FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1995003722> ?p ?o ?g. }

• W1995003722 endingPage "410" @default.
• W1995003722 startingPage "398" @default.
• W1995003722 abstract "Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid-rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans. Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid-rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans. Mutation of acs-3 disrupts C. elegans lipid storage acs-3 functions in a tissue distinct from major sites of fat storage Loss of fatty acyl-CoA-utilizing enzymes suppresses acs-3 phenotypes acs-3 regulates metabolism through the conserved nuclear hormone receptor nhr-25 The ability to store nutrients, primarily in the form of triacylglycerides, is necessary to provide energy during periods when energy demands exceed caloric intake; however, excessive diversion of nutrients for storage can impair growth and reduce reproductive fitness and is associated with adverse health effects (Guh et al., 2009Guh D.P. Zhang W. Bansback N. Amarsi Z. Birmingham C.L. Anis A.H. The incidence of co-morbidities related to obesity and overweight: a systematic review and meta-analysis.BMC Public Health. 2009; 9: 88Crossref PubMed Scopus (2053) Google Scholar, Reeves et al., 2007Reeves G.K. Pirie K. Beral V. Green J. Spencer E. Bull D. Million Women Study CollaborationCancer incidence and mortality in relation to body mass index in the Million Women Study: cohort study.BMJ. 2007; 335: 1134Crossref PubMed Scopus (952) Google Scholar). Numerous studies in organisms ranging from bacteria to humans have identified enzymatic components of metabolism as well as its neural and endocrine regulators. Although many of these components have already been characterized at the biochemical and structural levels, understanding the precise mechanisms by which metabolic activities of various tissues are coordinated and how dysregulation of these processes may result in aberrant metabolic regulation remains a significant challenge. One complexity of metabolism is that many enzyme families are composed of multiple and seemingly redundant members. For instance, at least 26 known or predicted acyl-CoA synthase (ACS) genes are found in the human genome (Watkins et al., 2007Watkins P.A. Maiguel D. Jia Z. Pevsner J. Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome.J. Lipid Res. 2007; 48: 2736-2750Crossref PubMed Scopus (207) Google Scholar). The ACS family of enzymes catalyzes conversation of free fatty acids to acyl-CoA derivates. Although the ACS enzymes have been classified based on their substrate chain length preference into very-long-chain, long-chain, medium-chain, and short-chain families, the precise physiological roles of most of these enzymes are unknown. Another complexity of metabolism is that the same metabolic intermediates can, depending on the pathway into which they are channeled, have vastly different fates. For instance, acyl-CoAs can be substrates for fat breakdown through mitochondrial or peroxisomal β oxidation, or alternatively, they may be substrates for synthesis pathways, generating triglycerides for storage or structural components of membranes such as phospholipids (Coleman et al., 2002Coleman R.A. Lewin T.M. Van Horn C.G. Gonzalez-Baró M.R. Do long-chain acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways?.J. Nutr. 2002; 132: 2123-2126Crossref PubMed Scopus (229) Google Scholar). Additionally, acyl-CoAs serve as substrates for lipid modification of proteins and function as signaling molecules either on their own or as components of more complex molecules, such as phosphoinositols and sphingolipids (Bartke and Hannun, 2009Bartke N. Hannun Y.A. Bioactive sphingolipids: metabolism and function.J. Lipid Res. 2009; 50: S91-S96Crossref PubMed Scopus (439) Google Scholar, Black and DiRusso, 2007Black P.N. DiRusso C.C. Yeast acyl-CoA synthetases at the crossroads of fatty acid metabolism and regulation.Biochim. Biophys. Acta. 2007; 1771: 286-298Crossref PubMed Scopus (140) Google Scholar, Faergeman and Knudsen, 1997Faergeman N.J. Knudsen J. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling.Biochem. J. 1997; 323: 1-12Crossref PubMed Scopus (557) Google Scholar, Payrastre et al., 2001Payrastre B. Missy K. Giuriato S. Bodin S. Plantavid M. Gratacap M. Phosphoinositides: key players in cell signalling, in time and space.Cell. Signal. 2001; 13: 377-387Crossref PubMed Scopus (186) Google Scholar). Thus, the consequences of misregulation of an individual ACS are quite difficult to predict even when the biochemical activity of that ACS is known. C. elegans is an emerging model for the study of metabolism in the context of intact animals (Jones and Ashrafi, 2009Jones K.T. Ashrafi K. Caenorhabditis elegans as an emerging model for studying the basic biology of obesity.Dis Model Mech. 2009; 2: 224-229Crossref PubMed Scopus (62) Google Scholar, Mullaney and Ashrafi, 2009Mullaney B.C. Ashrafi K. C. elegans fat storage and metabolic regulation.Biochim. Biophys. Acta. 2009; 1791: 474-478Crossref PubMed Scopus (69) Google Scholar, Watts, 2009Watts J.L. Fat synthesis and adiposity regulation in Caenorhabditis elegans.Trends Endocrinol. Metab. 2009; 20: 58-65Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Like most free-living organisms, C. elegans normally live in environments where food availability is dynamic. Thus, they are able to sense nutrient levels and coordinate their behavioral, physiological, and metabolic responses accordingly. Despite obvious differences, many known mammalian fat regulatory mechanisms are well conserved in C. elegans. These include fat and sugar uptake, transport, breakdown, and synthesis pathways; transcriptional regulators such as nuclear hormone receptors (NHRs) and sterol response element binding protein (SREBP); energy sensing mechanisms such as AMP-activated kinase and target of rapamycin (TOR) kinase; as well as endocrine regulators such as insulin (Ashrafi, 2007Ashrafi, K. (2007). Obesity and the regulation of fat metabolism. In WormBook, The C. elegans Research Community, ed. 10.1895/wormbook.1.130.1, http://www.wormbook.org.Google Scholar). The genetic tractability of C. elegans allows for the study of metabolic regulation through use of suppressor and enhancer screens to identify components of complex pathways (Hodgkin, 2005Hodgkin, J. (2005). Genetic suppression. In WormBook, The C. elegans Research Community, ed. 10.1895/wormbook.1.59.1, http://www.wormbook.org.Google Scholar). Thus far, of the more than 20 ACSs encoded by the C. elegans genome, phenotypes have been reported for only a few: loss-of-function mutations in acs-20 and acs-22 cause defective cuticle formation (Kage-Nakadai et al., 2010Kage-Nakadai E. Kobuna H. Kimura M. Gengyo-Ando K. Inoue T. Arai H. Mitani S. Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans.PLoS ONE. 2010; 5: e8857Crossref PubMed Scopus (46) Google Scholar), while acs-4 and acs-5 are required for serotonin-induced fat reduction (Srinivasan et al., 2008Srinivasan S. Sadegh L. Elle I.C. Christensen A.G. Faergeman N.J. Ashrafi K. Serotonin regulates C. elegans fat and feeding through independent molecular mechanisms.Cell Metab. 2008; 7: 533-544Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In this study, we show that reduction of function of acs-3, encoding a long-chain ACS, causes altered patterns of neutral lipid deposition and increased rates of fatty acid uptake and de novo synthesis. We show that acs-3 function in epidermal seam cells is sufficient for restoration of wild-type (WT) lipid deposition to acs-3 mutants. Since seam cells are anatomically distinct from sites of fat uptake, de novo synthesis, and storage, our findings suggest that acs-3 must exert its effects on whole-animal fat metabolism through mediators that can act cell nonautonomously. To uncover these mechanisms, we conducted suppressor screens and found that the acs-3 mutant phenotypes could be reverted to WT by additional mutations in metabolic enzymes that consume acyl-CoAs and by nhr-25, the only C. elegans member of the NR5A family of NHRs that includes mammalian steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1). Our genetic and biochemical analyses suggest a model whereby ACS-3-generated products ultimately modulate the function of NHR-25, which in turn could regulate an endocrine program of fat uptake and synthesis. To identify genes important in the regulation of fat storage in C. elegans, we performed an unbiased mutagenesis screen and isolated mutants with elevated staining of Nile Red, a solvatochromatic vital dye that has been used in mammalian, Drosophila, C. elegans, and other experimental systems to identify and characterize metabolic pathways involved in fat metabolism (Chen et al., 2009Chen W. Zhang C. Song L. Sommerfeld M. Hu Q. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae.J. Microbiol. Methods. 2009; 77: 41-47Crossref PubMed Scopus (499) Google Scholar, Flynn et al., 2009Flynn 3rd, E.J. Trent C.M. Rawls J.F. Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio).J. Lipid Res. 2009; 50: 1641-1652Crossref PubMed Scopus (148) Google Scholar, Fowler and Greenspan, 1985Fowler S.D. Greenspan P. Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O.J. Histochem. Cytochem. 1985; 33: 833-836Crossref PubMed Scopus (319) Google Scholar, Jones et al., 2008Jones K.S. Alimov A.P. Rilo H.L. Jandacek R.J. Woollett L.A. Penberthy W.T. A high throughput live transparent animal bioassay to identify non-toxic small molecules or genes that regulate vertebrate fat metabolism for obesity drug development.Nutr. Metab. (Lond). 2008; 5: 23Crossref PubMed Scopus (86) Google Scholar, McKay et al., 2003McKay R.M. McKay J.P. Avery L. Graff J.M. C elegans: a model for exploring the genetics of fat storage.Dev. Cell. 2003; 4: 131-142Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, Siloto et al., 2009Siloto R.M. Truksa M. He X. McKeon T. Weselake R.J. Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system.Lipids. 2009; 44: 963-973Crossref PubMed Scopus (53) Google Scholar, Suh et al., 2007Suh J.M. Zeve D. McKay R. Seo J. Salo Z. Li R. Wang M. Graff J.M. Adipose is a conserved dosage-sensitive antiobesity gene.Cell Metab. 2007; 6: 195-207Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, Van Gilst et al., 2005Van Gilst M.R. Hadjivassiliou H. Jolly A. Yamamoto K.R. Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans.PLoS Biol. 2005; 3: e53Crossref PubMed Scopus (255) Google Scholar). We found a recessive mutant, ft5, that exhibited dramatically elevated staining but developed at nearly the same rate as WT animals (Figure 1A ). To verify that the phenotype was not specific to the Nile Red dye, we grew animals on bacteria containing BODIPY-labeled fatty acid and observed a similar increase in staining intensity (Figure 1A). Additionally, ft5 mutants exhibited large intestinal granules not seen in WT animals. These enlarged granules were visible with DIC microscopy (Figure 1B), stained with BODIPY-labeled fatty acids (Figure 1B), and stained in fixed animals with Sudan Black (Figure 1F), a diazo dye used for detection of lipids (Greer et al., 2008Greer E.R. Pérez C.L. Van Gilst M.R. Lee B.H. Ashrafi K. Neural and molecular dissection of a C. elegans sensory circuit that regulates fat and feeding.Cell Metab. 2008; 8: 118-131Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). They were also encircled by a GFP reporter fused to C. elegans ATGL, a lipase that in mammalian and Drosophila cells is important for hydrolysis of triglycerides from lipid droplets (Grönke et al., 2005Grönke S. Mildner A. Fellert S. Tennagels N. Petry S. Müller G. Jäckle H. Kühnlein R.P. Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila.Cell Metab. 2005; 1: 323-330Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, Zimmermann et al., 2004Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. Zechner R. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1366) Google Scholar) (Figure 1C). Similar morphological and ATGL localization phenotypes were recently described in C. elegans with increased fat accumulation due to disrupted peroxisomal fat breakdown (Zhang et al., 2010Zhang S.O. Box A.C. Xu N. Le Men J. Yu J. Guo F. Trimble R. Mak H.Y. Genetic and dietary regulation of lipid droplet expansion in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2010; 107: 4640-4645Crossref PubMed Scopus (95) Google Scholar). Unlike BODIPY-labeled fatty acids or dyes used for detection of lipids in fixed samples, an advantage of Nile Red is that its spectral properties are exquisitely sensitive to the polarity of the environment in which it resides (Fowler and Greenspan, 1985Fowler S.D. Greenspan P. Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O.J. Histochem. Cytochem. 1985; 33: 833-836Crossref PubMed Scopus (319) Google Scholar, Greenspan and Fowler, 1985Greenspan P. Fowler S.D. Spectrofluorometric studies of the lipid probe, nile red.J. Lipid Res. 1985; 26: 781-789Abstract Full Text PDF PubMed Google Scholar). For instance, in relatively polar phospholipid-rich environments, Nile Red's fluorescence emission spectrum exhibits a peak intensity at 629 nm, while in relatively neutral lipid-rich environments such as triglyceride-rich fat droplets, it emits yellow-gold fluorescence characterized by an intensity peak at 576 nm (Greenspan and Fowler, 1985Greenspan P. Fowler S.D. Spectrofluorometric studies of the lipid probe, nile red.J. Lipid Res. 1985; 26: 781-789Abstract Full Text PDF PubMed Google Scholar). Because of this property, Nile Red has been used in mammalian systems to distinguish between various particles that stain with lipophilic dyes to specifically define neutral lipid-rich deposits (Fowler and Greenspan, 1985Fowler S.D. Greenspan P. Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O.J. Histochem. Cytochem. 1985; 33: 833-836Crossref PubMed Scopus (319) Google Scholar). Nevertheless, use of Nile Red as a vital dye to interrogate C. elegans fat storage has recently been criticized, in part due to the claim that this dye fails to stain lipid depots in tissues such as the skin-like hypodermis or in developing embryos within the hermaphrodite gonad (Brooks et al., 2009Brooks K.K. Liang B. Watts J.L. The influence of bacterial diet on fat storage in C. elegans.PLoS ONE. 2009; 4: e7545Crossref PubMed Scopus (179) Google Scholar, O'Rourke et al., 2009O'Rourke E.J. Soukas A.A. Carr C.E. Ruvkun G. C. elegans major fats are stored in vesicles distinct from lysosome-related organelles.Cell Metab. 2009; 10: 430-435Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). We found that by simply increasing the concentration of Nile Red fed to living animals, it was possible to visualize yellow fluorescence in the enlarged droplets seen in ft5 mutants (Figure 1D) as well as in hypodermis and developing embryos (data not shown). Moreover, the yellow fluorescent granules emitted with peak intensity between 570 and 580 nm, indicative of an environment rich in neutral lipids (Figure 1E). To identify the causative mutation in ft5, we performed positional cloning followed by sequence analysis and found a G-to-A mutation in the T08B1.6 gene. This mutation results in a glycine to glutamic acid substitution at amino acid 118 of the predicted protein (Table 1). T08B1.6 encodes a predicted long-chain fatty ACS, which we named acs-3.Table 1Mutations IsolatedAlleleGene IDGene NameGene FunctionLocusMutationft5T08B1.6acs-3Acyl-CoA synthaseV: −16.58Gly118 → Gluft8Y45F3A.3acdh-11Acyl-CoA dehydrogenaseIII: 3.73Asp309 → Asnft12Y45F3A.3acdh-11Acyl-CoA dehydrogenaseIII: 3.73Ala244 → Thrft11T08G2.3acdh-10Acyl-CoA dehydrogenaseX: 24.09Gly259 → Gluft14F41H10.8elo-6Fatty acid elongaseIV: 1.64Gln205 → STOPft13Y46E12BL.4spsb-1UnknownII: 24.58Gly3 → GluMolecular identities of mutant alleles identified in this study. acs-3(ft5) was isolated from a screen for animals with altered Nile Red staining, while all other mutants were isolated from an acs-3(ft5) suppressor screen by identifying animals that do not arrest on 110 μM LY294002. Each allele identified in this screen also suppressed the fat storage phenotype of acs-3(ft5). Open table in a new tab Molecular identities of mutant alleles identified in this study. acs-3(ft5) was isolated from a screen for animals with altered Nile Red staining, while all other mutants were isolated from an acs-3(ft5) suppressor screen by identifying animals that do not arrest on 110 μM LY294002. Each allele identified in this screen also suppressed the fat storage phenotype of acs-3(ft5). To begin characterization of acs-3, we first generated transgenic animals carrying 2.5 kb of the acs-3 upstream regulatory sequence fused to a GFP reporter. GFP expression was observed, beginning during embryogenesis and continuing through adulthood. From larval stage L1 to L4, expression appeared restricted to epidermal seam cells, the excretory cell, vulva cells, and a subset of unidentified cells in the head and tail (Figures 2A, 2B , and S1). In addition to prominent seam cell expression, adult animals also exhibited moderate intestinal GFP expression. Tissue expression patterns observed by the promoter-reporter fusion were recapitulated by transgenic animals in which full-length acs-3 cDNA was tagged at the C terminus with GFP and driven by the same 2.5 kb upstream regulatory elements (Figure S1). Nile Red fat staining of these transgenic animals was restored to near WT levels, indicating that the 2.5 kb promoter is sufficient for regulation of lipid storage (Figures 2F and 2G). To determine whether ACS-3 function in a subset of tissues could be sufficient for fat storage regulation, we expressed full-length WT acs-3 using a variety of tissue-specific promoters in acs-3(ft5) mutants. Expression of acs-3 in seam cells, using the previously characterized wrt-2 and grd-10 seam-cell-specific promoters (Aspöck et al., 1999Aspöck G. Kagoshima H. Niklaus G. Bürglin T.R. Caenorhabditis elegans has scores of hedgehog-related genes: sequence and expression analysis.Genome Res. 1999; 9: 909-923Crossref PubMed Scopus (88) Google Scholar), was sufficient to almost fully rescue the fat storage phenotype of acs-3(ft5) animals (Figures 2F and 2G). We attempted numerous injections of an intestinally driven acs-3 construct, but they caused lethality in nearly all instances. In one case, a single line carrying intestinally expressed acs-3 was generated; however, the transgenic animals exhibited numerous phenotypes associated with sickness, such as slow growth or growth arrest, small body size, and clear intestine (data not shown). Expression of acs-3 specifically in body-wall muscle, a tissue in which we never observed expression with the acs-3 promoter, failed to alter excess Nile Red staining of acs-3(ft5) animals (Figures 2F and 2G). Taken together, these data indicated that reconstitution of acs-3 in seam cells was sufficient to confer WT fat staining to acs-3(ft5) mutants, but expression at similar or higher levels in another tissue did not rescue the altered lipid storage of these mutants. Within the seam cells, expression of full-length ACS-3 fused to GFP at the C terminus driven by the rescuing 2.5 kb promoter resulted in localization of the fusion protein throughout the cytoplasm. Expression of the same reporter fusion driven by the seam-cell-specific promoters wrt-2 or grd-10, which allowed for nearly full rescue of acs-3(ft5) fat storage phenotype, resulted in clear localization of the reporter protein to the plasma membrane of seam cells (Figures 2C and 2D). Based on its amino acid sequence, the ACS-3 protein is not predicted to have any transmembrane domains. Thus, ACS-3 is likely a cytosolic enzyme that is localized to the inner leaflet of the plasma membrane of seam cells, and the difference in subcellular localization using the indicated promoters is likely a consequence of differences in the extent of promoter activity. Consistent with this, there was no obvious overlap between reporter fusions to ACS-3 and CLK-1, a C. elegans mitochondrial protein (Felkai et al., 1999Felkai S. Ewbank J.J. Lemieux J. Labbé J.C. Brown G.G. Hekimi S. CLK-1 controls respiration, behavior and aging in the nematode Caenorhabditis elegans.EMBO J. 1999; 18: 1783-1792Crossref PubMed Scopus (206) Google Scholar) (Figure S1). Localization of ACS-3 to seam cells was unexpected, as these cells have not been previously implicated in fat storage or regulation. Seam cells are relatively poorly characterized cells that are part of the skin-like epidermis of C. elegans. During development, seam cells exhibit stem cell characteristics as they go through multiple rounds of division whereby a subset of daughter cells regenerates the seam cell population after each division, while the remainder differentiate into epidermal, glial, or neuronal cells (Sulston and Horvitz, 1977Sulston J.E. Horvitz H.R. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.Dev. Biol. 1977; 56: 110-156Crossref PubMed Scopus (2354) Google Scholar). To confirm the predicted function of ACS-3, we generated and purified recombinant protein. Using an in vitro colorimetric assay, we tested the activity of this recombinant ACS-3 protein along with a commercially available Pseudomonas ACS as a positive control. Pseudomonas ACS exhibited strong ACS activity and could use a broad range of fatty acid chain lengths as substrate, in accordance with previous findings (Hosaka et al., 1981Hosaka K. Kikuchi T. Mitsuhida N. Kawaguchi A. A new colorimetric method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA oxidase.J. Biochem. 1981; 89: 1799-1803Crossref PubMed Scopus (58) Google Scholar, Knoll et al., 1994Knoll L.J. Johnson D.R. Gordon J.I. Biochemical studies of three Saccharomyces cerevisiae acyl-CoA synthetases, Faa1p, Faa2p, and Faa3p.J. Biol. Chem. 1994; 269: 16348-16356Abstract Full Text PDF PubMed Google Scholar) (Figure S2). Recombinant C. elegans ACS-3 also exhibited ACS activity but was capable of activating a narrower range of fatty acid chain lengths. 18-carbon fatty acid was the preferred substrate chain length, but the enzyme exhibited significant activity on 20- and 22-carbon fatty acids, as well (Figure S2). These findings confirm the predicted long-chain fatty acyl-CoA (LCFA-CoA) synthase function of ACS-3. We next tested the activity of recombinant mutant ACS-3 protein in which we introduced the Glu118 to Gly mutation identified in acs-3(ft5) animals. Surprisingly, this protein exhibited activity equivalent to WT protein (Figure S2). Thus, we asked whether the Glu118 to Gly mutation disrupts other aspects of ACS-3 function, such as its localization or expression pattern. As assessed by the full-length GFP reporter, the mutant ACS-3 protein mislocalized to the nucleus (Figure 2E). The incorrect trafficking of the mutant protein raised the possibility that the mutant phenotype may arise from inappropriate ACS activity in the seam cell nucleus. However, the recessive nature of the ft5 mutation, coupled with the fact that acs-3(ft5) mutants can be rescued by expression of the WT cDNA, indicated that the mutant phenotype corresponded to acs-3 loss of function rather than neomorphic activity. Thus, the Glu118 to Gly mutation likely disrupts ACS-3 function by mislocalizing this enzyme to the nucleus. To better understand the altered fat storage phenotype of acs-3(ft5) animals, we assayed various behavioral and metabolic parameters of these mutants. C. elegans intake nutrients through pharyngeal pumping, the rate of which is subject to modulation by food availability and food quality as well as experience of starvation prior to food exposure (Avery and Horvitz, 1990Avery L. Horvitz H.R. Effects of starvation and neuroactive drugs on feeding in Caenorhabditis elegans.J. Exp. Zool. 1990; 253: 263-270Crossref PubMed Scopus (265) Google Scholar, You et al., 2008You Y.J. Kim J. Raizen D.M. Avery L. Insulin, cGMP, and TGF-beta signals regulate food intake and quiescence in C. elegans: a model for satiety.Cell Metab. 2008; 7: 249-257Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). We found that pumping rates in acs-3(ft5) and WT animals were equivalent. Similarly, brood size and rates of movement of acs-3(ft5) were indistinguishable from those of WT animals (Figures 3B and 3C and data not shown). Finally, we found that under conditions of full starvation, acs-3 mutants tended to survive slightly longer than WT animals, although the differences in overall survival rate were not statistically significant (Figure S1). The requirement of ACS enzymes for consumption of fatty acids suggested defective β oxidation as a likely explanation for the observed fat storage phenotype. To directly test this hypothesis, we fed animals radiolabeled oleic acid and measured rates of production of labeled water, a byproduct of fat oxidation. Surprisingly, acs-3(ft5) animals showed an increase in β oxidation rate (Figure 3A). Since this assay is dependent on uptake of radiolabeled oleic acid by whole animals, it raised the possibility that the observed enhanced rate of labeled water production could reflect increased rate of fatty acid absorption rather than increased fat oxidation. To evaluate this possibility, we adapted an assay for fatty acid uptake that has been previously utilized in yeast and mammalian cell culture-based experiments (Jia et al., 2007Jia Z. Moulson C.L. Pei Z. Miner J.H. Watkins P.A. Fatty acid transport protein 4 is the principal very long chain fatty acyl-CoA synthetase in skin fibroblasts.J. Biol. Chem. 2007; 282: 20573-20583Crossref PubMed Scopus (82) Google Scholar, Li et al., 2005aLi H. Black P.N. DiRusso C.C. A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors.Anal. Biochem. 2005; 336: 11-19Crossref PubMed Scopus (34) Google Scholar, Schaffer and Lodish, 1994Schaffer J.E. Lodish H.F. Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein.Cell. 1994; 79: 427-436Abstract Full Text PDF PubMed Scopus (713) Google Scholar) as well as recently in C. elegans (Spanier et al., 2009Spanier B. Lasch K. Marsch S. Benner J. Liao W. Hu H. Kienberger H. Eisenreich W. Daniel H. How the intestinal peptide transporter PEPT-1 contributes to an obesity phenotype in Caenorhabditits elegans.PLoS ONE. 2009; 4: e6279Crossref PubMed Scopus (47) Google Scholar). We empirically determined conditions whereby feeding animals a short pulse of the fluorescently labeled fatty acid C1-BODIPY 500/512 C12 allowed for comparison of rate of fatty acid uptake. Based on this assay, acs-3(ft5) animals exhibited a striking increase in uptake rate compared to WT animals (Figure 3D). After uptake of a pulse of fluorescently labeled fatty acids, the rate of diminishment of fluorescence was similar in WT and mutant animals (data not shown), indicating that the increased accumulation of labeled dietary fatty acids in acs-3(ft5) animals reflected increased rate of fat uptake rather than reduced efflux. Assuming that the assay conditions did not saturate the capacity of β oxidation enzymes, the increased rate of β oxidation of acs-3(ft5) mutants likel" @default.
• W1995003722 created "2016-06-24" @default.
• W1995003722 creator A5004417143 @default.
• W1995003722 creator A5007432897 @default.
• W1995003722 creator A5009069351 @default.
• W1995003722 creator A5022176940 @default.
• W1995003722 creator A5071874602 @default.
• W1995003722 creator A5079959585 @default.
• W1995003722 creator A5084971165 @default.
• W1995003722 creator A5085188064 @default.
• W1995003722 creator A5088525691 @default.
• W1995003722 date "2010-10-01" @default.
• W1995003722 modified "2023-10-17" @default.
• W1995003722 title "Regulation of C. elegans Fat Uptake and Storage by Acyl-CoA Synthase-3 Is Dependent on NR5A Family Nuclear Hormone Receptor nhr-25" @default.
• W1995003722 cites W1537040405 @default.
• W1995003722 cites W1763805809 @default.
• W1995003722 cites W1910630598 @default.
• W1995003722 cites W1944127002 @default.
• W1995003722 cites W1974663161 @default.
• W1995003722 cites W1976382531 @default.
• W1995003722 cites W1980216636 @default.
• W1995003722 cites W1983018467 @default.
• W1995003722 cites W1984099131 @default.
• W1995003722 cites W1984160651 @default.
• W1995003722 cites W1985119795 @default.
• W1995003722 cites W1986006829 @default.
• W1995003722 cites W1986097037 @default.
• W1995003722 cites W1988077081 @default.
• W1995003722 cites W1994014704 @default.
• W1995003722 cites W1998861353 @default.
• W1995003722 cites W2012383052 @default.
• W1995003722 cites W2017846611 @default.
• W1995003722 cites W2019326965 @default.
• W1995003722 cites W2021599875 @default.
• W1995003722 cites W2026927904 @default.
• W1995003722 cites W2030176019 @default.
• W1995003722 cites W2035429390 @default.
• W1995003722 cites W2043949040 @default.
• W1995003722 cites W2045519188 @default.
• W1995003722 cites W2046753623 @default.
• W1995003722 cites W2057455189 @default.
• W1995003722 cites W2062793520 @default.
• W1995003722 cites W2065731326 @default.
• W1995003722 cites W2067517788 @default.
• W1995003722 cites W2075139515 @default.
• W1995003722 cites W2075199074 @default.
• W1995003722 cites W2084043096 @default.
• W1995003722 cites W2084856509 @default.
• W1995003722 cites W2087072206 @default.
• W1995003722 cites W2093223217 @default.
• W1995003722 cites W2093913603 @default.
• W1995003722 cites W2094091679 @default.
• W1995003722 cites W2095037640 @default.
• W1995003722 cites W2096237954 @default.
• W1995003722 cites W2098188916 @default.
• W1995003722 cites W2104315543 @default.
• W1995003722 cites W2110839026 @default.
• W1995003722 cites W2111338419 @default.
• W1995003722 cites W2113017183 @default.
• W1995003722 cites W2114149712 @default.
• W1995003722 cites W2117731241 @default.
• W1995003722 cites W2127498339 @default.
• W1995003722 cites W2129762213 @default.
• W1995003722 cites W2131008301 @default.
• W1995003722 cites W2132350109 @default.
• W1995003722 cites W2136356529 @default.
• W1995003722 cites W2137353897 @default.
• W1995003722 cites W2142395012 @default.
• W1995003722 cites W2147515706 @default.
• W1995003722 cites W2147834083 @default.
• W1995003722 cites W2153514402 @default.
• W1995003722 cites W2158933183 @default.
• W1995003722 cites W2181530934 @default.
• W1995003722 cites W2235504444 @default.
• W1995003722 cites W72536532 @default.
• W1995003722 doi "https://doi.org/10.1016/j.cmet.2010.08.013" @default.
• W1995003722 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2992884" @default.
• W1995003722 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20889131" @default.
• W1995003722 hasPublicationYear "2010" @default.
• W1995003722 type Work @default.
• W1995003722 sameAs 1995003722 @default.
• W1995003722 citedByCount "56" @default.
• W1995003722 countsByYear W19950037222012 @default.
• W1995003722 countsByYear W19950037222013 @default.
• W1995003722 countsByYear W19950037222014 @default.
• W1995003722 countsByYear W19950037222015 @default.
• W1995003722 countsByYear W19950037222016 @default.
• W1995003722 countsByYear W19950037222017 @default.
• W1995003722 countsByYear W19950037222018 @default.
• W1995003722 countsByYear W19950037222019 @default.
• W1995003722 countsByYear W19950037222020 @default.
• W1995003722 countsByYear W19950037222021 @default.
• W1995003722 countsByYear W19950037222022 @default.
• W1995003722 countsByYear W19950037222023 @default.
• W1995003722 crossrefType "journal-article" @default.
• W1995003722 hasAuthorship W1995003722A5004417143 @default.
• W1995003722 hasAuthorship W1995003722A5007432897 @default.
• W1995003722 hasAuthorship W1995003722A5009069351 @default.

• next