capacity for lipid storage that, despite lipid loss over a 14-h period of fasting, intense Oil Red O staining persists. Lipid release from the L3 fat body during starvation correlates with lipid droplet aggregation. However, droplet aggregation is not a reliable indicator of fasting at some other larval ages and durations of fasting. In contrast to the fat body, regions of the gut (including the proventriculus and anterior midgut) staining strongly with Oil Red O under fed conditions have only limited storage capacity, losing most lipid droplets after 14 h of fasting. The third cell type, larval oenocytes (distinct from adult oenocytes but abbreviated hereafter as oenocytes), are large cells of unknown function that are attached to the basal surface of the lateral epidermis in clusters of ~6 cells per abdominal hemi-segment. L3 oenocytes do not stain strongly with Oil Red O under fed conditions but they do contain numerous large lipid droplets after a 14-h fast. This change in droplet abundance is consistent from oenocyte-to-oenocyte within one cluster and also from one cluster to another. Thus, oenocytes are highly atypical cells, in that they accumulate large numbers of lipid droplets specifically during fasting. As this is a hallmark of hepatocytes, the possibility is raised that insect oenocytes might process lipids in a similar way to the mammalian liver (Gutierrez, 2007).

Next, whether the accumulation of lipid droplets in oenocytes is regulated by the fat body nutrient sensor was tested. An antisense transgene directed against the amino-acid transporter Slimfast was expressed in the fat body (ppl-GAL4 driving UAS-slifAnti; hereafter called ppl>slifAnti). As reported previously (Colombani, 2003), it was observed that ppl>slifAnti larvae raised to L3 on a standard diet resemble starved wild-type larvae in that lipid droplets aggregate in the fat body. Notably, it was also found that oenocytes contain numerous lipid droplets, regardless of whether ppl>slifAnti larvae are fed on a standard diet or food-deprived for 14 h. This indicates that amino-acid monitoring via Slif in the fat body is required to ensure that lipid accumulation in oenocytes is kept low under standard nutritional conditions. TSC/TOR signalling, another component of the fat body nutrient sensor, is also involved; overexpressing TSC1 and TSC2 (ppl>Tsc1+2) leads to a marked accumulation of large lipid droplets in the oenocytes of 100% (n = 11) of fed larvae. Similarly, inhibiting the phosphatidylinositol-3 kinase pathway, which intersects with TOR signalling, by overexpressing the lipid phosphatase PTEN, also produces a build up of lipid droplets in the oenocytes of 89% of fed ppl>PTEN larvae. Hence, the fat body nutrient sensor regulates lipid accumulation in oenocytes but this could be directly via lipid release or indirectly, in response to a TSC/TOR-dependent signal (Gutierrez, 2007).

To assess directly the effect of lipid mobilization from the fat body, the balance between TAG storage and hydrolysis was altered in two ways. First, Brummer (Bmm) lipase, which is normally limiting for lipid release from the fat body, was overexpressed. This is sufficient to produce specific accumulation of lipid droplets in the oenocytes of 92% of fed ppl>bmm larvae. Second, TAG release from lipid droplets was decreased by overexpressing Lsd2. This reduces the accumulation of oenocyte lipid droplets in 78% of starved ppl>Lsd2 larvae, with ~4-fold fewer large droplets per oenocyte. A second driver, Lsp2-GAL4, was used that unlike ppl-GAL4 is activated in the fat body only at the mid-L3 stage. This temporally restricted driver nevertheless suffices to induce oenocyte lipid droplet accumulation in 100% of fed Lsp2>bmm larvae and also in fed Lsp2>slifAnti animals. Together, the Slif, TSC, PTEN, Lsd2 and Bmm results demonstrate metabolic regulation from the fat body to the oenocytes, although they do not exclude the involvement of intermediate tissues such as the gut. Either way, these results strongly suggest that, when nutrition is poor, falling amino-acid levels stimulate lipid release from the fat body and subsequent lipid uptake from the haemolymph by oenocytes (Gutierrez, 2007).

To determine the in vivo roles of oenocytes during fasting and normal development, a targeted binary cell ablation system was developed. Larvae carrying sal[BO,7.6kb]GAL4, a purpose-built oenocyte driver, and also UAS-reaper, an inducible pro-apoptotic transgene, lack 100% of oenocytes from L1 onwards and die before reaching pupariation (hereafter called BO>rpr larvae). As a specificity control, BO>rpr animals were rescued to viable adults by expressing Gal80, an inhibitor of Gal4, under the regulation of an independent oenocyte enhancer from seven up (svp). As svp[3kb]GAL80 suppresses sal[BO,7.6kb]GAL4 activity in oenocytes but not in secondary larval sites, BO>rpr lethality results from the ablation of oenocytes and not some other cell type (Gutierrez, 2007).

BO>rpr larvae raised on a standard diet attain a similar mass to UAS-rpr controls during L1 but, after the L1-to-L2 transition, they grow at a much slower rate. Notably, reduced growth correlates with aberrant feeding behaviour, with most BO>rpr larvae dispersing away from the yeast food source during L2. This dispersal is distinct from premature wandering behaviour; BO>rpr larvae enter and exit the yeast source multiple times, retain food in the gut and do not pupariate precociously. Since BO>rpr larvae spend less time in the food source and grow more slowly than L2 controls, whether they show increased mouth-hook contractions, a behavioural response to hunger, was investigated. However, reduced mouth-hook contractions were observed that are not significantly increased by the motivation of a 2-h period of food deprivation. Thus, rather than stimulating hunger-driven feeding behaviour, oenocyte ablation seems to block it, although this effect could be very indirect. Either way, reduced feeding is likely to contribute to the slow growth rate of BO>rpr larvae during L2 (Gutierrez, 2007).

Since reduced growth resulting from inadequate nutrition before 70 h after egg laying (just before the L2/L3 moult) is associated with larval arrest rather than smaller-than-normal adult flies, morphological criteria were used to stage oenocyte-ablated animals. It was observed that BO>rpr larvae arrest at several different stages after the L1/L2 transition, thus displaying a polyphasic lethality profile. Although arrested development can result from reduced signalling by ecdysteroids, the BO>rpr polyphasic lethality profile is not significantly altered by adding 20-hydroxyecdysone or its precursor ecdysone. Therefore, a deficiency in these ecdysteroids is not the sole reason for BO>rpr arrest, but the possibility cannot be excluded that it, together with some other oenocyte deficit, contributes to the moulting phenotype (Gutierrez, 2007).

Unlike many larval tissues, oenocytes persist for much of pupal development. To address whether oenocytes are required for metamorphosis, a temperature-sensitive version of Gal80 (GAL80ts ) was used to attenuate Gal4 activity, thus bypassing BO>rpr larval lethality. Combining tub-GAL80ts with BO>rpr suppresses apoptosis in approximately 50% of oenocytes at 25 °C (from L1 onwards) and allows developmental progression until pupal stages. However, no animals complete pupal development, with many failing to separate from the puparial case during eclosion. Together, the 50% and 100% oenocyte ablation phenotypes demonstrate that oenocytes are required for growth and developmental progression during both larval and pupal stages (Gutierrez, 2007).

Whether lipid metabolism is altered in larvae lacking all oenocytes was examined. At early L2, when BO>rpr larvae are the same size as controls, no significant abnormalities in TAG content or in the relative amounts of the major long-chain fatty acids were detected. The fat storage capacity of larvae at early L2 is much less than at L3 such that a 12-h period of food withdrawal is sufficient to deplete ~60% of stored TAG in control animals. However, during this same fasting period, BO>rpr larvae only lose ~10% of total TAG. This deficit in TAG depletion correlates with a higher density of fat-body lipid droplets in 100% of starved BO>rpr larvae compared to controls after fasting. Since ~80% of larval fatty acids are stored as TAG, the proportions of individual fatty acids were examined in fasting BO>rpr larvae. In early L2 controls, lauric (C12:0) and myristic (C14:0) acids are depleted more efficiently than longer-chain (C16-C20) fatty acids such that their mass, relative to stearic acid (C18:0), is reduced twofold after 12 h fasting. However, in fasted BO>rpr larvae, the C12:0/C18:0 and C14:0/C18:0 ratios remain close to those before starvation, corresponding to approximately twice the value of starved controls. Together, these results indicate that oenocytes are required for efficiently depleting fatty acids, stored largely in the fat body as TAG, during nutrient deprivation. With the previous Slif, TSC, PTEN, Bmm and Lsd2 results, it is proposed that lipid-metabolic coupling between the fat body and oenocytes is bidirectional (Gutierrez, 2007).

To identify the metabolic pathways processing lipids within oenocytes, 51 genes expressed selectively or exclusively in oenocytes were identified. About 40% of these encode orthologues of known human lipid-metabolizing/processing proteins. The high degree of conservation of most Drosophila proteins, together with some previous functional studies, suggests that oenocytes express lipid metabolic pathways strikingly similar to those of hepatocytes. By analogy, oenocytes would capture lipid from lipophorin in the haemolymph via LpR1 and LpR2, two Lipophorin receptors. Fatty acids released from lipid droplets by lipases such as the CG17292 product, could then be modified by a variety of enzymes, including the Desat1 and CG9743 acyl-CoA desaturases, the CG18609 and CG6921 fatty acid elongases and the microsomal lipid omega-hydroxylase, Cytochrome P450-4g1 (Cyp4g1). Fatty acids could also be chain shortened, at least partially, by the actions of peroxisomal β-oxidation components including those encoded by CG11151 (similar to Sterol carrier protein 2), CG12428 (Carnitine O-octanoyl transferase), CG9527 (Pristanoyl-CoA oxidase) and Catalase, the peroxisomal enzyme inactivating oxygen free radicals produced by pristanoyl-CoA oxidases. In addition, oenocytes strongly express Hnf4 and Svp, orthologues of the mammalian nuclear receptors Hnf4-α and COUP-TF, known regulators of hepatocyte differentiation and lipid-metabolic genes. Thus, the oenocyte/hepatocyte analogy includes a shared set of lipid-metabolizing genes and at least two of their transcriptional regulators (Gutierrez, 2007).

To explore the functions of fatty acid metabolism specifically within Drosophila oenocytes, two lethal protein-null alleles were generated for the predicted lipid omega-hydroxylase encoded by Cyp4g1. Cyp4g1 is known to be expressed in oenocytes, and it was found that this is the only site of detectable expression in embryos and larvae. Animals homozygous for either the Cyp4g1Delta4 or Cyp4g1Delta4-9 allele develop normally through larval and early pupal stages but arrest during mid-to-late pupal stages, with many failing during adult eclosion. This pupal phenotype is strikingly similar to the 50% oenocyte ablation phenotype. Moreover, although late-L3 Cyp4g1 mutant larvae appear morphologically indistinguishable from controls, they manifest a twofold increase in the oleic acid:stearic acid ratio (C18:1/C18:0). Notably, this imbalance in fatty acid desaturation is found in the TAG fraction but not in the phospholipid fraction. This selectivity strongly suggests that the Cyp4g1 defect is specific to fatty acids in metabolic storage form, most of which reside in the fat body, rather than fatty acids present in the structural lipids of all cell membranes. Taken together, the metabolic profiles of oenocyte-ablated and Cyp4g1 mutant larvae provide two independent lines of evidence that oenocytes regulate the lipid composition of the fat body (Gutierrez, 2007).

Functions of larval oenocytes, described in insects over 140 yr ago, have remained unclear, with largely descriptive studies implicating them in processes such as cuticle synthesis and the regulation of haemolymph composition. Using cell ablation to test their functions directly for the first time, clear requirements for larval growth and pupal development were found. Although the subset of oenocyte genes mediating the larval developmental functions remains to be identified, for pupal development it was shown that the lipid omega-hydroxylase Cyp4g1 is required. At least one important role of Cyp4g1 is to downregulate the ratio of oleic-to-stearic acid, widely used as a marker of SCD-1 activity in mammals. This prompts speculation that Cyp4g1 may repress the activity of stearoyl CoA-desaturases like Desat1, thereby inhibiting inappropriate monounsaturated fatty acid synthesis during long non-feeding periods such as late L3 and pupal stages (Gutierrez, 2007).

Four lines of evidence argue that at least some of the lipid-metabolizing roles of insect oenocytes are analogous to those of mammalian hepatocytes: (1) oenocytes express 22 orthologues of human fat-metabolizing genes expressed in hepatocytes; (2) like hepatocytes, they are atypical cells in that they accumulate fat droplets during starvation; (3) like the liver, oenocytes lie downstream of a nutrient sensor in a major fat depot; (4) Brummer lipase and Lsd2 in the fat body regulate oenocyte lipid content in a broadly similar way as ATGL and perilipin in adipose tissue regulate hepatic fat influx. However, whereas hepatocytes store large quantities of glycogen, this role in Drosophila is primarily carried out by the fat body. Thus, mammalian liver functions in glycogen storage and lipid processing seem to be divided in Drosophila between the fat body and oenocytes (Gutierrez, 2007).

This study suggests the existence of two-way metabolic coupling between the fat body and oenocytes. Analogous to the mammalian adipose-liver axis, lipid mobilization from the fat body during starvation produces lipid droplet accumulation in oenocytes, a metabolic change resembling hepatic steatosis. In Drosophila, a reciprocal regulation was also found, namely that oenocytes are required for efficiently depleting lipid from the fat body during fasting. This suggests a feedback mechanism for matching lipid supply to demand, whereby oenocytes keep haemolymph lipids low and also promote lipid mobilization from the fat body. Thus, in oenocyte-less larvae, excess circulating lipids might underlie the behavioural syndrome of larval dispersal and reduced feeding in a similar way as reported for elevated amino-acid levels (Zinke, 1999). Central to the proposed feedback model is the signal acting on the lipogenesis/lipolysis balance within the fat body. The data presented in this study are equally compatible with this signal corresponding to a haemolymph lipid/metabolite or to a separate signal generated by oenocytes. Regarding the latter possibility, it is interesting that recent work in mammals indicates that the liver secretes signalling factors (hepatokines) that promote lipolysis in adipose tissue (Oike, 2005). This suggests that Drosophila may prove useful, not only for modelling hepatic steatosis, but also some regulatory roles of the liver in metabolic homeostasis (Gutierrez, 2007).

Oogenesis

Because intense deposition of lipids is known to occur during oogenesis in the female germ line, whether Lsd2 is expressed in this tissue was investigated. Whole-mount in situ hybridization on ovaries has shown that Lsd2 mRNA is detected from mid-oogenesis (stages 7/8), where it accumulates in the oocyte. From stage 10 on, Lsd2 mRNA expression is greatly increased throughout the germ line, as shown by the higher cytoplasmic staining in both the nurse cells and the oocyte. This accumulation is consistent with the previous detection of a high level of Lsd2 mRNA during the first stages of embryogenesis (Teixeira, 2003).

The expression of Lsd2 protein was also examined in the female germ line. Western blot analysis showed that the two previously described Lsd2 forms, at ~50 and ~45 kDa, are detected in extracts from wild type and EP(X)1614 ovaries, but are absent from Lsd21 ovaries. The distribution of Lsd2 in ovaries was examined by immunofluorescence. A specific signal was first detected in the wild-type oocyte around mid-oogenesis. Later, in stage 10 egg chambers, Lsd2 level increases in the cytoplasm of the nurse cells, reflecting the accumulation of Lsd2 mRNA observed at this stage. However, Lsd2 was detected to a lower level in the oocyte despite the abundance of mRNA (Teixeira, 2003).

To investigate the subcellular distribution of Lsd2 in the germ line, electron microscopy was performed on ultrathin sections on stage 10 wild-type egg chambers. Both in nurse cells and in the oocyte, Lsd2 is mainly enriched at the surface of neutral lipid droplets. It was also observed in the oocyte, but not in nurse cells, that lipid droplets are frequently connected to membrane-surrounded tubules. The membrane of these tubules were labeled by the 1D3 monoclonal antibody recognizing the last 12 amino acids of protein disulphide isomerase, an endoplasmic reticulum (ER) resident enzyme. The low electron-density content of these tubules together with their connection with lipid droplets suggests that they also contain lipids. However, Lsd2 was not detected on these tubules (Teixeira, 2003).

The expression of Lsd2 in the female germ line and its localization to neutral lipid droplets prompted further examination of the process of lipid accumulation in the germ line. To visualize neutral lipids, wild-type ovaries were stained with Nile red, a fluorescent probe known to label neutral lipids. Neutral lipids could be detected at a low level in both nurse cells and in the oocyte from stages 7/8. Whereas evenly dispersed in the oocyte, neutral lipids are enriched in a network surrounding the nuclei in the nurse cells. This pattern of accumulation in nurse cells is similar to the organization of the ER at these stages. Indeed, in a co-detection with a marker of the ER lumen, neutral lipids appear distributed similarly to the ER network, although they do not perfectly co-localize (Teixeira, 2003).

At stage 10, a higher level of punctuate and evenly distributed lipid droplets was visible in the cytoplasm of nurse cells and, to a lower extent, in the oocyte. At the onset of stage 11, the cytoplasmic content of the nurse cells is progressively delivered into the oocyte through a process called dumping, causing massive growth of the oocyte and resulting in higher fluorescent Nile red staining of the ooplasm compared to previous stages. At the end of stage 12, dumping is complete and nurse cells have transferred their cytoplasm into the oocyte. At this stage, traces of neutral lipid droplets were still visible, surrounding the nucleus, of the apoptotic nurse cells. At stage 14, the intense fluorescence visible throughout the oocyte cytoplasm revealed the high content of lipid droplets deposited at the end of oogenesis in the mature egg. Taken together, these results show that Lsd2 expression coincides with the accumulation of neutral lipid droplets during oogenesis (Teixeira, 2003).

The protein phosphatase PP2A-B' subunit Widerborst is a negative regulator of cytoplasmic activated Akt and lipid metabolism in Drosophila

Inappropriate regulation of the PI3-kinase/PTEN/Akt kinase-signalling cassette, a key downstream target of insulin/insulin-like growth factor signalling (IIS), is associated with several major human diseases such as diabetes, obesity and cancer. In Drosophila, studies have recently revealed that different subcellular pools of activated, phosphorylated Akt can modulate different IIS-dependent processes. For example, a specific pool of activated Akt within the cytoplasm alters aspects of lipid metabolism, a process that is misregulated in both obesity and diabetes. However, it remains unclear how this pool is regulated. The protein phosphatase PP2A-B' regulatory subunit Widerborst (Wdb), which coimmunoprecipitates with Akt in vivo, selectively modulates levels of activated Akt in the cytoplasm. It alters lipid droplet size and expression of the lipid storage perilipin-like protein LSD2 in the Drosophila ovary, but not in epithelial cells of the eye imaginal discs. It is concluded that isoforms of PP2A-B' can act as subcellular-compartment-specific regulators of PI3-kinase/PTEN/Akt kinase signalling and IIS, potentially providing new targets for modulating individual subcellular pools of activated Akt in insulin-linked disease (Vereshchagina, 2008).

The signalling cassette involving Class I phosphatidylinositol 3-kinase (PI3K), phosphatase and tensin homologue on chromosome 10 (PTEN) and Akt (also known as protein kinase B or PKB) is part of a major intracellular kinase cascade that regulates multiple cellular functions including metabolism, growth, proliferation and survival. It responds to a variety of stimuli, such as insulin, other growth factors including PDGF and FGF, and attachment to the extracellular matrix. Upon activation, PI3K catalyses the formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. PtdIns(3,4,5)P3 is a lipid second messenger, which in turn recruits the PH-domain-containing Akt protein kinase from the cytosol to the plasma membrane. Here it is activated through phosphorylation at Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and at Ser473 (or Ser505 in the unique Drosophila Akt kinase, Akt1) by PDK2, which is thought to be the Rictor-mTOR complex. Once activated, Akt subsequently phosphorylates multiple targets, leading to its numerous downstream effects (Vereshchagina, 2008).

Misregulation of Akt and its cellular targets is linked to several major human diseases. For example, cellular insulin resistance is associated with reduced signalling by the PI3K/PTEN/Akt cassette and is an important defect in individuals suffering from Type 2 diabetes. By contrast, hyperactivation of this cassette, most notably through loss-of-function mutations in the tumour suppressor PTEN, which converts PtdIns(3,4,5)P3 back to PtdIns(4,5)P2, is strongly associated with many forms of human cancer (Vereshchagina, 2008 and references therein).

Molecular genetic studies in Drosophila have given rise to several fundamental insights into the regulation and functions of the PI3K/PTEN/Akt-signalling cassette. Not only has this work highlighted the central importance of nutrient-regulated insulin/insulin-like growth factor signalling (IIS) in controlling the activity of this cassette and cell growth, but it has also revealed a critical downstream link with the nutrient-sensitive mTOR-signalling cascade, which regulates several cellular processes including protein translation and autophagy. Furthermore, studies in invertebrates have indicated roles for PI3K/PTEN/Akt and mTOR in ageing, cell polarity and neurodegeneration, functions that all appear to be conserved in mammals and which might involve a combination of cellular and metabolic defects (Vereshchagina, 2008).

If the role of PI3K/PTEN/Akt in insulin-linked diseases is to be fully understood, it is essential to determine how this single signalling cassette regulates so many different cellular functions. One important part of the explanation is presumably the existence of cell-type-specific downstream-signalling targets that perform different roles. However, recent work, much of it again initiated in flies, has indicated that Akt activity can also be differentially regulated in specific subcellular domains and that these subcellular pools of activated Akt can control different processes. For example, precise regulation of Akt activity at the apical membrane of epithelial cells by localised PTEN is required for normal apical morphology in higher eukaryotes. By contrast, cytoplasmic activated Akt appears to be required for transcription of specific IIS target genes and regulation of lipid metabolism and droplet size in nurse cells of the Drosophila female germ line (Vereshchagina, 2006). These observations have highlighted the importance of finding the molecules that regulate different pools of activated Akt in vivo, because their modulation might alter specific functions of IIS in health and disease more selectively (Vereshchagina, 2008).

In a screen for novel phosphatase regulators of IIS, Widerborst (Wdb), one of the B' regulatory subunits of the protein phosphatase PP2A, was identified as a negative regulator of the PI3K/PTEN/Akt-signalling cassette. Although wdb is essential for cell viability in some tissues, wdb mutant cells in the germ line and follicular epithelium of the ovary are viable and display phenotypes that are similar to those seen in PTEN mutant ovaries. This study shows that Wdb and Drosophila Akt1 physically interact in the ovary, and that within this tissue, Wdb regulates the subcellular pool of activated Akt1 in the cytoplasm. This study therefore highlights an important new function for PP2A-B' subunits in selectively modulating certain IIS-dependent processes by controlling signalling in a specific subcompartment of the cell (Vereshchagina, 2008).

Several lines of evidence confirm that Wdb controls IIS activity and Akt1 phosphorylation state. First, when overexpressed, wdb genetically modifies phenotypes produced by altered IIS signalling, rescuing a lethal PTEN mutant combination and modifying the effects of FOXO in the eye. Second, loss-of-function wdb mutations produce very similar phenotypes to PTEN mutations in nurse cells, elevating levels of cytoplasmic pAkt1 and LSD2 [a Perilipin/ADRP homologue that regulates lipid metabolism, and inducing an abnormal accumulation of lipid droplets. Third, although wdb mutations do not independently appear to have strong effects on growth, they do suppress growth phenotypes produced by reduced Akt1 signalling both in mutant follicle cells homozygous for the Akt11 allele and in animals carrying a hypomorphic viable combination of Akt1 alleles. Genetic interactions with the PP2A catalytic subunit Mts in the eye indicate that these effects are dependent on the PP2A regulatory activity of Wdb (Vereshchagina, 2008).

Coimmunoprecipitation experiments revealed that Akt1 and Wdb form a complex in ovaries, the tissue in which the most obvious effects of wdb on pAkt1 levels are seen. The data suggest that one isoform of Wdb affects IIS within a complex containing Akt1, presumably by directly modulating the phosphorylation state of this molecule. This regulatory interaction appears to be evolutionarily conserved, because several studies in mammalian cell culture have shown that a PP2A-type activity controls Akt phosphorylation at Ser473, the equivalent position to Ser505 in Drosophila Akt1. PP2A-B' activity has been implicated in this process. Furthermore, mammalian PP2A can dephosphorylate Akt in vitro. The phosphorylation state of Thr308 might also be affected by PP2A. However, current tools do not allow determination of the phosphorylation state of Thr342 (the equivalent position to Thr308 in mammalian Akt) in wdb mutant cells in ovaries. Nevertheless, this study adds to the current understanding of the effects of PP2A on Akt by showing for the first time that at least one PP2A-B' isoform can act as a pool-specific suppressor of activated Akt. It is thought that that this property is likely to be shared by some mammalian PP2A-B' isoforms (Vereshchagina, 2008).

Unlike several other previously characterised components of the IIS cascade, the effects of wdb mutations on IIS appear to be tissue specific. Although pAkt1 levels are strongly upregulated in wdb mutant nurse cells and follicle cells, they appear unaffected in clones within the eye. PP2A is a broad-specificity protein phosphatase, which is selectively targeted to specific signalling molecules by regulatory subunits such as Wdb. Wdb has already been shown to be involved in several signalling events, including those regulating apoptosis and the Hedgehog (Hh) pathway, pathways that might be implicated in the wdb mutant phenotype observed in the eye imaginal disc (Vereshchagina, 2008).

How can Wdb have such a central IIS-regulatory role in the ovary, but show no detectable effect on this pathway in the developing eye? It seems unlikely that wdb mutant cells in the eye die too rapidly to observe changes in Akt1 phosphorylation, because wdb clones are seen in posterior positions within eye imaginal discs, which must have formed many hours previously. The IIS cascade is active in this tissue, because mutations altering IIS produce significant effects on growth in the eye disc. However, unlike in nurse cells, activation of IIS in the developing eye primarily leads to cell surface accumulation of pAkt1, at least in pupae. Surface-localised activated Akt1 may normally be sufficient to promote eye growth, since a myristoylated membrane-anchored form of Akt1 dominantly induces overgrowth in this and other tissues. One possible explanation for the data is therefore that cytoplasmic pAkt1 levels in the eye are restricted by other unknown molecules in addition to Wdb in this tissue, so loss of wdb here has little effect, whereas increased expression can still modify the FOXO phenotype (Vereshchagina, 2008).

In this context, at least two other phosphatases might be involved in Akt1 regulation. First, there is a second isoform of PP2A-B' in flies [called PP2A-B', CG7913 or Well-rounded (Wrd); that is most closely related to mammalian PP2A-B'γ isoforms. Simian virus 40 small t antigen acts as a specific inhibitor of mammalian PP2A-B'γ, stimulating phosphorylation of Akt and other targets, and thereby promoting growth. Reduced PP2A-B'γ activity has also been linked to the establishment and progression of melanomas (Vereshchagina, 2008).

Surprisingly, a recent report suggests Wrd is nonessential. Unless it acts redundantly with Wdb, it cannot therefore play a significant role in growth regulation). Analysis of the PP2A catalytic subunit Mts, using a dominant-negative construct, indicates that this enzyme enhances the effects of FOXO and is important in normal growth regulation in the eye, perhaps consistent with the idea that the two PP2A-B' isoforms do act redundantly. Alternatively, Mts may perform some of its growth regulatory functions independently of PP2A-B' (Vereshchagina, 2008 and references therein).

A second candidate negative regulator of Akt is the novel phosphatase PHLPP, which directly dephosphorylates human Akt at Ser473 and Drosophila Akt1 at Ser505 in cell culture, a function that may be disrupted in some tumours. Drosophila PHLPP could therefore control pAkt1 accumulation at the cell surface and perhaps reduce the amount of pAkt1 that can diffuse into the cytoplasm in tissues such as the eye. Since loss of wdb in either follicle cells or nurse cells is sufficient to elevate levels of cytoplasmic pAkt1, PHLPP presumably does not play such an important role in these cell types (microarray data suggest that PHLLP is not expressed at detectable levels in the adult ovary) (Vereshchagina, 2008).

Interestingly, the data in the ovary suggest further variable tiers of pAkt1 control. In nurse cells, loss of PTEN leads to accumulation of pAkt1 and LSD2 in the cytoplasm, but most PTEN mutant follicle cell clones do not show these phenotypes, presumably because other pAkt1 regulators such as Wdb play a more dominant role in these cells. No good explanation is available for how genetically identical clones can show such phenotypic variability. There is no obvious correlation with clone size or position in the small minority of PTEN-mutant follicular clones where pAkt1 and LSD2 upregulation is observed (Vereshchagina, 2008).

Because perilipin, the mammalian LSD2 orthologue, is thought to be regulated via insulin-dependent transcriptional and post-translational mechanisms, it is proposed that the increased LSD2 expression seen in PTEN mutant nurse cell clones results from similar effects of IIS on this molecule in flies. An alternative explanation is that increased IIS promotes excess triacylglyceride (TAG) synthesis and that LSD2 is only indirectly upregulated to permit proper packaging of these triacylglycerides into lipid droplets. Analysis of wdb mutant follicle cell clones does not support this latter model, since these clones strongly upregulate LSD2 expression, but do not show obvious changes in lipid droplet accumulation (Vereshchagina, 2008).

When wdb is overexpressed in the differentiating eye, the external structure of the eye becomes more disorganised and there is a slight reduction in overall eye size. Since this effect is not noticeably suppressed by co-overexpressing Akt1, it seems unlikely to be caused by reduced IIS. Unlike PTEN mutant follicle cells, wdb mutant follicle cells are not noticeably larger than their wild-type neighbours. Furthermore, although low level constitutive expression of Wdb in a pupal-lethal PTEN mutant background can rescue these flies to viability, the rescue may be explained by altered metabolism, because the rescued flies are still larger than normal. All these observations are consistent with the model that Wdb modulates cytoplasmic pAkt1 and has less of an effect on cell surface pAkt1, which is thought to be the primary regulator of normal growth. Wdb shows a relatively strong genetic interaction with the IIS-regulated transcription factor FOXO and this is completely suppressed by Akt1, raising the possibility that low levels of pAkt1 in the cytoplasm may play an important part in controlling FOXO activity (Vereshchagina, 2008).

Although wdb does not appear to modulate growth significantly under normal IIS-signalling conditions, mutations in wdb do enhance growth when Akt1 activity is reduced. Viable Akt1 mutant animals are larger in the presence of a heterozygous wdb mutation, while the Akt11 recessive growth phenotype in follicle cells is strongly suppressed by wdb. Interestingly, it has been reported that mutations in foxo have no effect on growth in otherwise normal animals, but that when IIS is reduced in chico mutants, which produce small adults, this phenotype is partially suppressed by loss of foxo function. The current data are consistent with this result, and may indicate that growth regulation in chico flies relies more on cytoplasmic pAkt1 and its effects on downstream targets like FOXO than it does in normal flies (Vereshchagina, 2008).

In conclusion, the identification of a PP2A-B' subunit as a novel cell-type-specific regulator of IIS within a specific subcellular compartment highlights the importance of studying the subcellular control of this signalling pathway in multiple cell types in vivo. Akt activation also promotes lipid synthesis and droplet formation in many mammalian cell types. This is likely to involve similar regulatory control mechanisms for cytoplasmic pAkt to those uncovered in flies. This work therefore raises new issues concerning the underlying causes of IIS-associated disease. For example, excess accumulation of lipid and obesity could be linked to selective changes in cytoplasmic pAkt control and might therefore be modulated by specific PP2A-B' subunits. Developing a better understanding of this form of regulation could therefore suggest new strategies for disease-specific treatments of IIS-linked disorders in the future (Vereshchagina, 2008).


EFFECTS OF MUTATION

Many cells store neutral lipids, as triacylglycerol and sterol esters, in droplets. PAT-domain proteins form a conserved family of proteins that are localized at the surface of neutral lipid droplets. Two mammalian members of this family, Perilipin and adipose differentiation-related protein, are involved in lipid storage and regulate lipolysis. This study describes the Drosophila PAT-family member Lsd2. Lsd2 is predominantly expressed in tissues engaged in high levels of lipid metabolism, the fat body and the germ line of females. Ultrastructural analysis in the germ line shows that Lsd2 localizes to the surface of lipid droplets. An Lsd2 mutant has been generated and its phenotype described. Mutant adults have a reduced level of neutral lipid content compared to wild type, showing that Lsd2 is required for normal lipid storage. In addition, ovaries from Lsd2 mutant females exhibit an abnormal pattern of accumulation of neutral lipids from mid-oogenesis, which results in reduced deposition of lipids in the egg. Consistent with its expression in the female germ line, Lsd2 is shown to be a maternal effect gene that is required for normal embryogenesis. This work demonstrates that Lsd2 has an evolutionarily conserved function in lipid metabolism and establishes Drosophila melanogaster as a new in vivo model for studies on the PAT-family of proteins (Teixeira, 2003).

To generate Lsd2 mutants, imprecise P element excisions were carried out on the EP(X)1614 line, in which an EP element is inserted approximately 600 bp upstream of the Lsd2 gene. Northern blot analysis of poly(A+)RNA isolated from homozygous flies from this line has revealed that the EP insertion does not prevent Lsd2 expression. An imprecise excision was isolated causing a small directional deletion of ~500 bp removing the genomic region located downstream of the original EP insertion site and extending towards, but not into, the 5' end of the longest expressed sequence tag (EST) available for Lsd2 from the BDGP. The homozygous stock carrying this deletion is viable and fertile, as is the original EP(X)1614 line. Nevertheless, because of its position in the putative regulatory region of Lsd2, this deletion might be expected to affect Lsd2 expression. To determine if that was the case, Western blot analysis of adult protein extracts were performed using a polyclonal antiserum raised against the longest open reading frame of 352 amino acids predicted for the Lsd2 protein. A major band was detected at ~50 kDa in crude extracts from wild type and EP(X)1614 flies. This size is slightly greater than the predicted molecular weight of 38 kDa for the Lsd2 longest EST. Nevertheless, expression of this open reading frame in Escherichia coli generated a peptide with an apparent molecular mass of ~50 kDa in SDS-PAGE. This band was absent in extract of the homozygous stock bearing the ~500 bp deletion upstream of Lsd2, even after maximal exposure of the blot. This demonstrates that the ~50 kDa form is encoded by Lsd2. A slightly faster-migrating band at ~45 kDa was also detected in wild type and EP(X)1614 adult extracts and could possibly correspond to a variant of Lsd2, since it was also absent in extract of Lsd21 homozygotes, although the possibility that it results from proteolytic degradation of the major ~50 kDa form cannot be excluded. The allele generated by the deletion is called Lsd21. Based on the lack of protein reactivity of the Lsd21 extract with anti-Lsd2 serum, it is concluded that Lsd21 is a strong hypomorphic or null allele (Teixeira, 2003).

Lsd21 mutant nurse cells reveal an altered pattern of neutral lipid accumulation beginning at stages 9/10. In contrast to the punctuated distribution in wild type, prominent patches of brightly stained neutral lipids were detected in the cytoplasm of mutant nurse cells. They often distribute in a radial pattern surrounding the nucleus of the nurse cells. During stages 11/12, neutral lipids aggregate in enlarged lipid structures, sequestered in the apoptotic nurse cells. At the end of oogenesis, these structures persist, confined near the respiratory appendages at the antero-dorsal side of the oocyte. An independent P-insertion called P{SUPor-P}Lsd2KG00149 was isolated in the Lsd2 5' UTR. Genetic complementation analysis between Lsd21 and P{SUPor-P}Lsd2KG00149 revealed a defect in the pattern of neutral lipid accumulation similar to Lsd21 or P{SUPor-P}Lsd2KG00149 homozygous nurse cells at stage 10. This demonstrates that Lsd21 and P{SUPor-P}Lsd2KG00149 mutations are allelic and the phenotype is specific for the Lsd2 gene. This genetic analysis shows that Lsd2 is required for normal neutral lipid droplet accumulation in the nurse cells (Teixeira, 2003).

Surprisingly, the aberrant pattern of neutral lipid accumulation observed in nurse cells was not seen in the oocyte. In addition, despite the obvious retention of neutral lipids in the nurse cells, the oocyte accumulates lipid droplets, as revealed by the increase of its fluorescence from stage 10 to 14. It is concluded that Lsd2 is not strictly required for neutral lipid droplet accumulation in the oocyte. However, to test whether the aberrant accumulation of lipids in the mutant nurse cells impairs normal deposition into the oocyte, neutral lipids were quantified in early embryos (0-1 h). 50% less TAG was detetected in embryos from mutant mothers compared to wild type, demonstrating that Lsd2 is required for normal deposition of neutral lipids in the oocyte (Teixeira, 2003).

Embryos in the Lsd21 homozygous stock have a significantly lower hatching rate than those of a wild type control stock, nearly ~95% in control and ~63% in mutant stocks. This defect is also seen in the progeny of homozygous mutant females crossed with wild-type males. This shows that the hatching defect is not suppressed by providing a wild type Lsd2 copy from males and indicates that it is dependent on the genotype of the mother, rather than of the zygote. Consistent with this, no defect was observed in the progeny of Lsd21 heterozygous females crossed with hemizygous mutant males in spite of the fact that half of the progeny was mutant. This demonstrates that Lsd2 is a maternal effect gene. To further investigate the hatching defect, embryos were collected from wild type and Lsd21 homozygous mothers and their development was examined at two time points. No significant difference was visible between the two populations after ~7 h of development, most embryos being at stage 11. However, after ~21 h, just before hatching, clear differences were observed between the wild type and the embryos of Lsd21 mothers. In the wild-type population, 99% of embryos presented an elongated larva-like morphology. In contrast, 78% of embryos from Lsd21 mothers ranged from a stage 17 embryo-shaped morphology to the wild type elongated larva-like morphology. Moreover, the remaining 22% of embryos from Lsd21 mothers appeared to have degenerated. Thus, the loss of viability of ~37% in the progeny of Lsd21 mothers results from developmental defects occurring after stage 11 (Teixeira, 2003).

A further examination of the Lsd21 homozygous stock revealed that whereas larvae develop normally in a rich diet, they were less opaque than wild-type larvae. This seemed to be due to the fact that the fat body of these larvae, easy to visualize because of the transparency of the body wall, is less developed than that of control larvae. To test whether the lipid storage function of the fat body is impaired in the mutant, the TAG content was quantified in adult flies. The level of TAG was observed to be 27% lower in the mutant than in the wild type. This demonstrates that Lsd2, similarly to Perilipin in the mouse, is required for normal storage of lipids in the fly (Teixeira, 2003).

PERILIPIN-dependent control of lipid droplet structure and fat storage in Drosophila

Lipid droplets are intracellular organelles enriched in adipose tissue that govern the body fat stores of animals. In mammals, members of the evolutionarily conserved PERILIPIN protein family are associated with the lipid droplet surface and participate in lipid homeostasis. This study shows that Drosophila mutants lacking the PERILIPIN PLIN1 are hyperphagic and suffer from adult-onset obesity. PLIN1 is a central and Janus-faced component of fat metabolism. It provides barrier function to storage lipid breakdown and acts as a key factor of stimulated lipolysis by modulating the access of proteins to the lipid droplet surface. It also shapes lipid droplet structure, transforming unilocular into multilocular fat cells. Flies were generated devoid of all PERILIPIN family members, and it was shown that they exhibit impaired yet functional body fat regulation. The data reveal the existence of a basal and possibly ancient lipid homeostasis system (Beller, 2010).

These results establish that Drosophila PLIN1 is a constitutive lipid droplet protein that is expressed from late embryonic stages onward predominantly in the fat body. It has a dual function in fat storage control as an essential component of the stimulated AKH/AKHR lipolysis pathway and by mediating the localization of lipid droplet-associated proteins such as the BMM lipase. PLIN1 also determines the size of lipid droplets in fat body cells. Its activity is dynamically regulated both at the transcriptional and posttranscriptional level to regulate the body fat content of the organism (Beller, 2010).

PLIN1 mutant flies show increased fat storage and hyperphagia. These effects are not unique for PLIN1 mutants but are characteristic for AKH/AKHR signaling pathway impairment. Downregulation of the AKHR-dependent cAMP-responsive transcription factor dCREB2 in fat body causes adiposity and increased food intake (Iijima, 2009). The mechanism of how the structural and physiological defects in the fat body are communicated to the central nervous system (CNS) to increase food intake is currently unknown. CNS neuron populations that participate in fat storage and food intake control have been identified (Al-Anzi, 2009). Moreover, a yet uncharacterized humoral signal of the larval fat body that triggers insulin-like peptide release from CNS has been described. These studies suggest that communication between fat body and CNS is a prerequisite for lipohomeostatic regulation. In this view, impaired storage lipid mobilization in PLIN1 mutants may interfere with an afferent fat body signal (e.g., an adipokine or metabolite), which is read out in the CNS to incessantly match food intake to energy demand (Beller, 2010).

Mammalian PLIN1 is largely restricted to adipocytes and subject to posttranscriptional regulation and regulation by altered physiological conditions. It executes a barrier function in basal lipolysis and serves as platform for the assembly of protein complexes that mediate stimulated lipolysis in a phosphorylation-dependent manner. Fly PLIN1 acts in the AKH/ AKHR signaling pathway as mammalian PLIN1 does in the corresponding β-adrenergic pathway. These intriguing parallels suggest that functional aspects of the PERILIPIN system are evolutionarily ancient and that PLIN1 acts as a conserved surface-associated module of lipid droplets that promotes stimulated lipolysis in response to cAMP/PKA signaling. The in vivo data on PLIN1 confirm in vitro and ex vivo studies showing that PKA-phosphorylation of PLIN1 enhances lipase activity on artificial and native lipid droplets. These data argue that PLIN1 can directly interact with/recruit TG lipase(s) and may act as a phosphorylation-dependent regulator of a lipase activator just as mammalian Perilipin 1 acts on of the ATGL activator CGI-58. In fact, the Drosophila genome encodes a functionally uncharacterized CGI-58 homolog. Both mechanisms, inappropriate lipase recruitment and failure of lipase activator interaction, would contribute to the increased adiposity of PLIN1 mutants. However, a structural change from multi- to unilocular fat cells, might also influence lipolysis and contribute to the fat storage increase of plin1 mutants. Lipid droplet association of the BMM lipase is increased in plin1 mutant flies, which are also more sensitive to fat mobilization when challenged by targeted BMM expression. This phenomenon was also observed in murine AML12 hepatocytes, when the two PERILIPINs of this cell type (PLIN2 and PLIN3) were cotargeted by RNAi. Their loss resulted in fewer and enlarged lipid droplets. The first engineered PERILIPIN-free organism, as presented in this study, shows that PERILIPINs, at least in flies, are dispensable for lipid droplet biogenesis but responsible for regulating lipid droplet size in vivo (Beller, 2010).

PLIN1 knockout mice have a severe lipometabolism phenotype and loss of PLIN2 activity causes triglyceride storage reduction in liver and resistance to diet-induced hepatic steatosis. Similarly, Mpl1 mutants of the ascomycete Metarhizium anisopliae as well as plin2 flies show lipid storage defects. These results underline a distinct role for PERILIPINs in lipometabolism control as shown in this study for plin1. However, other eukaryotic model systems for fat storage control such as the yeast S. cerevisiae or the nematode C. elegans have no PERILIPIN genes. This notion is consistent with the finding that PERILIPINs are not essential for basal lipometabolic activity but rather to increase its efficacy and to improve the effectiveness of lipometabolism management in some lineages that is not required in others. The existence of multiple and in part functionally redundant PERILIPINs in mammals and insects reflects therefore a positive selection of the ancestral PERILIPIN, followed by gene duplication and functional diversification events. The notion that Drosophila PLIN2 also serves as an adaptor protein for lipid droplet transport during early embryogenesis exemplifies that PERILIPINs can indeed adopt novel cellular functions (Beller, 2010).

The finding that PERILIPINs are not essential for survival under ad libitum feeding supports their role as potentiator of lipometabolism. In a natural environment, however, where food access for flies is variable or even limited, impairment of the PERILIPIN system might entail a substantial selective disadvantage. This speculation can be tested with the PERILIPIN-free plin1 plin2 double mutants, which also provide access to the conserved control system underlying basic lipid homeostasis, and thereby might reveal novel therapeutic targets for the treatment of human lipopathologies (Beller, 2010).


EVOLUTIONARY HOMOLOGS

Characterization of mammalian Perilipin and and identification of the PAT domain

The Perilipins are a family of intracellular neutral lipid droplet storage proteins that are responsive to acute protein kinase A-mediated, hormonal stimulation. Perilipin (Peri) expression appears to be limited to adipocytes and steroidogenic cells, in which intracellular neutral lipid hydrolysis is regulated by protein kinase A. cDNA sets and overlapping genomic fragments of the murine Peri locus were isolated and chromosomal location, transcription start sites, polyadenylylation sites, and intron/exon junctions were mapped. Data confirm that the Perilipins are encoded by a single-copy gene, with alternative and tissue-specific, mRNA splicing and polyadenylylation yielding four different protein species. The Perilipin proteins have identical (approximately 22-kDa) amino termini with distinct carboxyl terminal sequences of varying lengths. These genomic and transcriptional maps of murine Perilipin are also essential for evaluating presumptive endogenous and targeted mutations within the locus. The N-terminal identity region of the Perilipins defines a sequence motif, termed PAT, that is shared with the ADRP and TIP47 proteins; additionally, the PAT domain may represent a novel, conserved pattern for lipid storage droplet (LSD) proteins of vertebrates and invertebrates alike. Comparative genomics suggest the presence of related LSD genes in species as diverse as Drosophila and Dictyostelium (Lu, 2002).

Structural studies of Perilipin

The perilipins are the most abundant proteins coating the surfaces of lipid droplets in adipocytes and are found at lower levels surrounding lipid droplets in steroidogenic cells. Perilipins drive triacylglycerol storage in adipocytes by regulating the rate of basal lipolysis and are also required to maximize hormonally stimulated lipolysis. To map the domains that target and anchor perilipin A to lipid droplets, fragments of perilipin A were stabily expressed in 3T3-L1 fibroblasts. Immunofluorescence microscopy and immunoblotting of proteins from isolated lipid droplets revealed that neither the amino nor the carboxyl terminus is required to target perilipin A to lipid droplets; however, there are multiple, partially redundant targeting signals within a central domain including 25% of the primary amino acid sequence. A peptide composed of the central domain of perilipin A directs a fused green fluorescent protein to the surfaces of lipid droplets. Full-length perilipin A associates with lipid droplets via hydrophobic interactions, as shown by the persistence of perilipins on lipid droplets after centrifugation through an alkaline carbonate solution. Results of the mutagenesis studies indicate that the sequences responsible for anchoring perilipin A to lipid droplets are most likely domains of moderately hydrophobic amino acids located within the central 25% of the protein. Thus, it is concluded that the central 25% of the perilipin A sequence contains all of the amino acids necessary to target and anchor the protein to lipid droplets (Garcia, 2003).

Perilipin A is the most abundant lipid droplet-associated protein in adipocytes and serves important functions in regulating triacylglycerol levels by reducing rates of basal lipolysis and facilitating hormonally stimulated lipolysis. The central region of perilipin A targets and anchors it to lipid droplets, at least in part via three moderately hydrophobic sequences that embed the protein into the hydrophobic core of the droplet. The current study examines the roles of the amino and carboxyl termini of perilipin A in facilitating triacylglycerol storage. Amino- and carboxyl-terminal truncation mutations of mouse perilipin A were stably expressed in 3T3-L1 preadipocytes, which lack perilipins. Triacylglycerol content of the cells was quantified as a measure of perilipin function and was compared with that of cells expressing full-length perilipin A or control cells lacking perilipins. The amino-terminal sequence between amino acids 122 and 222, including four 10-11-amino acid sequences predicted to form amphipathic beta-strands and a consensus site for cAMP-dependent protein kinase, and the carboxyl terminus of 112 amino acids that is unique to perilipin A are critical to facilitate triacylglycerol storage. The precocious expression of full-length perilipin A in 3T3-L1 preadipocytes aided more rapid storage of triacylglycerol during adipose differentiation. By contrast, the expression of highly truncated amino- or carboxyl-terminal mutations of perilipin failed to serve a dominant negative function in lowering triacylglycerol storage during adipose differentiation. It has been concluded that the amino and carboxyl termini are critical to the function of perilipin A in facilitating triacylglycerol storage (Garciaw, 2004).

Effects of Perilipin mutation

Perilipin coats the lipid droplets of adipocytes and is thought to have a role in regulating triacylglycerol hydrolysis. To study the role of perilipin in vivo, a perilipin knockout mouse was created. Perilipin null (peri-/-) and wild-type (peri+/+) mice consume equal amounts of food, but the adipose tissue mass in the null animals is reduced to approximately 30% of that in wild-type animals. Isolated adipocytes of perilipin null mice exhibit elevated basal lipolysis because of the loss of the protective function of perilipin. They also exhibit dramatically attenuated stimulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity. Plasma leptin concentrations in null animals were greater than expected for the reduced adipose mass. The peri-/- animals have a greater lean body mass and increased metabolic rate but they also show an increased tendency to develop glucose intolerance and peripheral insulin resistance. When fed a high-fat diet, the perilipin null animals are resistant to diet-induced obesity but not to glucose intolerance. The data reveal a major role for perilipin in adipose lipid metabolism and suggest perilipin as a potential target for attacking problems associated with obesity (Tansey, 2001).

Obesity is a major risk factor for diabetes and heart disease. Inactivation of the gene for perilipin (plin), an adipocyte lipid droplet surface protein, produces lean and obesity-resistant mice. To dissect the underlying mechanisms involved, oligonucleotide microarrays were used to analyze the gene-expression profile of white adipose tissue (WAT), liver, heart, skeletal muscle, and kidney of plin-/- and plin+/+ mice. As compared with wild-type littermates, the WAT of plin-/- mice had 270 and 543 transcripts that were significantly up- or down-regulated. There was a coordinated upregulation of genes involved in beta-oxidation, the Krebs cycle, and the electron transport chain concomitant with a downregulation of genes involved in lipid biosynthesis. There was also a significant downregulation of the stearoyl CoA desaturase-1 gene, which has been associated with obesity resistance. Thus, in response to the constitutive activation of lipolysis associated with absence of perilipin, WAT activates pathways to rid itself of the products of lipolysis and activates pathways of energy expenditure that contribute to the observed obesity resistance. The biochemical pathways involved in obesity resistance in plin-/- mice identified in this study may represent potential targets for the treatment of obesity (Castro-Chavez, 2003).

Targeted disruption of the lipid droplet protein, perilipin, in mice, leads to constitutional lipolysis associated with marked reduction in white adipose tissue as a result of unbridled lipolysis. To investigate the metabolic adaptations in response to the constitutive lipolysis, perilipin-null (plin-/-) mice were studied in terms of their fatty acid oxidation and glycerol and glucose metabolism homeostasis by using dynamic biochemical testing and clamp and tracer infusion methods. plin-/- mice show increased beta-oxidation in muscle, liver, and adipose tissue resulting from a coordinated regulation of the enzymes and proteins involved in beta-oxidation. The increased beta-oxidation helped remove the extra free fatty acids created by the constitutive lipolysis. An increase in the expression of the transcripts for uncoupling proteins-2 and -3 also accompany this increase in fatty acid oxidation. Adult plin-/- mice have normal plasma glucose but a reduced basal hepatic glucose production (46% that of plin+/+). Insulin infusion during low dose hyperinsulinemic-euglycemic clamp further lowers the glucose production in plin-/- mice, but plin-/- mice also show a 36% decrease in glucose disposal rate during the low dose insulin clamp, indicating peripheral insulin resistance. However, compared with plin+/+ mice, 14-week-old plin-/- mice show no significant difference in glucose disposal rate during the high dose hyperinsulinemic clamp, whereas 42-week-old plin-/- mice display significant insulin resistance on high dose hyperinsulinemic clamp. Despite increasing insulin resistance with age, plin-/- mice at different ages maintain a normal glucose response during an intraperitoneal glucose tolerance curve, being compensated by the increased beta-oxidation and reduced hepatic glucose production. These experiments uncover the metabolic adaptations associated with the constitutional lipolysis in plin-/- mice that allow the mice to continue to exhibit normal glucose tolerance in the presence of peripheral insulin resistance (Saha, 2004).

Transcriptional regulation of Perilipin

In a systematic search for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) target genes, S3-12 and perilipin were identified as novel direct PPAR-gamma target genes. Together with adipophilin and tail-interacting protein of 47 kDa, these genes are lipid droplet-associating proteins with distinct expression pattern but overlapping expression in adipose tissue. The expression of S3-12 and perilipin is tightly correlated to the expression and activation of PPAR-gamma in adipocytes, and promoter characterization revealed that the S3-12 and the perilipin promoters contain three and one evolutionarily conserved PPAR response elements, respectively. The expression of S3-12 and perilipin is reduced in obese compared with lean Zucker rats, whereas the expression of adipophilin is increased. Perilipin has been shown to be an essential factor in the hormonal regulation of lipolysis of stored triglycerides within adipose tissue. The direct regulation of perilipin and S3-12 by PPAR-gamma therefore is likely to be an important mediator of the in vivo effects of prolonged treatment with PPAR-gamma activators: insulin sensitization, fatty acid trapping in adipose tissue, reduced basal adipose lipolysis, and weight gain (Dalen, 2004).

Most cis-acting regulatory elements have generally been assumed to activate a single nearby gene. However, many genes are clustered together, raising the possibility that they are regulated through a common element. A single peroxisome proliferator response element (PPRE), located between the mouse PEX11 alpha and perilipin genes, confers on both genes activation by peroxisome proliferator-activated receptor alpha (PPAR alpha) and PPAR gamma. A functional PPRE 8.4 kb downstream of the promoter of PEX11 alpha, a PPAR alpha target gene, was identified by a gene transfection study. This PPRE is positioned 1.9 kb upstream of the perilipin gene and also functions with the perilipin promoter. In addition, this PPRE, when combined with the natural promoters of the PEX11 alpha and perilipin genes, confers subtype-selective activation by PPAR alpha and PPAR gamma 2. The PPRE sequence specifically binds to the heterodimer of RXR alpha and PPAR alpha or PPAR gamma 2, as assessed by electrophoretic gel mobility shift assays. Furthermore, tissue-selective binding of PPAR alpha and PPAR gamma to the PPRE was demonstrated in hepatocytes and adipocytes, respectively, by chromatin immunoprecipitation assay. Hence, the expression of these genes is induced through the same PPRE in the liver and adipose tissue, where the two PPAR subtypes are specifically expressed (Shimizu, 2004).

Recent studies have shown that lipid droplets are covered with a proteinaceous coat, although the functions and identities of the component proteins have not yet been well elucidated. The first identified lipid droplet-specific proteins are the perilipins, a family of proteins coating the surfaces of lipid droplets of adipocytes. The generation of perilipin-null mice has revealed that although they consume more food than control mice, they have normal body weight and are resistant to diet-induced obesity. In one study it was reported that in an animal model obesity was reversible by breeding perilipin -/- alleles into Lepr db/db obese mice, ostensibly by increasing the metabolic rate of the mice. To understand the exact mechanisms that drive the exclusive expression of the perilipin gene in adipocytes, the 5'-flanking region of the mouse gene was examined. Treatment of differentiating 3T3-L1 adipocytes with an agonist of proliferator-activated receptor (PPAR) gamma, the putative 'master regulator' of adipocyte differentiation, significantly augments perilipin gene expression. Reporter assays using the -2.0-kb promoter revealed that this region contains a functional PPARgamma-responsive element. Gel mobility shift and chromatin immunoprecipitation assays showed that endogenous PPARgamma protein binds to the perilipin promoter. PPARgamma2, an isoform exclusively expressed in adipocytes, was found to be the most potent regulator from among the PPAR family members including PPARalpha and PPARgamma1. These results make evident the fact that perilipin gene expression in differentiating adipocytes is crucially regulated by PPARgamma2, providing new insights into the adipogenic action of PPARgamma2 and adipose-specific gene expression, as well as potential anti-obesity pharmaceutical agents targeted to a reduction of the perilipin gene product (Arimura, 2004).

Expression of FoxC2 blocks the capacity of 3T3-L1 preadipocytes to undergo adipogenesis in the presence of dexamethasone, isobutylmethylxanthine, and insulin. This block is characterized by an extensive decrease in the expression of proteins associated with the function of the mature fat cell, most notably C/EBPalpha, adiponectin, perilipin, and the adipose-specific fatty acid-binding protein, FABP4/aP2. Since the expression of these proteins lies downstream of PPARgamma, overexpressed PPARgamma was overexpressed in Swiss mouse fibroblasts to promote adipocyte differentiation. FoxC2 blocks the ability of PPARgamma to induce adipogenic gene expression in response to exposure of the cells to dexamethasone, isobutylmethylxanthine, insulin, and a PPARgamma ligand. Interestingly, the expression of aP2 escapes the inhibitory action of FoxC2 under conditions that promote maximum PPARgamma activity. In contrast, FoxC2 inhibits the expression of C/EBPalpha, perilipin, and adiponectin even in the presence of potent PPARgamma ligands. Finally, it has been shown that FoxC2 does not affect the ability of PPARgamma to bind to or transactivate from a PPARgamma response element. These data suggest that FoxC2 blocks adipogenesis by inhibiting the capacity of PPARgamma to promote the expression of a subset of adipogenic genes (Davis, 2004).

Perilipin, a family of phosphoproteins located around lipid droplets in adipocytes, is essential for enlargement of lipid droplets and lipolytic reaction by hormone-sensitive lipase. Thiazolidinediones, peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been shown to increase perilipin expression in fully differentiated adipocytes. However, the precise mechanism of transcriptional regulation of murine perilipin gene heretofore remains unclear. The transcription start site of murine perilipin gene was determined by RNA ligase-mediated rapid amplification of the cDNA ends method. Luciferase reporter gene constructs containing various lengths of the 5'-flanking region of the murine perilipin gene were generated and promoter/enhancer activities were assayed using differentiated 3T3-L1 adipocytes. A functional PPAR-responsive element (PPRE) was identified in the murine perilipin promoter, and this was confirmed by gel EMSAs using nuclear extracts from differentiated 3T3-L1 adipocytes. Furthermore, point mutations of the identified functional PPRE markedly reduce both the reporter gene activity in differentiated 3T3-L1 adipocytes and PPARgamma/thiazolidinedione-induced transactivation in NIH-3T3 fibroblasts. Real-time RT-PCR reveals that thiazolidinedione up-regulates endogenous perilipin mRNA levels. It is proposed that PPARgamma plays a significant role in the transcriptional regulation of murine perilipin gene via the PPRE in its promoter (Nagai, 2004).

Perilipin is targeted by PKA

Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, NIH 3T3 fibroblasts were used lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulate 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhances TG accumulation and inhibits lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduces TG accumulation and enhances lipolysis. Forskolin treatment induces Peri A hyperphosphorylation and abrogates the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduces Peri A hyperphosphorylation and blocks constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells (Souza, 2002).

Perilipin A coats the lipid storage droplets in adipocytes and is polyphosphorylated by protein kinase A (PKA); the fact that PKA activates lipolysis in adipocytes suggests a role for perilipins in this process. To assess whether perilipins participate directly in PKA-mediated lipolysis, constructs coding for native and mutated forms of the two major splice variants of the perilipin gene, perilipins A and B, were expressed in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, it is concluded that the expression of perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect (Tansey, 2003).

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, adenoviruses expressing truncated or mutated Peri A and HSL were introduced into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. Two lipase-selective functional domains were identified: (1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and (2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis reveals that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser433, Ser492, and Ser517). Peri 301-517 promotes PKA-stimulated lipolysis by HSL yet does not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulates lipolysis by HSL. These results suggest a model of Peri A function in which (1) lipase-specific 'barrier' domains block basal lipolysis by HSL and non-HSL, (2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and (3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity (Zhang, 2003).

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. Proteomic analysis was used to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase (Subramanian. 2004).

Lipid droplets (LDs) are a class of ubiquitous cellular organelles that are involved in lipid storage and metabolism. Although the mechanisms of the biogenesis of LDs are still unclear, a set of proteins called the PAT domain family have been characterized as factors associating with LDs. Perilipin, a member of this family, is expressed exclusively in the adipose tissue and regulates the breakdown of triacylglycerol in LDs via its phosphorylation. A yeast two-hybrid system was used to examine the potential function of perilipin. Direct interaction was found between perilipin and CGI-58, a deficiency of which correlated with the pathogenesis of Chanarin-Dorfman syndrome (CDS). Endogenous CGI-58 is distributed predominantly on the surface of LDs in differentiated 3T3-L1 cells, and its expression increases during adipocyte differentiation. Overexpressed CGI-58 tagged with GFP gathers at the surface of LDs and colocalizes with perilipin. This interaction seems physiologically important because CGI-58 mutants carrying an amino acid substitution identical to that found in CDS lost the ability to be recruited to LDs. These mutations significantly weakened the binding of CGI-58 with perilipin, indicating that the loss of this interaction is involved in the etiology of CDS. Furthermore, CGI-58 was identified as a binding partner of ADRP, another PAT domain protein expressed ubiquitously. GFP-CGI-58 expressed in non-differentiated 3T3-L1 or CHO-K1 cells colocalizes with ADRP, and CGI-58 mutants are not recruited to LDs carrying ADRP, indicating that CGI-58 may also cooperate with ADRP (Yamaguchi, 2004).

Perilipin and lipid storage

Adipocyte lipolysis was compared with hormone-sensitive lipase (HSL)/perilipin subcellular distribution and perilipin phosphorylation using Western blot analysis. Under basal conditions, HSL resides predominantly in the cytosol and unphosphorylated perilipin resides predominantly on the surface of the lipid droplet. Upon lipolytic stimulation of adipocytes isolated from young rats with the beta-adrenergic agonist, isoproterenol, HSL translocates from the cytosol to the lipid droplet, but there was no movement of perilipin from the droplet to the cytosol; however, perilipin phosphorylation was observed. By contrast, upon lipolytic stimulation and perilipin phosphorylation in cells from more mature rats, there was no HSL translocation but a significant movement of perilipin away from the lipid droplet. Adipocytes from younger rats have markedly greater rates of lipolysis than those from the older rats. Thus high rates of lipolysis require translocation of HSL to the lipid droplet and translocation of HSL and perilipin can occur independently of each other. A loss of the ability to translocate HSL to the lipid droplet probably contributes to the diminished lipolytic response to catecholamines with age (Clifford, 2000).

The perilipins are the most abundant proteins at the surfaces of lipid droplets in adipocytes and are also found in steroidogenic cells. To investigate perilipin function, perilipin A, the predominant isoform, was ectopically expressed in fibroblastic 3T3-L1 pre-adipocytes that normally lack the perilipins. In control cells, fluorescent staining of neutral lipids with Bodipy 493/503 show a few minute and widely dispersed lipid droplets, while in cells stably expressing perilipin A, the lipid droplets were more numerous and tightly clustered in one or two regions of the cytoplasm. Immunofluorescence microscopy revealed that the ectopic perilipin A localizes to the surfaces of the tiny clustered lipid droplets; subcellular fractionation of the cells using sucrose gradients confirmed that the perilipin A localizes exclusively to lipid droplets. Cells expressing perilipin A store 6- to 30-fold more triacylglycerol than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol is 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control cells, when triacylglycerol synthesis is blocked with 6 microm triacsin C. This stabilization of triacylglycerol is not due to the suppression of triacylglycerol lipase activity by the expression of perilipin A. It is concluded that perilipin A increases the triacylglycerol content of cells by forming a barrier that reduces the access of soluble lipases to stored lipids, thus inhibiting triacylglycerol hydrolysis. These studies suggest that perilipin A plays a major role in the regulation of triacylglycerol storage and lipolysis in adipocytes (Brasaemle, 2000).

A key step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the beta-adrenergic receptor-adenylyl cyclase complex. HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. The HSL translocation reaction was reconstructed in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A-phosphorylated HSL and perilipin A (Sztalryd, 2003).

Leptin interaction with perilipin levels

Transgenic mice overexpressing leptin (LepTg) exhibit substantial reductions in adipose mass. Since the binding of leptin to its receptor activates the sympathetic nervous system, it was reasoned that the lean state of the LepTg mice could be caused by chronic lipolysis. Instead, the LepTg mice exhibit a low basal lipolysis state and their lean phenotype is not dependent on the presence of beta3-adrenergic receptors. In their white adipose tissue, protein levels of protein kinase A, hormone-sensitive lipase, and ADRP are not impaired. However, compared to normal mice, perilipin, perilipin mRNA, and cAMP-stimulated PKA activity are significantly attenuated. Overall, it is demonstrated that the lean phenotype of the LepTg mice does not result in a chronically elevated lipolytic state, but instead in a low basal lipolysis state characterized by a decrease in perilipin and PKA activity in white fat (Ke, 2003).

Obesity is a disorder of energy balance. Hormone-sensitive lipase (HSL) mediates the hydrolysis of triacylglycerol, the major form of stored energy in the body. Perilipin (encoded by the gene Plin), an adipocyte protein, has been postulated to modulate HSL activity. Targeted disruption of Plin results in healthy mice that have constitutively activated fat-cell HSL. Plin-/- mice consume more food than control mice, but have normal body weight. They are much leaner and more muscular than controls, have 62% smaller white adipocytes, show elevated basal lipolysis that is resistant to beta-adrenergic agonist stimulation, and are cold-sensitive except when fed. They are also resistant to diet-induced obesity. Breeding the Plin-/- alleles into Leprdb/db mice reverses the obesity by increasing the metabolic rate of the mice. These results demonstrate a role for perilipin in reining in basal HSL activity and regulating lipolysis and energy balance; thus, agents that inactivate perilipin may prove useful as anti-obesity medications (Martinez-Botas, 2000).


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Search PubMed for articles about Drosophila Lipid storage droplet-1 & Lipid storage droplet-2

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Biological Overview

date revised: 25 June 2014

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