The Interactive Fly
Zygotically transcribed genes
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).
Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets enclosed in a monolayer of phospholipids and associated proteins. These dynamic organelles serve as the principal reservoirs for storing cellular energy and for the building blocks for membrane lipids. Excessive lipid accumulation in cells is a central feature of obesity, diabetes and atherosclerosis, yet remarkably little is known about lipid-droplet cell biology. This study shows, by means of a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells, that about 1.5% of all genes function in lipid-droplet formation and regulation. The phenotypes of the gene knockdowns sorted into five distinct phenotypic classes. Genes encoding enzymes of phospholipid biosynthesis proved to be determinants of lipid-droplet size and number, suggesting that the phospholipid composition of the monolayer profoundly affects droplet morphology and lipid utilization. A subset of the Arf1-COPI vesicular transport proteins also regulated droplet morphology and lipid utilization, thereby identifying a previously unrecognized function for this machinery. These phenotypes are conserved in mammalian cells, suggesting that insights from these studies are likely to be central to the understanding of human diseases involving excessive lipid storage (Guo, 2008).
Lipid-droplet formation was induced by incubation with 1 mM oleate for 24 h. Staining with 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY493/503) showed that droplet size, number and overall volume were increased; cellular triacylglycerol content increased sevenfold. Imaging this process by time-lapse microscopy of BODIPY-labelled cells after oleate addition showed that droplet formation occurred in steps. First, increased numbers of small droplets formed in dispersed locations throughout the cell. Next, droplets increased in size and finally aggregated into one or several large clusters, resembling grapes. Electron microscopy confirmed the tight clustering of the droplets, which were often near the nucleus (Guo, 2008).
To unravel the molecular mechanisms governing this progression of changes during lipid-droplet formation, a genome-wide RNAi screen was performed in S2 cells. Images were acquired and examined by two independent observers, who scored them for alterations in droplet number, size and dispersion. The same data were analysed computationally. From visual screening, both observers identified 847 candidate genes with altered lipid-droplet morphology. To verify these genes and to minimize the misidentification of genes from off-target effects of RNAi treatments, RNAi experiments for these genes were repeated with a second, distinct set of double-stranded (ds)RNAs. Visual analyses identified 132 genes whose knockdown consistently and repeatedly altered lipid-droplet morphology and an additional 48 genes for which knockdown phenotypes were scored in two of three rounds. Computational analysis confirmed 86 of these 180 genes and added 47 genes with altered lipid-droplet morphology. Thus, 227 genes (about 1.5% of the genome) were identified that affect lipid-droplet morphology (Guo, 2008).
The 132 genes with striking phenotypes were categorized into five distinct phenotypic classes, which were validated for selected knockdowns by electron microscopy. Class I genes showed reduced numbers of droplets and included midway (encoding a diacylglycerol acyl-transferase), subunits of the proteasome and the spliceosome, and several uncharacterized open reading frames. Class II genes gave smaller, more dispersed, droplets and included subunits of the COP9-signallosome complex, dynein, and RNA polymerase II subunits. Class III genes showed more dispersed droplets of slightly larger size and were members of the Arf1-COPI vesicular transport machinery. Class IV genes yielded highly condensed clusters of droplets and included members of the translational machinery. Class V genes contained one or a few very large droplets and included an orthologue of sterol regulatory element binding-protein (SREBP), a master transcriptional regulator of lipid metabolism, and SREBP cleavage activating protein (SCAP). In Drosophila, the SREBP pathway is sensitive to and regulates phospholipid biosynthesis. This class also included Cct1 and Cct2, which encode isoforms of phosphocholine cytidylyltransferase, the enzyme that catalyses the rate-limiting step in phosphatidylcholine synthesis, and CG2201, which is predicted to have choline kinase activity that phosphorylates and activates choline. Thus, most class V genes were linked directly or indirectly to phospholipid biosynthesis (Guo, 2008).
To further explore how phospholipid metabolism regulates lipid-droplet formation, the Cct1 and Cct2 knockdowns were characterized. Larger droplets in Cct knockdowns could arise from a failure to form new droplets, forcing newly synthesized neutral lipids into a few large droplets, or from the fusion of independently formed droplets. To distinguish between these possibilities, the dynamics of lipid-droplet formation were observed by time-lapse microscopy, and evidence was found that the droplets fuse (Guo, 2008).
Then, where CCT proteins act was examined. In untreated cells, mCherry-tagged Cct1 localized exclusively to the nucleus, similar to mammalian cytidylyltransferase-α (CT-α). After treatment with oleate, a significant portion of Cct1 localized to the lipid-droplet surface. By contrast, similarly tagged Cct2 localized to the cytoplasm but was also concentrated on droplet surfaces after treatment with oleate. This marked translocation of CCT enzymes to the droplet surface may serve to provide adequate phosphatidylcholine to the phospholipid monolayers of growing lipid droplets. If so, the ratio of surface phospholipids to core neutral lipids may regulate lipid-droplet morphology: when phospholipids are limiting (as in Cct1 or Cct2 knockdowns), fusion is induced to decrease the surface-to-volume ratio of droplets. In fact, the content of phosphatidylcholine in cells with Cct1 knockdown was decreased by about 60%, and the triacylglycerol content was increased by about 40%. The increase in triacylglycerol may reflect compensatory channelling of diacylglycerol into neutral lipids. A decreased phosphatidylcholine content would increase the relative amount of phosphatidylethanolamine in the droplet monolayer (as observed in flies lacking Cct1, which itself may directly promote droplet fusion. The results suggest a model in which phosphatidylcholine availability is a crucial regulator of lipid-droplet size and number (Guo, 2008).
Class III genes, whose knockdowns showed slightly larger and more dispersed droplets, were investigated. All class III genes were members of the Arf1-COPI machinery, including Arf79F, encoding an Arf1 family member, a gene encoding guanine nucleoside exchange factor (GEF), garz, and genes encoding components of the COPI coat. Similar effects were obtained by incubating cells with brefeldin A, a specific inhibitor of Arf1 exchange factors, and by expressing a dominant-negative version of Arf79F, encoding the T31N mutant, analogously to dominant-negative mutants for Ras or Ran. To test the specificity of this phenotype, RNAi knockdowns were separately repeated with dsRNAs for Drosophila genes encoding six ARF proteins, three GEFs, two GTPase-activating proteins, and all COPI subunits. Other coat proteins were also tested, such as clathrin subunits and components of the COPII coat. Only Arf79f, garz and six of eight members of the COPI coat (α-, β-, β′-, δ-, γ- and ζ-Cop) exhibited the class III phenotype, indicating that the screen identifies a highly specific subset of vesicular transport components. Arf102F knockdown gave a partial phenotype. This function of the Arf1-COPI machinery in lipid-droplet formation seems to have been evolutionarily conserved; similar phenotypes were found in yeast and human cells (Guo, 2008).
Next, attempts sere made to determine whether Arf79F acts directly on lipid droplets. ARF proteins exchange rapidly between active (GTP-bound) and inactive (GDP-bound) forms, making it difficult to localize only the active form. However, Arf79F(T31N) binds its exchange factor tightly, and the distribution of the exchange factor is predicted to reflect the localization of active ARF protein. Expressed Arf79F(T31N) appeared diffusely in the cytosol but was enriched at the droplet surface. Thus, Arf79F may act at the lipid-droplet surface where, as for other ARF proteins, it interacts with its GEF (presumably encoded by garz) and recruits COPI components. A recent in vitro study showed that Arf1 and several subunits of the COPI complex are recruited from the cytosol to purified lipid droplets in the presence of GTP-γS (Bartz, 2007). Although the recruitment of Arf1 to lipid droplets was reported to activate phospholipase D, no effect of phospholipase D knockdown on lipid-droplet formation was found in Drosophila cells (Guo, 2008).
The well-established functions of class III genes in Arf1-COPI-mediated vesicular transport implicate this machinery in a similar budding mechanism at the surface of lipid droplets, possibly to promote the budding-off of droplets during lipid mobilization. Lipolysis is associated with the break-up of larger droplets into smaller ones, presumably to provide more surface area for lipases. The effect of the Arf79F knockdown on lipolysis was examined by inducing lipid-droplet formation and then inducing lipid mobilization by incubation with serum-free medium lacking oleate. After 24 h, control cells had few droplets. By contrast, many droplets remained when Arf79F was inactivated. In addition, much less glycerol, a product of lipolysis, was released by cells lacking the Arf1-COPI machinery. Supporting a function of the Arf1-COPI machinery in lipolysis, more glycerol was released by cells expressing a dominant-active form of Arf79F encoding Arf79F(Q71L). These data indicate that the Arf1-COPI machinery is required for efficient lipolysis. The data agree with a report showing that lipolysis in murine adipocytes is accompanied by a brefeldin A-sensitive process that is required for the mobilization of cholesterol from storage pools in droplets (Guo, 2008).
Increased droplet surface area during lipolysis would require more phospholipids in the surrounding monolayer. Because Cct1 knockdown limits phosphatidylcholine amounts, its effect on lipolysis were tested. As predicted, Cct1 knockdown markedly decreased the efficiency of lipolysis, as seen by lipid-droplet staining. The effects of knockdowns of Arf79F and Cct1 on lipolysis were additive, suggesting that these genes function independently (Guo, 2008).
Arf1-COPI complexes mediate retrograde vesicular trafficking of membranes and proteins from the Golgi apparatus to the endoplasmic reticulum and are also involved in vesicular transport processes from the trans-Golgi network and endosomes. Notably, the members of the Arf1-COPI complex this study identified were recently found in a Drosophila screen for genes involved in protein secretion and Golgi organization (Bard, 2006). Although the primary defect in class III knockdowns is as yet unknown, the phenotype on lipid-droplet formation is not likely to be an indirect consequence of inhibition of protein secretion. The effects are highly specific and are not observed with knockdowns of other proteins mediating secretory transport (endoplasmic reticulum translocation, COPII and clathrin). In addition, Arf79F is recruited to the lipid-droplet surface, where it is presumably activated by the loading of GTP on its exchange factor (Guo, 2008).
This study provides an initial systematic examination of the Drosophila genome to identify genes involved in lipid-droplet formation and utilization. Many genes identified sort to distinct classes of morphological changes, with each class containing functionally related proteins. These classes potentially link diverse processes, such as protein synthesis and degradation, the cell cycle, and organelle movement, with lipid-droplet biology. The variety of genes identified lends support to the emerging view of lipid droplets as dynamic organelles that are functionally connected to a variety of organelles and cellular processes, including the replication of intra-cellular pathogens such as Chlamydia trachomatis and hepatitis C. Many components of these processes are likely to be highly conserved across species. These studies in S2 cells may therefore be directly relevant to cellular lipid storage in general, holding the promise of identifying pathways and mechanisms central to human diseases involving excessive lipid storage and to the engineering of cellular lipid storage in organisms for the improved production of oils and biofuels (Guo, 2008).
Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. A systems-level genetic dissection of adiposity regulation was performed using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo; hedgehog signaling was the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, fat-specific hedgehog-activation mutant mice were generated. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals (Pospisilik, 2010).
To assess the in vivo relevance of hedgehog signaling in mammalian adipogenesis, fat-specific Sufu knockout animals (aP2-SufuKO) were generated. Sufu is a potent endogenous inhibitor of hedgehog signaling in mammals. Sufuflox/flox mice were crossed to the adipose tissue deleting aP2-Cre transgenic line, and the resulting aP2- SufuKO animals were born healthy and at Mendelian ratios. PCR amplification revealed target deletion in both white adipose tissue (WAT) and brown adipose tissue (BAT). aP2-SufuKO mice displayed an immediate and obvious lean phenotype. MRI analysis revealed a significant and global reduction in white adipose tissue mass, including subcutaneous, perigonadal, and mesenteric depots. Intriguingly, though, in contrast to the gross loss of WAT, cross-sectional examination of the interscapular region revealed fully developed BAT depots of both normal size and lipid content. Direct measurement of WAT and BAT depot weights corroborated the divergent WAT/BAT phenotype, with an ~85% reduction in perigonadal fat pad mass in aP2-SufuKO mice concomitant with unaltered BAT mass. Tissue weight and histological analyses confirmed lack of any remarkable phenotype in multiple other tissues including pancreas and liver (no indication of steatosis), and muscle mass was unaffected. Cutaneous adipose was also markedly diminished. Whereas the morphology of Sufu-deficient BAT depots was largely indistinguishable from that of control animals, examination of multiple WAT pads revealed marked and significant reductions in both adipocyte size and total numbers in mutant animals. Of note, qPCR showed elevated Gli1, Gli2, and Ptch2 expression in both WAT and BAT verifying the intended pathway activation in both tissues. Thus, deletion of Sufu in fat tissue results in a markedly decreased white fat cell number and, remarkably, in normal brown adipose tissue (Pospisilik, 2010).
When the literature was cross-referenced focusing on adipogenesis, an impressive 18 of 65 key regulators of adipogenesis were found to be described as Gli targets in other systems. Intriguingly, when examined in 3T3-L1 preadipocytes, hedgehog activation induced a coordinated downregulation of the proadipogenic targets Bmp2, Bmp4, Egr2/Krox20, Sfrp1, and Sfrp2 by an average of ~50% after only 24 hr. In contrast, quantification of the antiadipogenic target set showed upregulation of the multiple critical repressors (Nr2f2, Gilz, Hes1, and Ncor2); the negative regulators Jag1 and Pref1 remained unchanged at this time point. Analysis of the master regulatory machinery downstream of these effectors revealed critical reductions in Pparg, Cebpb, and Cebpd and increases in the antiadipogenic factors Cebpg and Ddit3. Outside of this dramatic antiadipogenic profile, elevated levels of Cebpa were observed. Importantly, a similar coordinate downregulation of Pparg, Cebpb, Cebpd, as well as Cebpa was observed in WAT-derived primary murine adipocyte progenitors (stromal vascular cell, SVC, preparations) following genetic activation of hedgehog signaling (Pospisilik, 2010).
To establish a direct link between hedgehog activation and adipogenic block in white adipose, in silico predictions were used to identify clusters of probable Gli-binding sites in the highly SAG-responsive genes Ncor2, Nr2f2, Sfrp2, and Hes1. To assess the functionality of these putative binding sites, the relevant promoter fragments were cloned and luciferase reporter assays were performed. Gli1 and Gli2 induced activation of all Ncor2 and Nr2f2 reporter constructs, with the binding site clusters Ncor2_B, Nr2f2_A, and Nr2f2_B showing responses comparable to the hallmark target Ptch. Further, chromatin immunoprecipitations on 3T3-L1 preadipocytes using Gli2- and Gli3-specifc antibodies revealed increases in Gli2 and Gli3 binding within the endogenous Ncor2, Hes1, Nr2f2, and Sfrp2 regulatory regions following SAG treatment. Together, these findings demonstrate endogenous Gli2/Gli3 binding to multiple adipogenic loci and implicate direct modulation of Ncor2 and Nr2f2 in the dysregulation of adipogenesis (Pospisilik, 2010).
The power of D. melanogaster RNAi transgenics to probe gene function on a genome-wide scale has allowed screening of ~78% coverage of the Drosophila genome. One significant advantage of this inducible approach is the ability to interrogate the fat regulatory potential of the ~30% of the Drosophila genome that is developmentally lethal under classic mutation conditions. Indeed, the result that cell differentiation genes scored as the most enriched ontology subcategory substantiates the inducible strategy employed and identifies a large number of developmentally lethal genes with strong lipid storage regulatory potential. Consistent with a previous feeding-induced RNAi C. elegans screen, the fraction of candidate genes resulting in decreased fat content upon knockdown (360 of 516; 70%) exceeded that of obesity-causing candidates (216 of 516; 30%), which is consistent with the hypothesis that the major evolutionary pressures for animals have been to favor nutrient storage. The screen identified a large number of genes already known to play a key role in mammalian fat or lipid metabolism, including enzymes of membrane lipid biosynthesis, fatty acid and glucose metabolism, and sterol metabolism. Further, the whole-genome screen has uncovered a plethora of additional candidate genes of adiposity regulation, a large proportion of which had no previous annotated biological function. Moreover, multiple genes were identified that either positively or negatively regulate whole fly triglyceride levels when targeted specifically in neurons, the fly liver (oenocyte), the fat body, or muscle cells. Analyses of the hits allowed definition of either gene sets that function globally in all these tissues or others that display coordinate regulation of adiposity when targeted in metabolically linked organs such as the fat and the liver. Since >60% of the candidate genes are conserved across phyla to humans, this data set is a unique starting point for the elucidation of novel regulatory modalities in mammals (Pospisilik, 2010).
The top-scoring signal transduction pathway in the GO-based enrichment analysis was the hedgehog pathway. Tissue-specificity assessment revealed further that this enrichment was primarily derived from a pronounced fat-body restriction in function. Hedgehog signaling has been previously implicated in adipose tissue biology. In Drosophila larvae, hedgehog activation reduces lipid content consistent with what was found in adult flies and the fat-specific fly knockdown lines (Suh, 2006). Similarly, knockdown of the C. elegans equivalent of the inhibitory hedgehog receptor Ptch results in a prominent adiposity reducing phenotype in a feeding-based RNAi screen. Therefore this study homed into the hedgehog pathway to provide proof of principle for the fly screen and to translate Drosophila results directly into the mammalian context (Pospisilik, 2010).
Several reports exist describing systemic manipulation of hedgehog signaling, either by injection of ligand-depleting antibody or through examination of a systemic hypomorphic mutant, the Ptchmes/mes mouse. Indeed Ptchmes/mes mice display largely normal white adipose tissue depots albeit reduced in size (Li, 2008). Hedgehog signaling plays a crucial role in multiple organs systems including at least one intimately involved in nutrient storage and the etiologies of obesity and insulin resistance, namely, the pancreatic islet. In vitro and in vivo data using the adipose-specific Sufu mutant mice clearly show that hedgehog activation results in a complete and cell-autonomous inhibition of white adipocyte differentiation. The residual white adipose tissue observed in aP2-SufuKO mice is most likely due to late inefficient deletion and/or is due to developmental timing effects. Indeed, aP2 (and thus aP2-Cre) are expressed relatively late during adipocyte differentiation. The remarkable finding was that genetic activation of hedgehog signaling in vivo and in vitro blocks only white but not brown adipocyte differentiation (Pospisilik, 2010).
Fat is mainly stored in two cell types: WAT, which is the major storage site for triglycerides, and BAT, which, through the burning of lipids to heat (through uncoupling of mitochondrial oxidative phosphorylation), serves to regulate body temperature. Recent PET-CT data have revealed that adult humans contain functional BAT and that the amount of BAT is inversely correlated with body mass index. These new data in humans rekindle the notion that a functional BAT depot in humans could represent a potent therapeutic target in the context of obesity control. Lineage tracking and genetic studies have shown that WAT and interscapular BAT cells derive from two different but related progenitor pools. The current genetic data now demonstrate both in vitro and in vivo that hedgehog activation results in a virtually complete block of WAT development but leaves the differentiation process of brown adipocytes wholly intact. These data further support the concept that white and brown adipocytes are derived from distinct precursor cells (Pospisilik, 2010).
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