Mammalian nuclear hormone receptors (NHRs), such as liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs), precisely control energy metabolism. Consequently, these receptors are important targets for the treatment of metabolic diseases, including diabetes and obesity. A thorough understanding of NHR fat regulatory networks has been limited, however, by a lack of genetically tractable experimental systems. Here we show that deletion of the  Caenorhabditis elegans  NHR gene  nhr-49  yielded worms with elevated fat content and shortened life span. Employing a quantitative RT-PCR screen, we found that  nhr-49  influenced the expression of 13 genes involved in energy metabolism. Indeed,  nhr-49  served as a key regulator of fat usage, modulating pathways that control the consumption of fat and maintain a normal balance of fatty acid saturation. We found that the two phenotypes of the  nhr-49  knockout were linked to distinct pathways and were separable: The high-fat phenotype was due to reduced expression of enzymes in fatty acid β-oxidation, and the shortened adult life span resulted from impaired expression of a stearoyl-CoA desaturase. Despite its sequence relationship with the mammalian hepatocyte nuclear factor 4 receptor, the biological activities of  nhr-49  were most similar to those of the mammalian PPARs, implying an evolutionarily conserved role for NHRs in modulating fat consumption and composition. Our findings in  C. elegans  provide novel insights into how NHR regulatory networks are coordinated to govern fat metabolism.nhr-49nhr-49nhr-49nhr-49nhr-49

Introduction Due to their ability to interact with fatty acids and other lipids, nuclear hormone receptors (NHRs), including peroxisome proliferator-activated receptors (PPARs), liver X receptor, and farnesoid X receptor, are important regulators of mammalian fat metabolism; accordingly, these small-molecule-gated transcription factors have been favored targets for therapies designed to combat diabetes, obesity, and atherosclerosis [ 1 , 2 , 3 ]. Although the molecular mechanisms employed by NHRs have been intensively probed, their pleiotropic effects on overall animal physiology are only partially understood. Thus, developing a broader range of experimental systems would be useful for characterizing the fat regulatory networks integrated by NHRs and for revealing the breadth of their physiological influence. The study of invertebrates, such as  Caenorhabditis elegans , has facilitated the elucidation and interpretation of biological networks, particularly in the context of the whole animal. However, despite the fact that  C. elegans  contains 284 NHR genes, compared to only 48 in mammals, there is a notable absence of worm NHRs orthologous to mammalian receptors involved in fat metabolism [ 4 , 5 ]. In fact, it has been suggested that fat-regulating NHRs, along with several other NHR types, were lost from the  C. elegans  and  Drosophila  lineages as these organisms simplified their body plans [ 6 ]. Conceivably, then, the biological activities associated with these NHRs may also have vanished. Therefore, it is still not clear if worm receptors mediate control over fat metabolism comparable to that of mammalian NHRs. Whether or not  C. elegans  employs NHRs to regulate fat metabolism, the evolution of the  C. elegans  NHR family is intrinsically interesting. Only 15 of the 284 worm NHRs are members of the broadly conserved subfamilies found in mammals and other metazoans [ 4 , 5 ]. The biological activities of most of these “conserved” NHRs have been broadly defined, revealing roles in development, molting, dauer formation, and sex determination [ 7 , 8 ]. The remaining 269 “divergent” NHRs have thus far been found only in nematodes and are predicted to have originated from repeated duplication of an ancestral gene that also gave rise to the hepatocyte nuclear factor 4 (HNF4) family of receptors [ 6 ]. Although mammalian HNF4 receptors have been implicated in liver development and glucose homeostasis [ 9 ], virtually nothing is known about the functions of these 269  C. elegans  “nematode-specific” HNF4-like receptors. It will be interesting to determine if these divergent NHRs have adopted novel regulatory functions or whether they carry out physiological tasks similar to those of other metazoan NHRs. Elucidating the physiological responsibilities of even one of the worm HNF4-like receptors will likely advance our understanding of NHR-regulated gene networks and provide insight into the evolution and diversification of the NHR family in nematodes. To this end, we have been systematically investigating the function of HNF4-like receptors in  C. elegans.  In this study, we present a physiological characterization of  nhr-49  (K10C3.6), one of the  C. elegans  receptor genes most closely related to the mammalian HNF4. Our findings reveal a surprising role for this HNF4-like receptor in the regulation of fat storage and metabolism.
Introduction
Due to their ability to interact with fatty acids and other lipids, nuclear hormone receptors (NHRs), including peroxisome proliferator-activated receptors (PPARs), liver X receptor, and farnesoid X receptor, are important regulators of mammalian fat metabolism; accordingly, these small-molecule-gated transcription factors have been favored targets for therapies designed to combat diabetes, obesity, and atherosclerosis [ 1 , 2 , 3 ]. Although the molecular mechanisms employed by NHRs have been intensively probed, their pleiotropic effects on overall animal physiology are only partially understood. Thus, developing a broader range of experimental systems would be useful for characterizing the fat regulatory networks integrated by NHRs and for revealing the breadth of their physiological influence.The study of invertebrates, such as  Caenorhabditis elegans , has facilitated the elucidation and interpretation of biological networks, particularly in the context of the whole animal. However, despite the fact that  C. elegans  contains 284 NHR genes, compared to only 48 in mammals, there is a notable absence of worm NHRs orthologous to mammalian receptors involved in fat metabolism [ 4 , 5 ]. In fact, it has been suggested that fat-regulating NHRs, along with several other NHR types, were lost from the  C. elegans  and  Drosophila  lineages as these organisms simplified their body plans [ 6 ]. Conceivably, then, the biological activities associated with these NHRs may also have vanished. Therefore, it is still not clear if worm receptors mediate control over fat metabolism comparable to that of mammalian NHRs.Whether or not  C. elegans  employs NHRs to regulate fat metabolism, the evolution of the  C. elegans  NHR family is intrinsically interesting. Only 15 of the 284 worm NHRs are members of the broadly conserved subfamilies found in mammals and other metazoans [ 4 , 5 ]. The biological activities of most of these “conserved” NHRs have been broadly defined, revealing roles in development, molting, dauer formation, and sex determination [ 7 , 8 ]. The remaining 269 “divergent” NHRs have thus far been found only in nematodes and are predicted to have originated from repeated duplication of an ancestral gene that also gave rise to the hepatocyte nuclear factor 4 (HNF4) family of receptors [ 6 ]. Although mammalian HNF4 receptors have been implicated in liver development and glucose homeostasis [ 9 ], virtually nothing is known about the functions of these 269  C. elegans  “nematode-specific” HNF4-like receptors. It will be interesting to determine if these divergent NHRs have adopted novel regulatory functions or whether they carry out physiological tasks similar to those of other metazoan NHRs.Elucidating the physiological responsibilities of even one of the worm HNF4-like receptors will likely advance our understanding of NHR-regulated gene networks and provide insight into the evolution and diversification of the NHR family in nematodes. To this end, we have been systematically investigating the function of HNF4-like receptors in  C. elegans.  In this study, we present a physiological characterization of  nhr-49  (K10C3.6), one of the  C. elegans  receptor genes most closely related to the mammalian HNF4. Our findings reveal a surprising role for this HNF4-like receptor in the regulation of fat storage and metabolism.C. elegans.nhr-49Results
 nhr-49  Is Necessary for Normal Life Span In an RNA interference (RNAi) screen designed to identify the role of  C. elegans  HNF4-like receptors in worm development and longevity, we found that interference of  nhr-49  resulted in dramatically reduced life span ( Figure 1 A). For further characterization, we obtained a  C. elegans  strain,  nhr-49(nr2041),  which harbors a deletion in the  nhr-49  gene encompassing part of the DNA binding domain and more than half of the ligand binding domain (LBD); this deletion likely results in complete loss of function [ 10 ]. At 23 °C,  nhr-49(nr2041)  worms lived only 6–8 d as adults, significantly shorter than the 15 to 18-d life span of N2 wild-type (WT) animals ( Figure 1 A). Although  nhr-49  deletion did not noticeably affect development or fertility,  nhr-49(nr2041)  worms experienced rapid decline in function beginning around day 3 of adulthood, when vacuoles appeared in the intestine and gonad ( Figure 1 B). By days 4 and 5,  nhr-49(nr2041)  animals were significantly smaller than WT, vacuoles were ubiquitous, and there was widespread gonadal necrosis. By days 5–7, the gonad had completely deteriorated, and the worms died shortly thereafter. Thus,  nhr-49  function is not required for development or fertility, but is clearly essential for normal longevity. Even though the increased vacuole formation was consistent with reported aging characteristics [ 11 ], we have not determined whether the shortened life span of  nhr-49(nr2041)  reflects accelerated aging or an unrelated pathology. Figure 1
 nhr-49(nr2041)  Animals Have Reduced Life Span and Higher Fat Content (A) Adult life span of WT (black squares, solid line),  nhr-49(nr2041)  (black circles, dotted line), and  nhr-49  RNAi animals (black diamonds, dashed line). (B) Nomarksi images of  nhr-49(nr2041)  at days 3, 5, and 7 of adulthood. The arrow in the day 3 image points to a gonadal vacuole that is typical of day 3 worms; the arrow in the day 5 image shows the continued deterioration of oocytes in the gonad, and the arrow in the day 7 image points to the clearing that results from complete gonadal necrosis and collapse. (C) Nile Red intestinal fat staining of WT and  nhr-49(nr2041).  Each image displays two representative worms from a population of L4 animals.
 nhr-49(nr2041)  Animals Display Abnormally High Fat Content
 nhr-49  was recently identified in a  C. elegans  genomewide RNAi screen as one of 112 genes that, when knocked down by RNAi, resulted in abnormally high Nile Red fat staining [ 12 ]. To determine whether deletion of  nhr-49  also yielded a high-fat phenotype, we used a similar Nile Red assay to visualize fat content in  nhr-49(nr2041).  Indeed, we found that  nhr-49(nr2041)  animals stained more brightly with Nile Red than did WT worms ( Figure 1 C). The difference in fat content between WT and  nhr-49(nr2041)  animals was most pronounced in the L3 and L4 stages of larval development; by day 2 of adulthood there was only a slight change in Nile Red staining, suggesting that the effects of  nhr-49  on visible fat content may vary during development (data not shown).
 nhr-49  Regulates Genes Involved in Energy Metabolism The high-fat phenotype of the  nhr-49(nr2041)  mutant led us to suspect that  nhr-49  might regulate genes involved in energy metabolism. To test this hypothesis, we identified from a survey of the  C. elegans  genome 65 genes predicted to participate in fatty acid synthesis, β-oxidation, desaturation, elongation, and binding/transport, in addition to 16 genes expected to function in glycolysis, gluconeogenesis, and glucose transport, and eight genes involved in the glyoxylate pathway ( Figure 2 ). We then employed quantitative RT-PCR (QRT-PCR) to measure the expression of all 89 of these genes in WT and  nhr-49(nr2041)  mutant worms. For the purpose of our survey, gene expression was measured in all four larval stages (for a complete dataset, see  Table S1 ). Figure 2
 nhr-49  Deletion Affects the Expression of 13 Energy Metabolism Genes Putative  C. elegans  fatty acid, glucose, and glyoxylate metabolism pathways are shown here; each box represents a gene predicted by sequence homology to code for the enzyme indicated in the figure. The fatty acid desaturation elongation pathway is adapted from previously published studies [ 14 , 17 ]. Mitochondrial localization of β-oxidation enzymes was predicted by TargetP and PSORT (see  Materials and Methods ); peroxisomal β-oxidation enzymes all contain a carboxy-terminal peroxisomal localization signal. QRT-PCR was used to measure the expression of these 89 genes in WT and  nhr-49(nr2041).  Genes expressed at lower levels in  nhr-49(nr2041)  are shown in red (>32-fold lower); orange (8- to 32-fold lower); or yellow (2- to 8-fold lower); and genes expressed at higher levels are shown in light green (2- to 8-fold higher) or dark green (>8-fold higher). Online QRT-PCR data for this analysis are available in  Table S1 . Our screen revealed that deletion of  nhr-49  significantly altered the expression of 13 genes, including six genes predicted to be involved in fatty acid β-oxidation, three genes involved in fatty acid desaturation, two genes involved in fatty acid binding/transport, and two genes involved in the glyoxylate pathway ( Figure 2 ). Similar changes in gene expression were observed when  nhr-49  was knocked down in WT animals using RNAi (data not shown). Overall,  nhr-49  deletion displayed the most dramatic effects on two metabolic pathways, mitochondrial β-oxidation and fatty acid desaturation, as the expression of multiple genes in each of these pathways was significantly compromised by loss of  nhr-49  function ( Figure 2 ). In contrast,  nhr-49  deletion had smaller and mixed consequences for genes that participate in peroxisomal β-oxidation and lipid binding/transport.  nhr-49  knockout also marginally reduced expression of two enzymes involved in the glyoxylate pathway, a pathway that facilitates the conversion of fatty acid β-oxidation products to glucose ( Figure 2 ).  nhr-49  deletion did not, however, significantly affect the expression of any genes predicted to be involved in glucose metabolism. Thus, it is clear that  nhr-49  is extensively involved in the control of fatty acid metabolism, with a pronounced role in the promotion of mitochondrial β-oxidation and fatty acid desaturation.
 nhr-49(nr2041)  Animals are High in Fat Due to Reduced Expression of β-Oxidation Genes Three genes predicted to participate in mitochondrial β-oxidation,  ech-1  (C29F3.1), F09F3.9, and an acyl-CoA synthetase gene  (acs-2)  (F28F8.2), were expressed at significantly lower levels in  nhr-49(nr2041)  animals throughout development ( Figure 3 A).  acs-2  is predicted to encode a mitochondrial acyl-CoA synthetase, whereas F09F3.9 likely codes for a carnitine palmitoyl transferase, and  ech-1  appears to encode a mitochondrial β-oxidation trifunctional enzyme. Because mitochondrial β-oxidation facilitates the degradation of stored fats for the production of energy, we suspected that  nhr-49(nr2041)  worms might be high in fat due to the reduced expression of these key β-oxidation enzymes. To test this hypothesis, we used RNAi to reduce separately the expression of  acs-2,  F09F3.9, and  ech-1  in WT animals. Indeed, we found that RNAi of either  acs-2  or  ech-1  resulted in worms with elevated Nile Red fat staining ( Figure 3 B).  acs-2  was previously identified for a similar effect on fat storage [ 12 ]. We conclude that  nhr-49  promotes the expression of three β-oxidation genes, two of which, in WT worms, reduce overall fat storage. Thus,  nhr-49(nr2041)  mutant worms are likely to be high in fat due to the impaired expression of these genes. Figure 3 Regulation of β-Oxidation Gene Expression by  nhr-49
 (A) QRT-PCR measurement of  acs-2,  F09F9.3, and  ech-1  expression in WT (gray bars) and  nhr-49(nr2041)  animals (blue bars). Expression was measured in all four stages of larval development. Error bars represent standard error of measurement. (B) RNAi of  nhr-49, acs-2,  or  ech-1  resulted in increased Nile Red fat staining. Each image shows 3 or 4 representative worms from a population of L4 animals grown on plates containing RNAi bacteria and Nile Red fat-staining dye. (C)  acs-2::gfp  is expressed in many tissues including hypodermis (hyp), intestine (int), body wall muscle (bwm), neurons (neu), and pharynx (pha). ACS-2::GFP localizes to subcellular structures in a pattern similar to what has been reported for mitochondrial proteins [ 13 ]. (D) Expression of  acs-2::gfp  was lower in  nhr-49(nr2041)  mutant animals. (E) Expression of P nhr-49 ::gfp  promoter fusion in WT animals revealed that  nhr-49  is expressed in multiple tissues, including hypodermis (hyp), body wall muscle (bwm), pharynx (pha), and intestine (int). The animals shown here are genetically mosaic, harboring the P nhr-49 ::gfp  construct in only a fraction of total cells. (F) Ectopic expression of  acs-2::gfp  in  nhr-49(nr2041)  was sufficient to reduce Nile Red staining to WT levels. ACS-2 Is Expressed in Multiple Tissues and Localizes to Mitochondria Out of five predicted mitochondrial  acs  genes in  C. elegans, nhr-49  affected only the expression of  acs-2,  suggesting that  nhr-49  may only influence a subset of mitochondrial β-oxidation pathways in worms. To determine if  acs-2  was expressed in a specific set of tissues, we fused the full-length  acs-2  gene and promoter to  gfp (acs-2::gfp);  injection of this construct into WT worms revealed that the ACS-2::green fluorescent protein (GFP) was widely expressed in many cell types, including intestine, hypodermis, pharynx, body wall muscle, and several neurons ( Figure 3 C). Moreover, ACS-2::GFP expression was reduced in all of these tissues in  nhr-49(nr2041)  worms, indicating that the effects of  nhr-49  on  acs-2  expression were widespread ( Figure 3 D). Notably, the expression pattern of  acs-2  overlapped extensively with that of an  nhr-49  promoter/GFP fusion, consistent with the idea that  nhr-49  could control  acs-2  through direct transcriptional activation ( Figure 3 E). As predicted, ACS-2::GFP localized to subcellular structures in patterns similar to those reported for other mitochondrial proteins [ 13 ] ( Figure 3 C). Overexpression of ACS-2::GFP Is Sufficient to Suppress the High-Fat Phenotype of  nhr-49(nr2041)
 Strikingly, overexpression of the ACS-2::GFP fusion was able to suppress the high-fat phenotype of  nhr-49(nr2041)  animals ( Figure 3 F). These data suggest that  acs-2  expression is reduced in  nhr-49(nr2041)  such that it becomes a rate-limiting factor in the consumption of fat; thus, high fat levels can be overcome simply by increasing  acs-2  expression. Overexpression of  acs-2  in WT animals did not affect fat content, however, indicating that WT levels of  acs-2  expression are not rate limiting. These data further support the conclusion that reduced expression of  acs-2  is a significant contributor to the high-fat phenotype of the  nhr-49(nr2041)  mutant.
 nhr-49  Modulates Fatty Acid Composition by Promoting Expression of Δ9-Desaturases In addition to stimulating genes in the mitochondrial β-oxidation pathway,  nhr-49  also promoted the expression of three  C. elegans  fatty acid desaturases (see  Figure 2 ).  fat-5  (W06D12.3) and  fat-7  (F10D2.9) expression was dramatically lowered in  nhr-49(nr2041)  worms (>30-fold) in all four larval stages, whereas  fat-6  (VZK8221.1) expression was marginally reduced (approximately 2-fold) only in L3 and L4 animals ( Figure 4 A). Figure 4 Deletion of  nhr-49  Hinders Fatty Acid Desaturation (A) QRT-PCR measurement of  fat-5, fat-6, and fat-7  expression in WT (gray bars) and in  nhr-49(nr2041)  mutant worms (blue bars). Expression was measured in all four stages of larval development. Error bars represent standard error of measurement. (B) Relative abundance of individual fatty acid species expressed as percentage of total measured fatty acid. Fatty acids included in total fatty acid measurement but not shown in the figure include C14:0, C15:0, and C17:Δ. Fatty acids were isolated and quantified by GC/MS from WT (dark gray bars),  nhr-49(nr2041)  (blue bars),  fat-7  RNAi animals (red bars), and  nhr-49  RNAi animals (light gray bars). Error bars represent standard error. The fatty acid desaturation/elongation pathway, along with enzymes involved in desaturation and elongation, is shown in the inset; this pathway was adapted from previous studies [ 14 , 17 ]. Studies in yeast have shown that the  C. elegans fat-5, fat-6,  and  fat-7  genes encode Δ9-desaturases, which preferentially convert saturated C16:0 and C18:0 fatty acids to monounsaturated C16:1 and C18:1 fatty acids [ 14 ].  fat-5  is a palmitoyl-CoA desaturase, specifically acting on palmitic acid (C16:0), and  fat-6  and  fat-7  are stearoyl-CoA desaturases (SCDs), preferentially functioning to desaturate stearic acid (C18:0). Mammalian SCDs have been shown to be important for maintaining an appropriate level of fatty acid desaturation, vital for modulating membrane fluidity and for controlling lipid metabolism [ 15 , 16 ]. Furthermore, in  C. elegans,  SCDs are also involved in catalyzing the first step in the synthesis of polyunsaturated fatty acids (PUFAs) [ 17 ] (see  Figure 4 B inset). Because the expression of these three  C. elegans  Δ9-desaturases was significantly impaired by  nhr-49  deletion, we used gas chromatography/mass spectrometry (GC/MS) to determine whether overall fatty acid desaturation and PUFA synthesis were altered in  nhr-49(nr2041)  animals. The most significant consequence of  nhr-49  deletion on fatty acid composition was a marked increase in the abundance of stearic acid (C18:0) and a corresponding decrease in the level of oleic acid (C18:1n9) ( Figure 4 B). This result is consistent with a reduction in SCD activity. In mammals, a primary role of SCD is to control the ratio of fully saturated stearic acid to monounsaturated oleic acid (C18:0/C18:1n9), and this ratio has been employed as an indicator of the activity of the mammalian SCD1 [ 15 , 16 ]. In  C. elegans  we found that the ratio of stearic acid to oleic acid (C18:0/C18:1n9) was increased from 1.9 in WT animals to 4.3 in  nhr-49(nr2041)  ( Table 1 ). A similar but less pronounced alteration in the ratio of stearic acid to oleic acid was observed when  nhr-49  was knocked down in WT animals using RNAi ( Figure 4 B and  Table 1 ). This smaller influence on fatty acid desaturation likely resulted from the fact that  nhr-49  RNAi did not reduce Δ9-desaturase expression as dramatically as  nhr-49  knockout (data not shown). Table 1 Life Span, Fat Content, and Relative C18:0 Fatty Acid Abundance Fat content was determined by Nile Red staining. Life span indicated is the day when half of the worms in a given population expired. Fatty acid abundance is indicated by percent of total fatty acid measured by GC/MS
 C. elegans  SCDs are also important for the synthesis of PUFAs; however, we did not observe any measurable changes in PUFA abundance in  nhr-49(nr2041)  animals ( Figure 4 B). Therefore, despite the dramatic effect of  nhr-49  on SCD activity,  nhr-49  does not appear to play an appreciable role in overall PUFA synthesis. To determine which of the Δ9-desaturases accounted for the influence of  nhr-49  deletion on fatty acid composition, we used RNAi to individually knock down expression of  fat-5  and  fat-7  (because  fat-6  expression is only reduced by approximately 2-fold in  nhr-49[nr2041],  we did not examine  fat-6  RNAi animals). Although we did not observe a significant consequence of  fat-5  RNAi on fatty acid desaturation,  fat-7  RNAi altered fatty acid composition in a manner very similar to that of  nhr-49  deletion ( Figure 4 B and  Table 1 ).  fat-7  RNAi animals displayed a significant increase in the abundance of C18:0 fatty acid, as well as a decrease in the levels of C18:1n9 and C18:2n6 fatty acids. The influence of  fat-7  RNAi on fatty acid composition was stronger than that of  nhr-49  deletion, increasing the ratio of stearic to oleic acid (C18:0/C18:1n9) to 8.0.  fat-7  RNAi animals also exhibited a slight lowering of PUFA abundance. The stronger effect of  fat-7  RNAi on fatty acid composition suggests that  fat-7  expression was reduced to levels even lower than those observed in  nhr-49(nr2041).  Because  fat-7  shares significant homology (approximately 85% nucleotide identity) with  fat-6,  it was possible that  fat-7  RNAi also partially interfered with  fat-6  expression; however, we found by QRT-PCR that the overall levels of  fat-6  mRNA were not noticeably reduced by  fat-7  RNAi. (data not shown). Therefore, we conclude that by strongly promoting SCD expression,  nhr-49  influences fatty acid composition in  C. elegans.  Although it appears that the primary effect of  nhr-49  deletion is a modulation of stearic and oleic acid levels, not PUFA abundance, we cannot rule out the possibility that  nhr-49  influences PUFA synthesis in a more localized manner, such that the changes in PUFA composition are not detectable by our whole-worm analysis. Interference of  fat-7  Expression Reproduces Shortened Life-Span Phenotype Using RNAi to knock down the  nhr-49 -dependent genes identified in this study, we found that interference of only  fat-7  resulted in a shortened life-span phenotype similar to that of  nhr-49(nr2041)  (see  Table 1 ).  fat-7  RNAi animals displayed many of the same characteristics of the  nhr-49(nr2041)  mutant, including widespread vacuole formation and germ line necrosis ( Figure 5 ). Figure 5
 fat-7  Is Necessary for Normal Life Span and Inhibits β-Oxidation (A) Nomarski images of WT worms subjected to  fat-7  RNAi at days 1 and 3 of adulthood. The arrow in the day 1 image points to vacuole formation in the intestine, and the arrow in the day 3 image points to clearing that results from collapse of the gonad. These characteristics are nearly identical to those observed for  nhr-49(nr2041)  worms (see  Figure 1 B). (B) QRT-PCR measurement of  acs-2  and  ech-1  expression in WT and  nhr-49(nr2041)  L4 animals grown on control RNAi bacteria (dark gray bars) or on  fat-7  RNAi bacteria (blue bars). Error bars represent standard error of measurement. (C) RNAi knockdown of  fat-7  expression in WT animals reduced Nile Red fat staining (D) RNAi of  fat-7  in  nhr-49(nr2041)  also decreased fat staining. Interestingly, the effect of  fat-7  RNAi on life span was even more potent than  nhr-49  deletion, reducing adult life span to 3–5 d, as opposed to the 5- to 7-d life span of  nhr-49(nr2041)  (see  Table 1 ). This is consistent with the finding that SCD activity is more dramatically compromised in  fat-7  RNAi animals than it is in the  nhr-49(nr2041)  mutant. In fact, we observed a striking correlation between  fat-7  activity, as determined by the ratio of stearic to oleic acid in our various mutants, and life span (see  Table 1 ). WT animals (stearic/oleic = 1.9) lived approximately 16–18 d,  nhr-49 RNAi  animals (stearic/oleic = 2.3) lived approximately 10–12 d,  nhr-49(nr2041)  animals (stearic/oleic = 4.3) lived approximately 6–8 d, and  fat-7  RNAi animals (stearic/oleic = 8.0) lived approximately 3–5 d. Because  fat-7  expression is significantly compromised by  nhr-49  deletion, and because lowered  fat-7  expression can lead to shortened life span, we suggest that the reduced life-span phenotype of  nhr-49(nr2041)  likely reflects, at least in part, diminished  fat-7  expression. Stimulation of  fat-7  by  nhr-49  Feeds Back to Inhibit β-Oxidation By promoting β-oxidation gene expression,  nhr-49  stimulates fat consumption, and by enhancing  fat-7  expression,  nhr-49  modulates fat desaturation and ensures normal longevity. We next set out to determine whether  nhr-49  regulates these two pathways independently. Although RNAi of  acs-2  or  ech-1  was sufficient to cause a high-fat phenotype,  acs-2  or  ech-1  interference did not significantly impact life span, fatty acid composition, or desaturase gene expression, demonstrating that the effects of  nhr-49  deletion on  fat-7  expression and life span were not a downstream consequence of compromised β-oxidation or increased fat content (see  Table 1 ). In contrast, RNAi of  fat-7  significantly impacted fat storage and β-oxidation. The effect was paradoxical, however, as  fat-7  interference produced the opposite result of  nhr-49  deletion, causing  reduced  fat content and  increased  expression of genes in β-oxidation, including  acs-2  and  ech-1  ( Figure 5 ). As the influence of  fat-7  on the expression of β-oxidation genes is opposite that of  nhr-49,  it is clear that the stimulation of β-oxidation by  nhr-49  does not result from promotion of  fat-7  expression. In fact, by enhancing  fat-7  expression,  nhr-49  indirectly causes inhibition of the very same β-oxidation genes that it is independently stimulating ( Figure 6 ). Figure 6 Model for  nhr-49  Regulation of Fat Storage and Composition By stimulating expression of genes predicted to be involved in fatty acid β-oxidation,  nhr-49  promotes fat consumption; likely by facilitating the flow of fatty acids (FA) into the mitochondrial matrix. Through an independent pathway,  nhr-49  modulates fatty acid desaturation by enhancing the expression of the  fat-7  SCD. Stimulation of  fat-7  is necessary for normal adult life span. Finally,  fat-7  also feeds back to partially inhibit expression of β-oxidation genes. Because  fat-7  targets some of the same β-oxidation genes as  nhr-49,  we wondered if the effects of  fat-7  on fat storage might be dependent on  nhr-49;  for example, perhaps  fat-7  represses  ech-1  and  acs-2  expression by blocking the  nhr-49  mediated induction of these genes. However, we found that both  ech-1  and  acs-2  were induced when  fat-7  was knocked down even further in the  nhr-49(nr2041)  mutant using RNAi, indicating that  fat-7  hinders the expression of  ech-1  and  acs-2  through a mechanism that is independent of  nhr-49  (see  Figure 5 B). Furthermore, RNAi of  fat-7  reduced fat content in  nhr-49(nr2041)  worms, demonstrating that the stimulation of fat storage by  fat-7  is also not dependent upon  nhr-49  (see  Figure 5 D). Taken together, our findings demonstrate that  nhr-49  controls two distinct circuits within a gene network: The stimulation of β-oxidation gene expression is independent of  fat-7,  and the promotion of  fat-7  expression is independent of  nhr-49 's effects on β-oxidation. However, because  fat-7  expression inhibits the same β-oxidation genes induced by  nhr-49,  the regulation of  fat-7  by  nhr-49  connects these two circuits into a single regulatory network, serving to limit β-oxidation through an apparent feedback mechanism ( Figure 6 ).
Results
nhr-49  Is Necessary for Normal Life Span In an RNA interference (RNAi) screen designed to identify the role of  C. elegans  HNF4-like receptors in worm development and longevity, we found that interference of  nhr-49  resulted in dramatically reduced life span ( Figure 1 A). For further characterization, we obtained a  C. elegans  strain,  nhr-49(nr2041),  which harbors a deletion in the  nhr-49  gene encompassing part of the DNA binding domain and more than half of the ligand binding domain (LBD); this deletion likely results in complete loss of function [ 10 ]. At 23 °C,  nhr-49(nr2041)  worms lived only 6–8 d as adults, significantly shorter than the 15 to 18-d life span of N2 wild-type (WT) animals ( Figure 1 A). Although  nhr-49  deletion did not noticeably affect development or fertility,  nhr-49(nr2041)  worms experienced rapid decline in function beginning around day 3 of adulthood, when vacuoles appeared in the intestine and gonad ( Figure 1 B). By days 4 and 5,  nhr-49(nr2041)  animals were significantly smaller than WT, vacuoles were ubiquitous, and there was widespread gonadal necrosis. By days 5–7, the gonad had completely deteriorated, and the worms died shortly thereafter. Thus,  nhr-49  function is not required for development or fertility, but is clearly essential for normal longevity. Even though the increased vacuole formation was consistent with reported aging characteristics [ 11 ], we have not determined whether the shortened life span of  nhr-49(nr2041)  reflects accelerated aging or an unrelated pathology. Figure 1
 nhr-49(nr2041)  Animals Have Reduced Life Span and Higher Fat Content (A) Adult life span of WT (black squares, solid line),  nhr-49(nr2041)  (black circles, dotted line), and  nhr-49  RNAi animals (black diamonds, dashed line). (B) Nomarksi images of  nhr-49(nr2041)  at days 3, 5, and 7 of adulthood. The arrow in the day 3 image points to a gonadal vacuole that is typical of day 3 worms; the arrow in the day 5 image shows the continued deterioration of oocytes in the gonad, and the arrow in the day 7 image points to the clearing that results from complete gonadal necrosis and collapse. (C) Nile Red intestinal fat staining of WT and  nhr-49(nr2041).  Each image displays two representative worms from a population of L4 animals.
nhr-49  Is Necessary for Normal Life Span
nhr-49In an RNA interference (RNAi) screen designed to identify the role of  C. elegans  HNF4-like receptors in worm development and longevity, we found that interference of  nhr-49  resulted in dramatically reduced life span ( Figure 1 A). For further characterization, we obtained a  C. elegans  strain,  nhr-49(nr2041),  which harbors a deletion in the  nhr-49  gene encompassing part of the DNA binding domain and more than half of the ligand binding domain (LBD); this deletion likely results in complete loss of function [ 10 ]. At 23 °C,  nhr-49(nr2041)  worms lived only 6–8 d as adults, significantly shorter than the 15 to 18-d life span of N2 wild-type (WT) animals ( Figure 1 A). Although  nhr-49  deletion did not noticeably affect development or fertility,  nhr-49(nr2041)  worms experienced rapid decline in function beginning around day 3 of adulthood, when vacuoles appeared in the intestine and gonad ( Figure 1 B). By days 4 and 5,  nhr-49(nr2041)  animals were significantly smaller than WT, vacuoles were ubiquitous, and there was widespread gonadal necrosis. By days 5–7, the gonad had completely deteriorated, and the worms died shortly thereafter. Thus,  nhr-49  function is not required for development or fertility, but is clearly essential for normal longevity. Even though the increased vacuole formation was consistent with reported aging characteristics [ 11 ], we have not determined whether the shortened life span of  nhr-49(nr2041)  reflects accelerated aging or an unrelated pathology.nhr-49nhr-49(nr2041),nhr-49nhr-49(nr2041)nhr-49nhr-49(nr2041)nhr-49(nr2041)nhr-49nhr-49(nr2041)nhr-49(nr2041)  Animals Display Abnormally High Fat Content
 nhr-49  was recently identified in a  C. elegans  genomewide RNAi screen as one of 112 genes that, when knocked down by RNAi, resulted in abnormally high Nile Red fat staining [ 12 ]. To determine whether deletion of  nhr-49  also yielded a high-fat phenotype, we used a similar Nile Red assay to visualize fat content in  nhr-49(nr2041).  Indeed, we found that  nhr-49(nr2041)  animals stained more brightly with Nile Red than did WT worms ( Figure 1 C). The difference in fat content between WT and  nhr-49(nr2041)  animals was most pronounced in the L3 and L4 stages of larval development; by day 2 of adulthood there was only a slight change in Nile Red staining, suggesting that the effects of  nhr-49  on visible fat content may vary during development (data not shown).
nhr-49(nr2041)  Animals Display Abnormally High Fat Content
nhr-49(nr2041)nhr-49  was recently identified in a  C. elegans  genomewide RNAi screen as one of 112 genes that, when knocked down by RNAi, resulted in abnormally high Nile Red fat staining [ 12 ]. To determine whether deletion of  nhr-49  also yielded a high-fat phenotype, we used a similar Nile Red assay to visualize fat content in  nhr-49(nr2041).  Indeed, we found that  nhr-49(nr2041)  animals stained more brightly with Nile Red than did WT worms ( Figure 1 C). The difference in fat content between WT and  nhr-49(nr2041)  animals was most pronounced in the L3 and L4 stages of larval development; by day 2 of adulthood there was only a slight change in Nile Red staining, suggesting that the effects of  nhr-49  on visible fat content may vary during development (data not shown).nhr-49nhr-49nhr-49(nr2041).nhr-49(nr2041)nhr-49(nr2041)nhr-49nhr-49  Regulates Genes Involved in Energy Metabolism The high-fat phenotype of the  nhr-49(nr2041)  mutant led us to suspect that  nhr-49  might regulate genes involved in energy metabolism. To test this hypothesis, we identified from a survey of the  C. elegans  genome 65 genes predicted to participate in fatty acid synthesis, β-oxidation, desaturation, elongation, and binding/transport, in addition to 16 genes expected to function in glycolysis, gluconeogenesis, and glucose transport, and eight genes involved in the glyoxylate pathway ( Figure 2 ). We then employed quantitative RT-PCR (QRT-PCR) to measure the expression of all 89 of these genes in WT and  nhr-49(nr2041)  mutant worms. For the purpose of our survey, gene expression was measured in all four larval stages (for a complete dataset, see  Table S1 ). Figure 2
 nhr-49  Deletion Affects the Expression of 13 Energy Metabolism Genes Putative  C. elegans  fatty acid, glucose, and glyoxylate metabolism pathways are shown here; each box represents a gene predicted by sequence homology to code for the enzyme indicated in the figure. The fatty acid desaturation elongation pathway is adapted from previously published studies [ 14 , 17 ]. Mitochondrial localization of β-oxidation enzymes was predicted by TargetP and PSORT (see  Materials and Methods ); peroxisomal β-oxidation enzymes all contain a carboxy-terminal peroxisomal localization signal. QRT-PCR was used to measure the expression of these 89 genes in WT and  nhr-49(nr2041).  Genes expressed at lower levels in  nhr-49(nr2041)  are shown in red (>32-fold lower); orange (8- to 32-fold lower); or yellow (2- to 8-fold lower); and genes expressed at higher levels are shown in light green (2- to 8-fold higher) or dark green (>8-fold higher). Online QRT-PCR data for this analysis are available in  Table S1 . Our screen revealed that deletion of  nhr-49  significantly altered the expression of 13 genes, including six genes predicted to be involved in fatty acid β-oxidation, three genes involved in fatty acid desaturation, two genes involved in fatty acid binding/transport, and two genes involved in the glyoxylate pathway ( Figure 2 ). Similar changes in gene expression were observed when  nhr-49  was knocked down in WT animals using RNAi (data not shown). Overall,  nhr-49  deletion displayed the most dramatic effects on two metabolic pathways, mitochondrial β-oxidation and fatty acid desaturation, as the expression of multiple genes in each of these pathways was significantly compromised by loss of  nhr-49  function ( Figure 2 ). In contrast,  nhr-49  deletion had smaller and mixed consequences for genes that participate in peroxisomal β-oxidation and lipid binding/transport.  nhr-49  knockout also marginally reduced expression of two enzymes involved in the glyoxylate pathway, a pathway that facilitates the conversion of fatty acid β-oxidation products to glucose ( Figure 2 ).  nhr-49  deletion did not, however, significantly affect the expression of any genes predicted to be involved in glucose metabolism. Thus, it is clear that  nhr-49  is extensively involved in the control of fatty acid metabolism, with a pronounced role in the promotion of mitochondrial β-oxidation and fatty acid desaturation.
nhr-49  Regulates Genes Involved in Energy Metabolism
nhr-49The high-fat phenotype of the  nhr-49(nr2041)  mutant led us to suspect that  nhr-49  might regulate genes involved in energy metabolism. To test this hypothesis, we identified from a survey of the  C. elegans  genome 65 genes predicted to participate in fatty acid synthesis, β-oxidation, desaturation, elongation, and binding/transport, in addition to 16 genes expected to function in glycolysis, gluconeogenesis, and glucose transport, and eight genes involved in the glyoxylate pathway ( Figure 2 ). We then employed quantitative RT-PCR (QRT-PCR) to measure the expression of all 89 of these genes in WT and  nhr-49(nr2041)  mutant worms. For the purpose of our survey, gene expression was measured in all four larval stages (for a complete dataset, see  Table S1 ).nhr-49(nr2041)nhr-49nhr-49(nr2041)Our screen revealed that deletion of  nhr-49  significantly altered the expression of 13 genes, including six genes predicted to be involved in fatty acid β-oxidation, three genes involved in fatty acid desaturation, two genes involved in fatty acid binding/transport, and two genes involved in the glyoxylate pathway ( Figure 2 ). Similar changes in gene expression were observed when  nhr-49  was knocked down in WT animals using RNAi (data not shown).nhr-49nhr-49Overall,  nhr-49  deletion displayed the most dramatic effects on two metabolic pathways, mitochondrial β-oxidation and fatty acid desaturation, as the expression of multiple genes in each of these pathways was significantly compromised by loss of  nhr-49  function ( Figure 2 ). In contrast,  nhr-49  deletion had smaller and mixed consequences for genes that participate in peroxisomal β-oxidation and lipid binding/transport.  nhr-49  knockout also marginally reduced expression of two enzymes involved in the glyoxylate pathway, a pathway that facilitates the conversion of fatty acid β-oxidation products to glucose ( Figure 2 ).  nhr-49  deletion did not, however, significantly affect the expression of any genes predicted to be involved in glucose metabolism. Thus, it is clear that  nhr-49  is extensively involved in the control of fatty acid metabolism, with a pronounced role in the promotion of mitochondrial β-oxidation and fatty acid desaturation.nhr-49nhr-49nhr-49nhr-49nhr-49nhr-49nhr-49(nr2041)  Animals are High in Fat Due to Reduced Expression of β-Oxidation Genes Three genes predicted to participate in mitochondrial β-oxidation,  ech-1  (C29F3.1), F09F3.9, and an acyl-CoA synthetase gene  (acs-2)  (F28F8.2), were expressed at significantly lower levels in  nhr-49(nr2041)  animals throughout development ( Figure 3 A).  acs-2  is predicted to encode a mitochondrial acyl-CoA synthetase, whereas F09F3.9 likely codes for a carnitine palmitoyl transferase, and  ech-1  appears to encode a mitochondrial β-oxidation trifunctional enzyme. Because mitochondrial β-oxidation facilitates the degradation of stored fats for the production of energy, we suspected that  nhr-49(nr2041)  worms might be high in fat due to the reduced expression of these key β-oxidation enzymes. To test this hypothesis, we used RNAi to reduce separately the expression of  acs-2,  F09F3.9, and  ech-1  in WT animals. Indeed, we found that RNAi of either  acs-2  or  ech-1  resulted in worms with elevated Nile Red fat staining ( Figure 3 B).  acs-2  was previously identified for a similar effect on fat storage [ 12 ]. We conclude that  nhr-49  promotes the expression of three β-oxidation genes, two of which, in WT worms, reduce overall fat storage. Thus,  nhr-49(nr2041)  mutant worms are likely to be high in fat due to the impaired expression of these genes. Figure 3 Regulation of β-Oxidation Gene Expression by  nhr-49
 (A) QRT-PCR measurement of  acs-2,  F09F9.3, and  ech-1  expression in WT (gray bars) and  nhr-49(nr2041)  animals (blue bars). Expression was measured in all four stages of larval development. Error bars represent standard error of measurement. (B) RNAi of  nhr-49, acs-2,  or  ech-1  resulted in increased Nile Red fat staining. Each image shows 3 or 4 representative worms from a population of L4 animals grown on plates containing RNAi bacteria and Nile Red fat-staining dye. (C)  acs-2::gfp  is expressed in many tissues including hypodermis (hyp), intestine (int), body wall muscle (bwm), neurons (neu), and pharynx (pha). ACS-2::GFP localizes to subcellular structures in a pattern similar to what has been reported for mitochondrial proteins [ 13 ]. (D) Expression of  acs-2::gfp  was lower in  nhr-49(nr2041)  mutant animals. (E) Expression of P nhr-49 ::gfp  promoter fusion in WT animals revealed that  nhr-49  is expressed in multiple tissues, including hypodermis (hyp), body wall muscle (bwm), pharynx (pha), and intestine (int). The animals shown here are genetically mosaic, harboring the P nhr-49 ::gfp  construct in only a fraction of total cells. (F) Ectopic expression of  acs-2::gfp  in  nhr-49(nr2041)  was sufficient to reduce Nile Red staining to WT levels.
nhr-49(nr2041)  Animals are High in Fat Due to Reduced Expression of β-Oxidation Genes
nhr-49(nr2041)Three genes predicted to participate in mitochondrial β-oxidation,  ech-1  (C29F3.1), F09F3.9, and an acyl-CoA synthetase gene  (acs-2)  (F28F8.2), were expressed at significantly lower levels in  nhr-49(nr2041)  animals throughout development ( Figure 3 A).  acs-2  is predicted to encode a mitochondrial acyl-CoA synthetase, whereas F09F3.9 likely codes for a carnitine palmitoyl transferase, and  ech-1  appears to encode a mitochondrial β-oxidation trifunctional enzyme. Because mitochondrial β-oxidation facilitates the degradation of stored fats for the production of energy, we suspected that  nhr-49(nr2041)  worms might be high in fat due to the reduced expression of these key β-oxidation enzymes. To test this hypothesis, we used RNAi to reduce separately the expression of  acs-2,  F09F3.9, and  ech-1  in WT animals. Indeed, we found that RNAi of either  acs-2  or  ech-1  resulted in worms with elevated Nile Red fat staining ( Figure 3 B).  acs-2  was previously identified for a similar effect on fat storage [ 12 ]. We conclude that  nhr-49  promotes the expression of three β-oxidation genes, two of which, in WT worms, reduce overall fat storage. Thus,  nhr-49(nr2041)  mutant worms are likely to be high in fat due to the impaired expression of these genes.ech-1(acs-2)nhr-49(nr2041)acs-2ech-1nhr-49(nr2041)acs-2,ech-1acs-2ech-1acs-2nhr-49nhr-49(nr2041)ACS-2 Is Expressed in Multiple Tissues and Localizes to Mitochondria Out of five predicted mitochondrial  acs  genes in  C. elegans, nhr-49  affected only the expression of  acs-2,  suggesting that  nhr-49  may only influence a subset of mitochondrial β-oxidation pathways in worms. To determine if  acs-2  was expressed in a specific set of tissues, we fused the full-length  acs-2  gene and promoter to  gfp (acs-2::gfp);  injection of this construct into WT worms revealed that the ACS-2::green fluorescent protein (GFP) was widely expressed in many cell types, including intestine, hypodermis, pharynx, body wall muscle, and several neurons ( Figure 3 C). Moreover, ACS-2::GFP expression was reduced in all of these tissues in  nhr-49(nr2041)  worms, indicating that the effects of  nhr-49  on  acs-2  expression were widespread ( Figure 3 D). Notably, the expression pattern of  acs-2  overlapped extensively with that of an  nhr-49  promoter/GFP fusion, consistent with the idea that  nhr-49  could control  acs-2  through direct transcriptional activation ( Figure 3 E). As predicted, ACS-2::GFP localized to subcellular structures in patterns similar to those reported for other mitochondrial proteins [ 13 ] ( Figure 3 C).
ACS-2 Is Expressed in Multiple Tissues and Localizes to Mitochondria
Out of five predicted mitochondrial  acs  genes in  C. elegans, nhr-49  affected only the expression of  acs-2,  suggesting that  nhr-49  may only influence a subset of mitochondrial β-oxidation pathways in worms. To determine if  acs-2  was expressed in a specific set of tissues, we fused the full-length  acs-2  gene and promoter to  gfp (acs-2::gfp);  injection of this construct into WT worms revealed that the ACS-2::green fluorescent protein (GFP) was widely expressed in many cell types, including intestine, hypodermis, pharynx, body wall muscle, and several neurons ( Figure 3 C). Moreover, ACS-2::GFP expression was reduced in all of these tissues in  nhr-49(nr2041)  worms, indicating that the effects of  nhr-49  on  acs-2  expression were widespread ( Figure 3 D). Notably, the expression pattern of  acs-2  overlapped extensively with that of an  nhr-49  promoter/GFP fusion, consistent with the idea that  nhr-49  could control  acs-2  through direct transcriptional activation ( Figure 3 E). As predicted, ACS-2::GFP localized to subcellular structures in patterns similar to those reported for other mitochondrial proteins [ 13 ] ( Figure 3 C).acsC. elegans, nhr-49acs-2,nhr-49acs-2acs-2gfp (acs-2::gfp);nhr-49(nr2041)nhr-49acs-2acs-2nhr-49nhr-49acs-2Overexpression of ACS-2::GFP Is Sufficient to Suppress the High-Fat Phenotype of  nhr-49(nr2041)
 Strikingly, overexpression of the ACS-2::GFP fusion was able to suppress the high-fat phenotype of  nhr-49(nr2041)  animals ( Figure 3 F). These data suggest that  acs-2  expression is reduced in  nhr-49(nr2041)  such that it becomes a rate-limiting factor in the consumption of fat; thus, high fat levels can be overcome simply by increasing  acs-2  expression. Overexpression of  acs-2  in WT animals did not affect fat content, however, indicating that WT levels of  acs-2  expression are not rate limiting. These data further support the conclusion that reduced expression of  acs-2  is a significant contributor to the high-fat phenotype of the  nhr-49(nr2041)  mutant.
Overexpression of ACS-2::GFP Is Sufficient to Suppress the High-Fat Phenotype of  nhr-49(nr2041)
nhr-49(nr2041)Strikingly, overexpression of the ACS-2::GFP fusion was able to suppress the high-fat phenotype of  nhr-49(nr2041)  animals ( Figure 3 F). These data suggest that  acs-2  expression is reduced in  nhr-49(nr2041)  such that it becomes a rate-limiting factor in the consumption of fat; thus, high fat levels can be overcome simply by increasing  acs-2  expression. Overexpression of  acs-2  in WT animals did not affect fat content, however, indicating that WT levels of  acs-2  expression are not rate limiting. These data further support the conclusion that reduced expression of  acs-2  is a significant contributor to the high-fat phenotype of the  nhr-49(nr2041)  mutant.nhr-49(nr2041)acs-2nhr-49(nr2041)acs-2acs-2acs-2acs-2nhr-49(nr2041)nhr-49  Modulates Fatty Acid Composition by Promoting Expression of Δ9-Desaturases In addition to stimulating genes in the mitochondrial β-oxidation pathway,  nhr-49  also promoted the expression of three  C. elegans  fatty acid desaturases (see  Figure 2 ).  fat-5  (W06D12.3) and  fat-7  (F10D2.9) expression was dramatically lowered in  nhr-49(nr2041)  worms (>30-fold) in all four larval stages, whereas  fat-6  (VZK8221.1) expression was marginally reduced (approximately 2-fold) only in L3 and L4 animals ( Figure 4 A). Figure 4 Deletion of  nhr-49  Hinders Fatty Acid Desaturation (A) QRT-PCR measurement of  fat-5, fat-6, and fat-7  expression in WT (gray bars) and in  nhr-49(nr2041)  mutant worms (blue bars). Expression was measured in all four stages of larval development. Error bars represent standard error of measurement. (B) Relative abundance of individual fatty acid species expressed as percentage of total measured fatty acid. Fatty acids included in total fatty acid measurement but not shown in the figure include C14:0, C15:0, and C17:Δ. Fatty acids were isolated and quantified by GC/MS from WT (dark gray bars),  nhr-49(nr2041)  (blue bars),  fat-7  RNAi animals (red bars), and  nhr-49  RNAi animals (light gray bars). Error bars represent standard error. The fatty acid desaturation/elongation pathway, along with enzymes involved in desaturation and elongation, is shown in the inset; this pathway was adapted from previous studies [ 14 , 17 ]. Studies in yeast have shown that the  C. elegans fat-5, fat-6,  and  fat-7  genes encode Δ9-desaturases, which preferentially convert saturated C16:0 and C18:0 fatty acids to monounsaturated C16:1 and C18:1 fatty acids [ 14 ].  fat-5  is a palmitoyl-CoA desaturase, specifically acting on palmitic acid (C16:0), and  fat-6  and  fat-7  are stearoyl-CoA desaturases (SCDs), preferentially functioning to desaturate stearic acid (C18:0). Mammalian SCDs have been shown to be important for maintaining an appropriate level of fatty acid desaturation, vital for modulating membrane fluidity and for controlling lipid metabolism [ 15 , 16 ]. Furthermore, in  C. elegans,  SCDs are also involved in catalyzing the first step in the synthesis of polyunsaturated fatty acids (PUFAs) [ 17 ] (see  Figure 4 B inset). Because the expression of these three  C. elegans  Δ9-desaturases was significantly impaired by  nhr-49  deletion, we used gas chromatography/mass spectrometry (GC/MS) to determine whether overall fatty acid desaturation and PUFA synthesis were altered in  nhr-49(nr2041)  animals. The most significant consequence of  nhr-49  deletion on fatty acid composition was a marked increase in the abundance of stearic acid (C18:0) and a corresponding decrease in the level of oleic acid (C18:1n9) ( Figure 4 B). This result is consistent with a reduction in SCD activity. In mammals, a primary role of SCD is to control the ratio of fully saturated stearic acid to monounsaturated oleic acid (C18:0/C18:1n9), and this ratio has been employed as an indicator of the activity of the mammalian SCD1 [ 15 , 16 ]. In  C. elegans  we found that the ratio of stearic acid to oleic acid (C18:0/C18:1n9) was increased from 1.9 in WT animals to 4.3 in  nhr-49(nr2041)  ( Table 1 ). A similar but less pronounced alteration in the ratio of stearic acid to oleic acid was observed when  nhr-49  was knocked down in WT animals using RNAi ( Figure 4 B and  Table 1 ). This smaller influence on fatty acid desaturation likely resulted from the fact that  nhr-49  RNAi did not reduce Δ9-desaturase expression as dramatically as  nhr-49  knockout (data not shown). Table 1 Life Span, Fat Content, and Relative C18:0 Fatty Acid Abundance Fat content was determined by Nile Red staining. Life span indicated is the day when half of the worms in a given population expired. Fatty acid abundance is indicated by percent of total fatty acid measured by GC/MS
 C. elegans  SCDs are also important for the synthesis of PUFAs; however, we did not observe any measurable changes in PUFA abundance in  nhr-49(nr2041)  animals ( Figure 4 B). Therefore, despite the dramatic effect of  nhr-49  on SCD activity,  nhr-49  does not appear to play an appreciable role in overall PUFA synthesis. To determine which of the Δ9-desaturases accounted for the influence of  nhr-49  deletion on fatty acid composition, we used RNAi to individually knock down expression of  fat-5  and  fat-7  (because  fat-6  expression is only reduced by approximately 2-fold in  nhr-49[nr2041],  we did not examine  fat-6  RNAi animals). Although we did not observe a significant consequence of  fat-5  RNAi on fatty acid desaturation,  fat-7  RNAi altered fatty acid composition in a manner very similar to that of  nhr-49  deletion ( Figure 4 B and  Table 1 ).  fat-7  RNAi animals displayed a significant increase in the abundance of C18:0 fatty acid, as well as a decrease in the levels of C18:1n9 and C18:2n6 fatty acids. The influence of  fat-7  RNAi on fatty acid composition was stronger than that of  nhr-49  deletion, increasing the ratio of stearic to oleic acid (C18:0/C18:1n9) to 8.0.  fat-7  RNAi animals also exhibited a slight lowering of PUFA abundance. The stronger effect of  fat-7  RNAi on fatty acid composition suggests that  fat-7  expression was reduced to levels even lower than those observed in  nhr-49(nr2041).  Because  fat-7  shares significant homology (approximately 85% nucleotide identity) with  fat-6,  it was possible that  fat-7  RNAi also partially interfered with  fat-6  expression; however, we found by QRT-PCR that the overall levels of  fat-6  mRNA were not noticeably reduced by  fat-7  RNAi. (data not shown). Therefore, we conclude that by strongly promoting SCD expression,  nhr-49  influences fatty acid composition in  C. elegans.  Although it appears that the primary effect of  nhr-49  deletion is a modulation of stearic and oleic acid levels, not PUFA abundance, we cannot rule out the possibility that  nhr-49  influences PUFA synthesis in a more localized manner, such that the changes in PUFA composition are not detectable by our whole-worm analysis.
nhr-49  Modulates Fatty Acid Composition by Promoting Expression of Δ9-Desaturases
nhr-49In addition to stimulating genes in the mitochondrial β-oxidation pathway,  nhr-49  also promoted the expression of three  C. elegans  fatty acid desaturases (see  Figure 2 ).  fat-5  (W06D12.3) and  fat-7  (F10D2.9) expression was dramatically lowered in  nhr-49(nr2041)  worms (>30-fold) in all four larval stages, whereas  fat-6  (VZK8221.1) expression was marginally reduced (approximately 2-fold) only in L3 and L4 animals ( Figure 4 A).nhr-49fat-5fat-7nhr-49(nr2041)fat-6Studies in yeast have shown that the  C. elegans fat-5, fat-6,  and  fat-7  genes encode Δ9-desaturases, which preferentially convert saturated C16:0 and C18:0 fatty acids to monounsaturated C16:1 and C18:1 fatty acids [ 14 ].  fat-5  is a palmitoyl-CoA desaturase, specifically acting on palmitic acid (C16:0), and  fat-6  and  fat-7  are stearoyl-CoA desaturases (SCDs), preferentially functioning to desaturate stearic acid (C18:0). Mammalian SCDs have been shown to be important for maintaining an appropriate level of fatty acid desaturation, vital for modulating membrane fluidity and for controlling lipid metabolism [ 15 , 16 ]. Furthermore, in  C. elegans,  SCDs are also involved in catalyzing the first step in the synthesis of polyunsaturated fatty acids (PUFAs) [ 17 ] (see  Figure 4 B inset).C. elegans fat-5, fat-6,fat-7fat-5fat-6fat-7C. elegans,Because the expression of these three  C. elegans  Δ9-desaturases was significantly impaired by  nhr-49  deletion, we used gas chromatography/mass spectrometry (GC/MS) to determine whether overall fatty acid desaturation and PUFA synthesis were altered in  nhr-49(nr2041)  animals. The most significant consequence of  nhr-49  deletion on fatty acid composition was a marked increase in the abundance of stearic acid (C18:0) and a corresponding decrease in the level of oleic acid (C18:1n9) ( Figure 4 B). This result is consistent with a reduction in SCD activity.nhr-49nhr-49(nr2041)nhr-49In mammals, a primary role of SCD is to control the ratio of fully saturated stearic acid to monounsaturated oleic acid (C18:0/C18:1n9), and this ratio has been employed as an indicator of the activity of the mammalian SCD1 [ 15 , 16 ]. In  C. elegans  we found that the ratio of stearic acid to oleic acid (C18:0/C18:1n9) was increased from 1.9 in WT animals to 4.3 in  nhr-49(nr2041)  ( Table 1 ). A similar but less pronounced alteration in the ratio of stearic acid to oleic acid was observed when  nhr-49  was knocked down in WT animals using RNAi ( Figure 4 B and  Table 1 ). This smaller influence on fatty acid desaturation likely resulted from the fact that  nhr-49  RNAi did not reduce Δ9-desaturase expression as dramatically as  nhr-49  knockout (data not shown).nhr-49(nr2041)nhr-49nhr-49nhr-49Life Span, Fat Content, and Relative C18:0 Fatty Acid Abundance
Fat content was determined by Nile Red staining. Life span indicated is the day when half of the worms in a given population expired. Fatty acid abundance is indicated by percent of total fatty acid measured by GC/MSC. elegans  SCDs are also important for the synthesis of PUFAs; however, we did not observe any measurable changes in PUFA abundance in  nhr-49(nr2041)  animals ( Figure 4 B). Therefore, despite the dramatic effect of  nhr-49  on SCD activity,  nhr-49  does not appear to play an appreciable role in overall PUFA synthesis.nhr-49(nr2041)nhr-49nhr-49To determine which of the Δ9-desaturases accounted for the influence of  nhr-49  deletion on fatty acid composition, we used RNAi to individually knock down expression of  fat-5  and  fat-7  (because  fat-6  expression is only reduced by approximately 2-fold in  nhr-49[nr2041],  we did not examine  fat-6  RNAi animals). Although we did not observe a significant consequence of  fat-5  RNAi on fatty acid desaturation,  fat-7  RNAi altered fatty acid composition in a manner very similar to that of  nhr-49  deletion ( Figure 4 B and  Table 1 ).  fat-7  RNAi animals displayed a significant increase in the abundance of C18:0 fatty acid, as well as a decrease in the levels of C18:1n9 and C18:2n6 fatty acids. The influence of  fat-7  RNAi on fatty acid composition was stronger than that of  nhr-49  deletion, increasing the ratio of stearic to oleic acid (C18:0/C18:1n9) to 8.0.  fat-7  RNAi animals also exhibited a slight lowering of PUFA abundance. The stronger effect of  fat-7  RNAi on fatty acid composition suggests that  fat-7  expression was reduced to levels even lower than those observed in  nhr-49(nr2041).  Because  fat-7  shares significant homology (approximately 85% nucleotide identity) with  fat-6,  it was possible that  fat-7  RNAi also partially interfered with  fat-6  expression; however, we found by QRT-PCR that the overall levels of  fat-6  mRNA were not noticeably reduced by  fat-7  RNAi. (data not shown).nhr-49fat-5fat-7fat-6nhr-49[nr2041],fat-6fat-5fat-7nhr-49fat-7fat-7nhr-49fat-7fat-7fat-7nhr-49(nr2041).fat-7fat-6,fat-7fat-6fat-6fat-7Therefore, we conclude that by strongly promoting SCD expression,  nhr-49  influences fatty acid composition in  C. elegans.  Although it appears that the primary effect of  nhr-49  deletion is a modulation of stearic and oleic acid levels, not PUFA abundance, we cannot rule out the possibility that  nhr-49  influences PUFA synthesis in a more localized manner, such that the changes in PUFA composition are not detectable by our whole-worm analysis.nhr-49C. elegans.nhr-49nhr-49Interference of  fat-7  Expression Reproduces Shortened Life-Span Phenotype Using RNAi to knock down the  nhr-49 -dependent genes identified in this study, we found that interference of only  fat-7  resulted in a shortened life-span phenotype similar to that of  nhr-49(nr2041)  (see  Table 1 ).  fat-7  RNAi animals displayed many of the same characteristics of the  nhr-49(nr2041)  mutant, including widespread vacuole formation and germ line necrosis ( Figure 5 ). Figure 5
 fat-7  Is Necessary for Normal Life Span and Inhibits β-Oxidation (A) Nomarski images of WT worms subjected to  fat-7  RNAi at days 1 and 3 of adulthood. The arrow in the day 1 image points to vacuole formation in the intestine, and the arrow in the day 3 image points to clearing that results from collapse of the gonad. These characteristics are nearly identical to those observed for  nhr-49(nr2041)  worms (see  Figure 1 B). (B) QRT-PCR measurement of  acs-2  and  ech-1  expression in WT and  nhr-49(nr2041)  L4 animals grown on control RNAi bacteria (dark gray bars) or on  fat-7  RNAi bacteria (blue bars). Error bars represent standard error of measurement. (C) RNAi knockdown of  fat-7  expression in WT animals reduced Nile Red fat staining (D) RNAi of  fat-7  in  nhr-49(nr2041)  also decreased fat staining. Interestingly, the effect of  fat-7  RNAi on life span was even more potent than  nhr-49  deletion, reducing adult life span to 3–5 d, as opposed to the 5- to 7-d life span of  nhr-49(nr2041)  (see  Table 1 ). This is consistent with the finding that SCD activity is more dramatically compromised in  fat-7  RNAi animals than it is in the  nhr-49(nr2041)  mutant. In fact, we observed a striking correlation between  fat-7  activity, as determined by the ratio of stearic to oleic acid in our various mutants, and life span (see  Table 1 ). WT animals (stearic/oleic = 1.9) lived approximately 16–18 d,  nhr-49 RNAi  animals (stearic/oleic = 2.3) lived approximately 10–12 d,  nhr-49(nr2041)  animals (stearic/oleic = 4.3) lived approximately 6–8 d, and  fat-7  RNAi animals (stearic/oleic = 8.0) lived approximately 3–5 d. Because  fat-7  expression is significantly compromised by  nhr-49  deletion, and because lowered  fat-7  expression can lead to shortened life span, we suggest that the reduced life-span phenotype of  nhr-49(nr2041)  likely reflects, at least in part, diminished  fat-7  expression.
Interference of  fat-7  Expression Reproduces Shortened Life-Span Phenotype
fat-7Using RNAi to knock down the  nhr-49 -dependent genes identified in this study, we found that interference of only  fat-7  resulted in a shortened life-span phenotype similar to that of  nhr-49(nr2041)  (see  Table 1 ).  fat-7  RNAi animals displayed many of the same characteristics of the  nhr-49(nr2041)  mutant, including widespread vacuole formation and germ line necrosis ( Figure 5 ).nhr-49fat-7nhr-49(nr2041)fat-7nhr-49(nr2041)Interestingly, the effect of  fat-7  RNAi on life span was even more potent than  nhr-49  deletion, reducing adult life span to 3–5 d, as opposed to the 5- to 7-d life span of  nhr-49(nr2041)  (see  Table 1 ). This is consistent with the finding that SCD activity is more dramatically compromised in  fat-7  RNAi animals than it is in the  nhr-49(nr2041)  mutant. In fact, we observed a striking correlation between  fat-7  activity, as determined by the ratio of stearic to oleic acid in our various mutants, and life span (see  Table 1 ). WT animals (stearic/oleic = 1.9) lived approximately 16–18 d,  nhr-49 RNAi  animals (stearic/oleic = 2.3) lived approximately 10–12 d,  nhr-49(nr2041)  animals (stearic/oleic = 4.3) lived approximately 6–8 d, and  fat-7  RNAi animals (stearic/oleic = 8.0) lived approximately 3–5 d.fat-7nhr-49nhr-49(nr2041)fat-7nhr-49(nr2041)fat-7nhr-49 RNAinhr-49(nr2041)fat-7Because  fat-7  expression is significantly compromised by  nhr-49  deletion, and because lowered  fat-7  expression can lead to shortened life span, we suggest that the reduced life-span phenotype of  nhr-49(nr2041)  likely reflects, at least in part, diminished  fat-7  expression.fat-7nhr-49fat-7nhr-49(nr2041)fat-7Stimulation of  fat-7  by  nhr-49  Feeds Back to Inhibit β-Oxidation By promoting β-oxidation gene expression,  nhr-49  stimulates fat consumption, and by enhancing  fat-7  expression,  nhr-49  modulates fat desaturation and ensures normal longevity. We next set out to determine whether  nhr-49  regulates these two pathways independently. Although RNAi of  acs-2  or  ech-1  was sufficient to cause a high-fat phenotype,  acs-2  or  ech-1  interference did not significantly impact life span, fatty acid composition, or desaturase gene expression, demonstrating that the effects of  nhr-49  deletion on  fat-7  expression and life span were not a downstream consequence of compromised β-oxidation or increased fat content (see  Table 1 ). In contrast, RNAi of  fat-7  significantly impacted fat storage and β-oxidation. The effect was paradoxical, however, as  fat-7  interference produced the opposite result of  nhr-49  deletion, causing  reduced  fat content and  increased  expression of genes in β-oxidation, including  acs-2  and  ech-1  ( Figure 5 ). As the influence of  fat-7  on the expression of β-oxidation genes is opposite that of  nhr-49,  it is clear that the stimulation of β-oxidation by  nhr-49  does not result from promotion of  fat-7  expression. In fact, by enhancing  fat-7  expression,  nhr-49  indirectly causes inhibition of the very same β-oxidation genes that it is independently stimulating ( Figure 6 ). Figure 6 Model for  nhr-49  Regulation of Fat Storage and Composition By stimulating expression of genes predicted to be involved in fatty acid β-oxidation,  nhr-49  promotes fat consumption; likely by facilitating the flow of fatty acids (FA) into the mitochondrial matrix. Through an independent pathway,  nhr-49  modulates fatty acid desaturation by enhancing the expression of the  fat-7  SCD. Stimulation of  fat-7  is necessary for normal adult life span. Finally,  fat-7  also feeds back to partially inhibit expression of β-oxidation genes. Because  fat-7  targets some of the same β-oxidation genes as  nhr-49,  we wondered if the effects of  fat-7  on fat storage might be dependent on  nhr-49;  for example, perhaps  fat-7  represses  ech-1  and  acs-2  expression by blocking the  nhr-49  mediated induction of these genes. However, we found that both  ech-1  and  acs-2  were induced when  fat-7  was knocked down even further in the  nhr-49(nr2041)  mutant using RNAi, indicating that  fat-7  hinders the expression of  ech-1  and  acs-2  through a mechanism that is independent of  nhr-49  (see  Figure 5 B). Furthermore, RNAi of  fat-7  reduced fat content in  nhr-49(nr2041)  worms, demonstrating that the stimulation of fat storage by  fat-7  is also not dependent upon  nhr-49  (see  Figure 5 D). Taken together, our findings demonstrate that  nhr-49  controls two distinct circuits within a gene network: The stimulation of β-oxidation gene expression is independent of  fat-7,  and the promotion of  fat-7  expression is independent of  nhr-49 's effects on β-oxidation. However, because  fat-7  expression inhibits the same β-oxidation genes induced by  nhr-49,  the regulation of  fat-7  by  nhr-49  connects these two circuits into a single regulatory network, serving to limit β-oxidation through an apparent feedback mechanism ( Figure 6 ).
Stimulation of  fat-7  by  nhr-49  Feeds Back to Inhibit β-Oxidation
fat-7nhr-49By promoting β-oxidation gene expression,  nhr-49  stimulates fat consumption, and by enhancing  fat-7  expression,  nhr-49  modulates fat desaturation and ensures normal longevity. We next set out to determine whether  nhr-49  regulates these two pathways independently.nhr-49fat-7nhr-49nhr-49Although RNAi of  acs-2  or  ech-1  was sufficient to cause a high-fat phenotype,  acs-2  or  ech-1  interference did not significantly impact life span, fatty acid composition, or desaturase gene expression, demonstrating that the effects of  nhr-49  deletion on  fat-7  expression and life span were not a downstream consequence of compromised β-oxidation or increased fat content (see  Table 1 ).acs-2ech-1acs-2ech-1nhr-49fat-7In contrast, RNAi of  fat-7  significantly impacted fat storage and β-oxidation. The effect was paradoxical, however, as  fat-7  interference produced the opposite result of  nhr-49  deletion, causing  reduced  fat content and  increased  expression of genes in β-oxidation, including  acs-2  and  ech-1  ( Figure 5 ). As the influence of  fat-7  on the expression of β-oxidation genes is opposite that of  nhr-49,  it is clear that the stimulation of β-oxidation by  nhr-49  does not result from promotion of  fat-7  expression. In fact, by enhancing  fat-7  expression,  nhr-49  indirectly causes inhibition of the very same β-oxidation genes that it is independently stimulating ( Figure 6 ).fat-7fat-7nhr-49reducedincreasedacs-2ech-1fat-7nhr-49,nhr-49fat-7fat-7nhr-49Because  fat-7  targets some of the same β-oxidation genes as  nhr-49,  we wondered if the effects of  fat-7  on fat storage might be dependent on  nhr-49;  for example, perhaps  fat-7  represses  ech-1  and  acs-2  expression by blocking the  nhr-49  mediated induction of these genes. However, we found that both  ech-1  and  acs-2  were induced when  fat-7  was knocked down even further in the  nhr-49(nr2041)  mutant using RNAi, indicating that  fat-7  hinders the expression of  ech-1  and  acs-2  through a mechanism that is independent of  nhr-49  (see  Figure 5 B). Furthermore, RNAi of  fat-7  reduced fat content in  nhr-49(nr2041)  worms, demonstrating that the stimulation of fat storage by  fat-7  is also not dependent upon  nhr-49  (see  Figure 5 D).fat-7nhr-49,fat-7nhr-49;fat-7ech-1acs-2nhr-49ech-1acs-2fat-7nhr-49(nr2041)fat-7ech-1acs-2nhr-49fat-7nhr-49(nr2041)fat-7nhr-49Taken together, our findings demonstrate that  nhr-49  controls two distinct circuits within a gene network: The stimulation of β-oxidation gene expression is independent of  fat-7,  and the promotion of  fat-7  expression is independent of  nhr-49 's effects on β-oxidation. However, because  fat-7  expression inhibits the same β-oxidation genes induced by  nhr-49,  the regulation of  fat-7  by  nhr-49  connects these two circuits into a single regulatory network, serving to limit β-oxidation through an apparent feedback mechanism ( Figure 6 ).nhr-49fat-7,fat-7nhr-49fat-7nhr-49,fat-7nhr-49Discussion Here we demonstrated that deletion of the  C. elegans nhr-49  gene produced two global phenotypes: shortened life span and high fat content. Using a QRT-PCR strategy, we found 13 genes involved in energy metabolism that depend upon  nhr-49  for expression. The phenotypes of  nhr-49(nr2041)  were attributable to deficiencies in two different metabolic pathways, fatty acid β-oxidation and fatty acid desaturation. The high-fat phenotype is explained by lowered expression of β-oxidation enzymes, and the shortened life-span phenotype results from reduced expression of the  fat-7  SCD. Interestingly, these pathways are linked by an apparent feedback mechanism, as  fat-7  also serves to inhibit the expression of fatty acid β-oxidation genes ( Figure 6 ). These results represent the first detailed characterization of a divergent HNF4-like receptor in  C. elegans  and provide the first description of an NHR-controlled fat-regulatory network in invertebrates. Due to their evolutionary relationship to  nhr-49,  it seems conceivable that many of the  C. elegans  HNF4-like NHRs might participate in the regulation of fat metabolism. However, we have found that several other worm HNF4-like receptors, including those previously implicated for affecting overall fat storage [ 12 ], do not significantly impact the expression of fatty acid metabolism genes (M. R. Van Gilst and K. R. Yamamoto, unpublished data). Thus, the regulation of fat metabolism does not appear to be a general mechanism common to all of the divergent NHRs; whether or not  nhr-49  is unique in its control of  C. elegans  fat metabolism remains to be determined. The mechanism by which  nhr-49  influences fat storage is likely to be through modulation of β-oxidation gene expression ( Figure 6 ). Overexpression of  acs-2  alone was sufficient to suppress the high-fat phenotype of  nhr-49(nr2041),  indicating that  acs-2  expression is rate-limiting in  nhr-49  deletion mutants, thus leading to reduced fat breakdown and excessive fat accumulation. Acyl-CoA synthetases activate fatty acids for many different metabolic pathways; in particular, mitochondrial acyl-CoA synthetases are likely to activate fatty acids for transport into the mitochondrial matrix, where the fatty acids are then subject to β-oxidation [ 18 , 19 ]. Notably, mitochondrial acyl-CoA synthetases are commonly regulatory targets for factors that control fat consumption [ 18 ]. We propose that by stimulating  acs-2  expression,  nhr-49  likely acts by increasing the flow of fatty acids into the mitochondria for β-oxidation ( Figure 6 ). Out of 11  acs  genes in  C. elegans,  five of which are predicted to be mitochondrial,  nhr-49  specifically affected the expression of only  acs-2,  suggesting that  nhr-49  may be promoting transport of a specific subset of fatty acids into mitochondria. These subsets may differ by fatty acid chain length or tissue distribution. As both  acs-2  and  nhr-49  are expressed in numerous  C. elegans  tissues, regulation of fat consumption by  nhr-49  may occur throughout the animal. Although  acs-2  is related (with approximately 25% identity) to multiple mammalian acyl-CoA synthetases, mammals contain a particular homolog with approximately 41% identity to the  C. elegans  ACS-2 protein; this mammalian ACS protein is yet to be characterized. It will be interesting to determine whether the mammalian counterpart of  acs-2  also serves as an important focal point for gene networks that regulate fat utilization in mammals. Interestingly, we also found that  nhr-49  stimulates expression of a carnitine palmitoyl transferase (F09F3.9). Carnitine palmitoyl transferases execute the step immediately downstream of acyl-CoA synthetase in mitochondrial β-oxidation, directly shuttling activated acyl-CoAs into the mitochondrial matrix. Thus,  nhr-49  appears to target multiple enzymes involved in transporting fatty acids across the mitochondrial membrane for β-oxidation ( Figure 6 ). RNAi of F09F3.9 did not detectably alter overall fat content, however, suggesting that interference of F09F3.9 expression alone is not sufficient to affect overall fat consumption under the conditions surveyed in our study. In contrast, we found that RNAi of  ech-1,  another β-oxidation gene targeted by  nhr-49,  was sufficient to increase overall fat content; thus it is likely that the reduced expression of  ech-1  also contributes, in some fashion, to the high-fat phenotype of  nhr-49(nr2041).  Thus, we have demonstrated that  nhr-49  strongly promotes the expression of three separate enzymes predicted to participate in mitochondrial β-oxidation. Consequently, we suggest that  nhr-49  plays a prominent role in promoting the breakdown of fatty acids in mitochondria, and that, through control of β-oxidation,  nhr-49  influences overall fat storage. In addition to its effect on β-oxidation, we found that  nhr-49  helps to maintain the balance of saturated and monounsaturated fatty acids. By stimulating the expression of three fatty acid Δ9-desaturase genes,  nhr-49  facilitates the conversion of saturated fatty acids to monounsaturated fatty acids, and consequently,  nhr-49(nr2041)  mutant animals are high in saturated fat and lower in monounsaturated fat. In  C. elegans,  the FAT-6 and FAT-7 SCDs may also be involved in the synthesis of PUFAs [ 17 ]. However, a dramatic reduction in  fat-7  expression, either by  nhr-49(nr2041)  deletion or by  fat-7  RNAi, did not significantly affect PUFA synthesis. This result suggests that a normal level of FAT-7 expression is not necessary for PUFA synthesis, likely because FAT-6 is the principle enzyme in this pathway, or because FAT-6 is capable of substituting for reduced FAT-7 activity. In either event, we propose that the primary effect of  nhr-49  on fatty acid composition is a modulation of the ratio of C18:0 to C18:1n9 fatty acid ( Figure 6 ). However, we cannot rule out the possibility that  nhr-49  and  fat-7  also affect PUFA synthesis in a more localized manner, for example, specific cell types, such that changes in PUFA abundance are not detected in our whole-worm analysis. Our results also indicate that the short life-span phenotype of  nhr-49(nr2041)  is due, at least in part, to reduced expression of  fat-7.  The strong correlation between the increase in C18:0 saturated fatty acid and the shortening of life span is compelling. Several reports in mammals have described changes in fatty acid composition during aging, and it has been argued that an alteration of fatty acid saturation ratios in mitochondrial membranes may affect the rate of aging [ 20 ]. Additionally, imbalance of fatty acid saturation has also been linked to numerous pathological conditions [ 15 , 16 ]. Although SCDs have not yet been linked to life span in mammals, studies in  C. elegans  have identified  fat-7  as one of the targets of the mechanisms that extend longevity through the insulin pathway [ 21 ]. Thus, it will be interesting to determine if the shortened life-span phenotype of  nhr-49(nr2041)  and  fat-7 RNAi  animals results from an improper ratio of saturated and monounsaturated fats, and if the mechanistic cause of the reduced life span is related to accelerated aging or to another pathology. Although it is clear that  nhr-49  independently promotes the expression of genes in fatty acid β-oxidation and desaturation, we found that downstream components of these pathways do indeed communicate with each other. By inhibiting the expression of genes involved in β-oxidation, including the  nhr-49 -dependent genes  acs-2  and  ech-1, fat-7  works against the actions of  nhr-49,  acting to hinder fat consumption ( Figure 6 ). Thus, our results reveal an elegant feedback mechanism:  nhr-49  induces the expression of β-oxidation enzymes—thus promoting fat consumption—yet by independently stimulating  fat-7  expression,  nhr-49  tempers the expression of the very same β-oxidation genes ( Figure 6 ). Thus, we suggest that  nhr-49  serves as a key regulator of fat usage, governing pathways that consume fat for energy and partition fat for storage, potentially shifting the balance in response to changing energy needs. Indeed, we have found that in response to starvation,  nhr-49  selectively modulates the expression of these two pathways in order to increase fat consumption (M. R. Van Gilst and K. R. Yamamoto, unpublished data). As the predominant function of  fat-7  is to convert C18:0 to C18:1n9 fatty acid, it is tempting to speculate that one of these two fatty acid species serves as a signal to modulate the feedback effect of  fat-7  on fatty acid β-oxidation ( Figure 6 ). In this scenario, C18:0 could be serving as an activator of β-oxidation gene expression, C18:1n9 could be functioning as a repressor, or both ( Figure 6 ). Although it seems reasonable to propose that an NHR would communicate this fatty acid signal, we found that the effects of  fat-7  on fatty acid β-oxidation were not dependent on  nhr-49;  hence, one of the other 283  C. elegans  NHRs may mediate this effect. Despite its strong sequence similarity with the mammalian HNF4 receptors, the overall biological effects of  nhr-49  on metabolism, fat storage, and life span are remarkably similar to those of the mammalian PPARs. Perhaps most striking is the finding that  nhr-49  and the PPARs, particularly PPARα and PPARδ, positively influence similar genes in multiple metabolic processes, including fatty acid β-oxidation, fatty acid desaturation, and fatty acid binding/transport [ 22 , 23 ]. Moreover, knockout of PPARα or PPARδ can lead to high-fat phenotypes [ 23 , 24 ], demonstrating that the physiological effects of these receptors on fat storage are similar to those of  nhr-49.
 The regulation of SCDs by  nhr-49  also parallels the regulation of the SCD1 gene by PPARα. In mammals, the Δ9-desaturase SCD1 is a lipogenic enzyme that is controlled by several key regulators of fat storage, including PPARα, leptin, and sterol response element-binding protein [ 15 , 25 , 26 ]. When SCD1 expression is left unchecked, as occurs in leptin knockout mice, severe obesity can result. Similar to what we found for the  C. elegans fat-7  gene, mammalian SCD1 affects fat storage by inhibiting β-oxidation [ 27 ]. Thus, because PPARα also promotes fat consumption by stimulating β-oxidation gene expression[ 28 ], it is clear that PPARα is independently regulating pathways that promote fat consumption and stimulate fat storage. In fact, a similar feedback mechanism to what we have described here for  nhr-49  has been proposed for PPARα and SCD1 [ 27 , 29 ]. Taken together, these results demonstrate that the multicircuited regulatory networks governing fat usage appear well conserved between  C. elegans nhr-49  and the mammalian PPARα. Although we have not yet determined whether or not the  nhr-49 -dependent genes identified in this study are direct transcriptional targets, each is homologous to known transcriptional targets of the PPARs [ 22 , 23 ]. Because NHR-49 and the PPARs are clearly regulating similar physiological processes and gene networks, we believe that they are likely to be using similar molecular mechanisms, including direct transcriptional regulation. However, it is possible that  nhr-49  controls fatty acid β-oxidation and desaturation through an indirect mechanism, functioning upstream of the transcription factors that directly act on the  nhr-49 -dependent genes identified in this study. The latter possibility would also be intriguing, as it would demonstrate NHR involvement in fat regulation at a point upstream of a PPAR-like factor. As the physiological functions and regulatory targets of  nhr-49  are highly similar to those of the mammalian PPARs, it seems reasonable to speculate that, like the PPARs, NHR-49 may be regulated by fatty acid ligands. By binding directly to ingested lipids and/or their metabolic products, NHR-49 could serve both as a sensor of intracellular fatty acid levels and an effector, responding to changes in fatty acid composition through modulation of fatty acid β-oxidation and desaturation. Interestingly, the mammalian HNF4 receptors, whose LBDs share 33% identity with the NHR-49 LBD, can bind to several saturated and monounsaturated fatty acids, including stearic and oleic acid [ 30 , 31 ]; the mammalian PPARs also bind to many types of fatty acids, although they interact preferentially with PUFAs [ 22 ]. It will be interesting to determine if mammalian HNF4 receptors harbor an undiscovered capacity to regulate fat metabolism in mammals, or if PPARs have adopted their regulatory functions by evolving from an ancestral HNF4 gene that had properties similar to  nhr-49  and the modern PPARs. Alternatively, the PPARs and  nhr-49  may have acquired their activities through convergent evolution. Precise regulation of fatty acid β-oxidation and desaturation is crucial for physiological homeostasis; imbalance in these metabolic pathways in humans can lead to disorders such as diabetes, obesity, atherosclerosis, and accelerated aging. However, although NHRs are important targets for drugs that moderate these metabolic disorders, the NHR-mediated regulatory networks that govern fat usage are notably complex and poorly understood. We showed here that many features of  C. elegans nhr-49  regulation of fat metabolism are closely analogous to those of the mammalian PPARs, key targets for the treatment of metabolic disease. Moreover, our  C. elegans  studies revealed that  nhr-49,  like PPARα, oversees a branch point for two separate circuits within a network that controls fatty acid consumption and composition. The extent of the functional homology between  nhr-49  and PPARα remains to be determined, that is, whether or not  nhr-49  also functions by binding to fatty acid ligands and directly regulating gene transcription. Nevertheless, our finding that  nhr-49  is a key regulator of fat metabolism in  C. elegans  represents a considerable advance in our understanding of NHR control of fat storage and composition in invertebrates. Consequently, we suggest that these and future studies in  C. elegans  will continue to enhance our understanding of the complex roles of NHRs and their ligands on gene networks governing fat physiology.
Discussion
Here we demonstrated that deletion of the  C. elegans nhr-49  gene produced two global phenotypes: shortened life span and high fat content. Using a QRT-PCR strategy, we found 13 genes involved in energy metabolism that depend upon  nhr-49  for expression. The phenotypes of  nhr-49(nr2041)  were attributable to deficiencies in two different metabolic pathways, fatty acid β-oxidation and fatty acid desaturation. The high-fat phenotype is explained by lowered expression of β-oxidation enzymes, and the shortened life-span phenotype results from reduced expression of the  fat-7  SCD. Interestingly, these pathways are linked by an apparent feedback mechanism, as  fat-7  also serves to inhibit the expression of fatty acid β-oxidation genes ( Figure 6 ).C. elegans nhr-49nhr-49nhr-49(nr2041)fat-7fat-7These results represent the first detailed characterization of a divergent HNF4-like receptor in  C. elegans  and provide the first description of an NHR-controlled fat-regulatory network in invertebrates. Due to their evolutionary relationship to  nhr-49,  it seems conceivable that many of the  C. elegans  HNF4-like NHRs might participate in the regulation of fat metabolism. However, we have found that several other worm HNF4-like receptors, including those previously implicated for affecting overall fat storage [ 12 ], do not significantly impact the expression of fatty acid metabolism genes (M. R. Van Gilst and K. R. Yamamoto, unpublished data). Thus, the regulation of fat metabolism does not appear to be a general mechanism common to all of the divergent NHRs; whether or not  nhr-49  is unique in its control of  C. elegans  fat metabolism remains to be determined.nhr-49,nhr-49The mechanism by which  nhr-49  influences fat storage is likely to be through modulation of β-oxidation gene expression ( Figure 6 ). Overexpression of  acs-2  alone was sufficient to suppress the high-fat phenotype of  nhr-49(nr2041),  indicating that  acs-2  expression is rate-limiting in  nhr-49  deletion mutants, thus leading to reduced fat breakdown and excessive fat accumulation. Acyl-CoA synthetases activate fatty acids for many different metabolic pathways; in particular, mitochondrial acyl-CoA synthetases are likely to activate fatty acids for transport into the mitochondrial matrix, where the fatty acids are then subject to β-oxidation [ 18 , 19 ]. Notably, mitochondrial acyl-CoA synthetases are commonly regulatory targets for factors that control fat consumption [ 18 ]. We propose that by stimulating  acs-2  expression,  nhr-49  likely acts by increasing the flow of fatty acids into the mitochondria for β-oxidation ( Figure 6 ).nhr-49acs-2nhr-49(nr2041),acs-2nhr-49acs-2nhr-49Out of 11  acs  genes in  C. elegans,  five of which are predicted to be mitochondrial,  nhr-49  specifically affected the expression of only  acs-2,  suggesting that  nhr-49  may be promoting transport of a specific subset of fatty acids into mitochondria. These subsets may differ by fatty acid chain length or tissue distribution. As both  acs-2  and  nhr-49  are expressed in numerous  C. elegans  tissues, regulation of fat consumption by  nhr-49  may occur throughout the animal.acsC. elegans,nhr-49acs-2,nhr-49acs-2nhr-49nhr-49Although  acs-2  is related (with approximately 25% identity) to multiple mammalian acyl-CoA synthetases, mammals contain a particular homolog with approximately 41% identity to the  C. elegans  ACS-2 protein; this mammalian ACS protein is yet to be characterized. It will be interesting to determine whether the mammalian counterpart of  acs-2  also serves as an important focal point for gene networks that regulate fat utilization in mammals.acs-2acs-2Interestingly, we also found that  nhr-49  stimulates expression of a carnitine palmitoyl transferase (F09F3.9). Carnitine palmitoyl transferases execute the step immediately downstream of acyl-CoA synthetase in mitochondrial β-oxidation, directly shuttling activated acyl-CoAs into the mitochondrial matrix. Thus,  nhr-49  appears to target multiple enzymes involved in transporting fatty acids across the mitochondrial membrane for β-oxidation ( Figure 6 ). RNAi of F09F3.9 did not detectably alter overall fat content, however, suggesting that interference of F09F3.9 expression alone is not sufficient to affect overall fat consumption under the conditions surveyed in our study.nhr-49nhr-49In contrast, we found that RNAi of  ech-1,  another β-oxidation gene targeted by  nhr-49,  was sufficient to increase overall fat content; thus it is likely that the reduced expression of  ech-1  also contributes, in some fashion, to the high-fat phenotype of  nhr-49(nr2041).  Thus, we have demonstrated that  nhr-49  strongly promotes the expression of three separate enzymes predicted to participate in mitochondrial β-oxidation. Consequently, we suggest that  nhr-49  plays a prominent role in promoting the breakdown of fatty acids in mitochondria, and that, through control of β-oxidation,  nhr-49  influences overall fat storage.ech-1,nhr-49,ech-1nhr-49(nr2041).nhr-49nhr-49nhr-49In addition to its effect on β-oxidation, we found that  nhr-49  helps to maintain the balance of saturated and monounsaturated fatty acids. By stimulating the expression of three fatty acid Δ9-desaturase genes,  nhr-49  facilitates the conversion of saturated fatty acids to monounsaturated fatty acids, and consequently,  nhr-49(nr2041)  mutant animals are high in saturated fat and lower in monounsaturated fat. In  C. elegans,  the FAT-6 and FAT-7 SCDs may also be involved in the synthesis of PUFAs [ 17 ]. However, a dramatic reduction in  fat-7  expression, either by  nhr-49(nr2041)  deletion or by  fat-7  RNAi, did not significantly affect PUFA synthesis. This result suggests that a normal level of FAT-7 expression is not necessary for PUFA synthesis, likely because FAT-6 is the principle enzyme in this pathway, or because FAT-6 is capable of substituting for reduced FAT-7 activity. In either event, we propose that the primary effect of  nhr-49  on fatty acid composition is a modulation of the ratio of C18:0 to C18:1n9 fatty acid ( Figure 6 ). However, we cannot rule out the possibility that  nhr-49  and  fat-7  also affect PUFA synthesis in a more localized manner, for example, specific cell types, such that changes in PUFA abundance are not detected in our whole-worm analysis.nhr-49nhr-49nhr-49(nr2041)C. elegans,fat-7nhr-49(nr2041)fat-7nhr-49nhr-49fat-7Our results also indicate that the short life-span phenotype of  nhr-49(nr2041)  is due, at least in part, to reduced expression of  fat-7.  The strong correlation between the increase in C18:0 saturated fatty acid and the shortening of life span is compelling. Several reports in mammals have described changes in fatty acid composition during aging, and it has been argued that an alteration of fatty acid saturation ratios in mitochondrial membranes may affect the rate of aging [ 20 ]. Additionally, imbalance of fatty acid saturation has also been linked to numerous pathological conditions [ 15 , 16 ]. Although SCDs have not yet been linked to life span in mammals, studies in  C. elegans  have identified  fat-7  as one of the targets of the mechanisms that extend longevity through the insulin pathway [ 21 ]. Thus, it will be interesting to determine if the shortened life-span phenotype of  nhr-49(nr2041)  and  fat-7 RNAi  animals results from an improper ratio of saturated and monounsaturated fats, and if the mechanistic cause of the reduced life span is related to accelerated aging or to another pathology.nhr-49(nr2041)fat-7.fat-7nhr-49(nr2041)fat-7 RNAiAlthough it is clear that  nhr-49  independently promotes the expression of genes in fatty acid β-oxidation and desaturation, we found that downstream components of these pathways do indeed communicate with each other. By inhibiting the expression of genes involved in β-oxidation, including the  nhr-49 -dependent genes  acs-2  and  ech-1, fat-7  works against the actions of  nhr-49,  acting to hinder fat consumption ( Figure 6 ). Thus, our results reveal an elegant feedback mechanism:  nhr-49  induces the expression of β-oxidation enzymes—thus promoting fat consumption—yet by independently stimulating  fat-7  expression,  nhr-49  tempers the expression of the very same β-oxidation genes ( Figure 6 ). Thus, we suggest that  nhr-49  serves as a key regulator of fat usage, governing pathways that consume fat for energy and partition fat for storage, potentially shifting the balance in response to changing energy needs. Indeed, we have found that in response to starvation,  nhr-49  selectively modulates the expression of these two pathways in order to increase fat consumption (M. R. Van Gilst and K. R. Yamamoto, unpublished data).nhr-49nhr-49acs-2ech-1, fat-7nhr-49,nhr-49fat-7nhr-49nhr-49nhr-49As the predominant function of  fat-7  is to convert C18:0 to C18:1n9 fatty acid, it is tempting to speculate that one of these two fatty acid species serves as a signal to modulate the feedback effect of  fat-7  on fatty acid β-oxidation ( Figure 6 ). In this scenario, C18:0 could be serving as an activator of β-oxidation gene expression, C18:1n9 could be functioning as a repressor, or both ( Figure 6 ). Although it seems reasonable to propose that an NHR would communicate this fatty acid signal, we found that the effects of  fat-7  on fatty acid β-oxidation were not dependent on  nhr-49;  hence, one of the other 283  C. elegans  NHRs may mediate this effect.fat-7fat-7fat-7nhr-49;Despite its strong sequence similarity with the mammalian HNF4 receptors, the overall biological effects of  nhr-49  on metabolism, fat storage, and life span are remarkably similar to those of the mammalian PPARs. Perhaps most striking is the finding that  nhr-49  and the PPARs, particularly PPARα and PPARδ, positively influence similar genes in multiple metabolic processes, including fatty acid β-oxidation, fatty acid desaturation, and fatty acid binding/transport [ 22 , 23 ]. Moreover, knockout of PPARα or PPARδ can lead to high-fat phenotypes [ 23 , 24 ], demonstrating that the physiological effects of these receptors on fat storage are similar to those of  nhr-49.nhr-49nhr-49nhr-49.The regulation of SCDs by  nhr-49  also parallels the regulation of the SCD1 gene by PPARα. In mammals, the Δ9-desaturase SCD1 is a lipogenic enzyme that is controlled by several key regulators of fat storage, including PPARα, leptin, and sterol response element-binding protein [ 15 , 25 , 26 ]. When SCD1 expression is left unchecked, as occurs in leptin knockout mice, severe obesity can result. Similar to what we found for the  C. elegans fat-7  gene, mammalian SCD1 affects fat storage by inhibiting β-oxidation [ 27 ]. Thus, because PPARα also promotes fat consumption by stimulating β-oxidation gene expression[ 28 ], it is clear that PPARα is independently regulating pathways that promote fat consumption and stimulate fat storage. In fact, a similar feedback mechanism to what we have described here for  nhr-49  has been proposed for PPARα and SCD1 [ 27 , 29 ]. Taken together, these results demonstrate that the multicircuited regulatory networks governing fat usage appear well conserved between  C. elegans nhr-49  and the mammalian PPARα.nhr-49C. elegans fat-7nhr-49C. elegans nhr-49Although we have not yet determined whether or not the  nhr-49 -dependent genes identified in this study are direct transcriptional targets, each is homologous to known transcriptional targets of the PPARs [ 22 , 23 ]. Because NHR-49 and the PPARs are clearly regulating similar physiological processes and gene networks, we believe that they are likely to be using similar molecular mechanisms, including direct transcriptional regulation. However, it is possible that  nhr-49  controls fatty acid β-oxidation and desaturation through an indirect mechanism, functioning upstream of the transcription factors that directly act on the  nhr-49 -dependent genes identified in this study. The latter possibility would also be intriguing, as it would demonstrate NHR involvement in fat regulation at a point upstream of a PPAR-like factor.nhr-49nhr-49nhr-49As the physiological functions and regulatory targets of  nhr-49  are highly similar to those of the mammalian PPARs, it seems reasonable to speculate that, like the PPARs, NHR-49 may be regulated by fatty acid ligands. By binding directly to ingested lipids and/or their metabolic products, NHR-49 could serve both as a sensor of intracellular fatty acid levels and an effector, responding to changes in fatty acid composition through modulation of fatty acid β-oxidation and desaturation. Interestingly, the mammalian HNF4 receptors, whose LBDs share 33% identity with the NHR-49 LBD, can bind to several saturated and monounsaturated fatty acids, including stearic and oleic acid [ 30 , 31 ]; the mammalian PPARs also bind to many types of fatty acids, although they interact preferentially with PUFAs [ 22 ]. It will be interesting to determine if mammalian HNF4 receptors harbor an undiscovered capacity to regulate fat metabolism in mammals, or if PPARs have adopted their regulatory functions by evolving from an ancestral HNF4 gene that had properties similar to  nhr-49  and the modern PPARs. Alternatively, the PPARs and  nhr-49  may have acquired their activities through convergent evolution.nhr-49nhr-49nhr-49Precise regulation of fatty acid β-oxidation and desaturation is crucial for physiological homeostasis; imbalance in these metabolic pathways in humans can lead to disorders such as diabetes, obesity, atherosclerosis, and accelerated aging. However, although NHRs are important targets for drugs that moderate these metabolic disorders, the NHR-mediated regulatory networks that govern fat usage are notably complex and poorly understood. We showed here that many features of  C. elegans nhr-49  regulation of fat metabolism are closely analogous to those of the mammalian PPARs, key targets for the treatment of metabolic disease. Moreover, our  C. elegans  studies revealed that  nhr-49,  like PPARα, oversees a branch point for two separate circuits within a network that controls fatty acid consumption and composition.C. elegans nhr-49nhr-49,The extent of the functional homology between  nhr-49  and PPARα remains to be determined, that is, whether or not  nhr-49  also functions by binding to fatty acid ligands and directly regulating gene transcription. Nevertheless, our finding that  nhr-49  is a key regulator of fat metabolism in  C. elegans  represents a considerable advance in our understanding of NHR control of fat storage and composition in invertebrates. Consequently, we suggest that these and future studies in  C. elegans  will continue to enhance our understanding of the complex roles of NHRs and their ligands on gene networks governing fat physiology.nhr-49nhr-49nhr-49Materials and Methods RNAi constructs All of the RNAi constructs used in the NHR screen were created by cloning full-length NHR cDNAs into the vector L4440 (Andy Fire, Stanford University). The  fat-7  RNAi vector was a gift from Jennifer Watts (Washington State University). All of the other RNAi constructs were obtained from the Ahringer RNAi library [ 32 ]. Life span assays Approximately 30–40 starved L1 worms were transferred to RNAi bacteria (HT115 transformed with RNAi clone) and life-span assays were carried out at 23 °C as described previously [ 33 ].  nhr-49(nr2041)  and WT life-span assays were carried out at 23 °C on OP50 bacteria. Nile Red assays Nile Red fat-staining assays were carried out as described previously [ 12 ]. Selection of fatty acid and glucose metabolism genes Most of the fatty acid and glucose metabolism candidate genes examined in this study were initially identified using the KEGG pathway database ( http://www.genome.ad.jp/kegg/pathway.html ). Other genes were identified using BLAST searches designed to find  C. elegans  open reading frames that were highly related to known mammalian glucose and fat metabolism enzymes, or to plant glyoxylate cycle genes. For prediction of β-oxidation enzyme subcellular localization, we used the following online prediction tools. TargetP server version 1.01 ( http://www.cbs.dtu.dk/services/TargetP/ ) [ 34 ] and PSORT ( http://psort.nibb.ac.jp/form.html ). Preparation of nematode total RNA Both  C. elegans  N2-Bristol (WT) and  nhr-49(nr2041)  were grown at 23 °C on high-growth plates seeded with OP50 bacteria. Gravid adults from 30 10-cm plates were bleached, and embryos were dispersed onto 15-cm nematode growth media (NGM)-lite plates seeded with OP50. The worms were distributed as follows: for L1 harvest, 80,000 embryos/plate; for L2 harvest, 40,000 embryos/plate; for L3 harvest, 20,000 embryos/plate; and for L4 harvest, 10,000 embryos/plate. The remaining embryos were saved for embryonic RNA preparation. Worms were harvested at the appropriate stage, washed twice with M9, and frozen in liquid nitrogen. For RNAi experiments, 10,000 embryos were placed onto NGM-lite plates containing 8 mM IPTG and 100 μg/ml carbenicillin seeded with control bacteria (HT115 transformed with empty L4440 vector) or  nhr-49  RNAi bacteria (HT115 transformed with L4440- nhr-49 ). Worms were harvested at the L4 stage of development, washed twice with M9, and frozen in liquid nitrogen. For RNA preparation, worms were thawed at 65 °C for 10 min, and RNA was isolated using the Tri-Reagent Kit (Molecular Research Center, Cincinnati, Ohio, United States). Isolated total RNA was subjected to DNAase treatment and further purification using RNAeasy (Qiagen, Valencia, California, United States). QRT-PCR cDNA was prepared from 5 μg of total RNA in a 100-μl reaction using the Protoscript cDNA preparation kit (New England Biolabs, Beverly, Massachusetts, United States). Primer pairs (primer sequences available upon request) were diluted into 96-well cell culture plates at a concentration of 3 μM. Next, 30-μl PCR reactions were prepared in 96-well plates. Each PCR reaction was carried out with  Taq DNA Polymerase (Invitrogen, Carlsbad, California, United States) and consisted of the following reaction mixture: 0.3 μM primers, 1/500th of the cDNA reaction (corresponds to cDNA derived from 10 ng of total RNA), 125 μM dNTPs, 1.5 mM MgCl 2 , and 1X reaction buffer (20 mM Tris pH 8.4, 50 mM KCl). 0.15 μl (0.75 units) of  Taq DNA Polymerase was used for each reaction. Formation of double-stranded DNA product was monitored using SYBR-Green (Molecular Probes, Eugene, Oregon, United States). All QRT-PCR reactions were carried out and analyzed on a DNA Engine-Opticon 2 (MJ Research, Waltham, Massachusetts, United States). Data were collected using RNA from at least three independent  C. elegans  growths. To determine the relationship between mRNA abundance and PCR cycle number, all primer sets were calibrated using serial dilutions of cDNA preparations. Primer sets were also calibrated by performing QRT-PCR reactions on serial dilutions of  C. elegans  genomic DNA. Relative abundance is reported as the mRNA abundance of each gene relative to the mRNA abundance of several control genes, which are expressed at constant levels throughout development. GC/MS Fatty acids were isolated from 10,000 L4 animals grown on a single 15-cm NGM-Lite plate. For WT and  nhr-49(nr2041)  experiments, worms were grown at 23 °C on a lawn of OP50. For RNAi, 10,000 embryos were placed on control bacteria, or on  fat-5, fat-7, ech-1, acs-2,  or  nhr-49  RNAi bacteria. Fatty acid extract was prepared as described previously [ 17 ]. GC/MS spectra were collected on an HP 6890 gas chromatograph outfitted with a J&W DB-XLB column. The mass spectrometer was an HP MSD 5973 (Agilent, Palo Alto, California, United States) and data were analyzed using Chemstation version A.03.00 software (Agilent). Peaks were assigned using fatty acid standards. GFP reporter analysis and ectopic expression of ACS-2::GFP To make the P nhr-49 GFP plasmid, a genomic fragment including 3.5 kb of the  nhr-49  upstream sequence and 88 bp of the first exon of  nhr-49  was ligated into the L3691 GFP expression vector (a gift of A. Fire). Germ-line transformation was performed following standard procedures [ 35 ]. P nhr-49 GFP was injected at the following concentrations: 1 ng/μl, 5 ng/μl and 20 ng/μl along with the co-injection marker pRF4  rol-6(su1006)  injected at 60 ng/μl. At all three P nhr-49 GFP concentrations, we were unable to obtain stably transmitting lines. Animals used for GFP imaging were derived from the F1 generation and were mosaic, hosting the P nhr-49 GFP transgene in only a subset of tissues. To construct  acs-2::gfp,  a 4-kb genomic fragment, including the full-length  acs-2  gene plus 2 kb of upstream sequence, was cloned into the vector L3691. Germ-line transformation was performed using 50 ng/μl of  acs:2::gfp  and 50 ng/μl pRF4  rol-6(su1006).  For N2/ acs-2::gfp  and  nhr-49(nr2041)/acs-2::gfp,  over ten independent transmitting lines were obtained and analyzed by GFP microscopy. GFP reporter expression was observed using a Zeiss Axiovert S100 fluorescence microscope (Carl Zeiss, Thornwood, New York, United States) under a 40X objective. For rescue experiments, 50–100 ng/μl of  acs-2::gfp  was injected into  nhr-49(nr2041)  and WT animals along with 50 ng/μl of pRF4 rol-6(su1006). Transgenic adults, selected for rolling behavior, were placed on Nile Red plates and assayed for fat content as described above. pRF4  rol-6(su1006)  was injected into  nhr-49(nr2041)  in combination with unrelated plasmids to demonstrate that the lowered fat content was not a result of hosting a transgenic array or caused by expression of the  rol-6(su1006)  marker gene.
Materials and Methods
RNAi constructs All of the RNAi constructs used in the NHR screen were created by cloning full-length NHR cDNAs into the vector L4440 (Andy Fire, Stanford University). The  fat-7  RNAi vector was a gift from Jennifer Watts (Washington State University). All of the other RNAi constructs were obtained from the Ahringer RNAi library [ 32 ]. Life span assays Approximately 30–40 starved L1 worms were transferred to RNAi bacteria (HT115 transformed with RNAi clone) and life-span assays were carried out at 23 °C as described previously [ 33 ].  nhr-49(nr2041)  and WT life-span assays were carried out at 23 °C on OP50 bacteria. Nile Red assays Nile Red fat-staining assays were carried out as described previously [ 12 ]. Selection of fatty acid and glucose metabolism genes Most of the fatty acid and glucose metabolism candidate genes examined in this study were initially identified using the KEGG pathway database ( http://www.genome.ad.jp/kegg/pathway.html ). Other genes were identified using BLAST searches designed to find  C. elegans  open reading frames that were highly related to known mammalian glucose and fat metabolism enzymes, or to plant glyoxylate cycle genes. For prediction of β-oxidation enzyme subcellular localization, we used the following online prediction tools. TargetP server version 1.01 ( http://www.cbs.dtu.dk/services/TargetP/ ) [ 34 ] and PSORT ( http://psort.nibb.ac.jp/form.html ). Preparation of nematode total RNA Both  C. elegans  N2-Bristol (WT) and  nhr-49(nr2041)  were grown at 23 °C on high-growth plates seeded with OP50 bacteria. Gravid adults from 30 10-cm plates were bleached, and embryos were dispersed onto 15-cm nematode growth media (NGM)-lite plates seeded with OP50. The worms were distributed as follows: for L1 harvest, 80,000 embryos/plate; for L2 harvest, 40,000 embryos/plate; for L3 harvest, 20,000 embryos/plate; and for L4 harvest, 10,000 embryos/plate. The remaining embryos were saved for embryonic RNA preparation. Worms were harvested at the appropriate stage, washed twice with M9, and frozen in liquid nitrogen. For RNAi experiments, 10,000 embryos were placed onto NGM-lite plates containing 8 mM IPTG and 100 μg/ml carbenicillin seeded with control bacteria (HT115 transformed with empty L4440 vector) or  nhr-49  RNAi bacteria (HT115 transformed with L4440- nhr-49 ). Worms were harvested at the L4 stage of development, washed twice with M9, and frozen in liquid nitrogen. For RNA preparation, worms were thawed at 65 °C for 10 min, and RNA was isolated using the Tri-Reagent Kit (Molecular Research Center, Cincinnati, Ohio, United States). Isolated total RNA was subjected to DNAase treatment and further purification using RNAeasy (Qiagen, Valencia, California, United States). QRT-PCR cDNA was prepared from 5 μg of total RNA in a 100-μl reaction using the Protoscript cDNA preparation kit (New England Biolabs, Beverly, Massachusetts, United States). Primer pairs (primer sequences available upon request) were diluted into 96-well cell culture plates at a concentration of 3 μM. Next, 30-μl PCR reactions were prepared in 96-well plates. Each PCR reaction was carried out with  Taq DNA Polymerase (Invitrogen, Carlsbad, California, United States) and consisted of the following reaction mixture: 0.3 μM primers, 1/500th of the cDNA reaction (corresponds to cDNA derived from 10 ng of total RNA), 125 μM dNTPs, 1.5 mM MgCl 2 , and 1X reaction buffer (20 mM Tris pH 8.4, 50 mM KCl). 0.15 μl (0.75 units) of  Taq DNA Polymerase was used for each reaction. Formation of double-stranded DNA product was monitored using SYBR-Green (Molecular Probes, Eugene, Oregon, United States). All QRT-PCR reactions were carried out and analyzed on a DNA Engine-Opticon 2 (MJ Research, Waltham, Massachusetts, United States). Data were collected using RNA from at least three independent  C. elegans  growths. To determine the relationship between mRNA abundance and PCR cycle number, all primer sets were calibrated using serial dilutions of cDNA preparations. Primer sets were also calibrated by performing QRT-PCR reactions on serial dilutions of  C. elegans  genomic DNA. Relative abundance is reported as the mRNA abundance of each gene relative to the mRNA abundance of several control genes, which are expressed at constant levels throughout development. GC/MS Fatty acids were isolated from 10,000 L4 animals grown on a single 15-cm NGM-Lite plate. For WT and  nhr-49(nr2041)  experiments, worms were grown at 23 °C on a lawn of OP50. For RNAi, 10,000 embryos were placed on control bacteria, or on  fat-5, fat-7, ech-1, acs-2,  or  nhr-49  RNAi bacteria. Fatty acid extract was prepared as described previously [ 17 ]. GC/MS spectra were collected on an HP 6890 gas chromatograph outfitted with a J&W DB-XLB column. The mass spectrometer was an HP MSD 5973 (Agilent, Palo Alto, California, United States) and data were analyzed using Chemstation version A.03.00 software (Agilent). Peaks were assigned using fatty acid standards. GFP reporter analysis and ectopic expression of ACS-2::GFP To make the P nhr-49 GFP plasmid, a genomic fragment including 3.5 kb of the  nhr-49  upstream sequence and 88 bp of the first exon of  nhr-49  was ligated into the L3691 GFP expression vector (a gift of A. Fire). Germ-line transformation was performed following standard procedures [ 35 ]. P nhr-49 GFP was injected at the following concentrations: 1 ng/μl, 5 ng/μl and 20 ng/μl along with the co-injection marker pRF4  rol-6(su1006)  injected at 60 ng/μl. At all three P nhr-49 GFP concentrations, we were unable to obtain stably transmitting lines. Animals used for GFP imaging were derived from the F1 generation and were mosaic, hosting the P nhr-49 GFP transgene in only a subset of tissues. To construct  acs-2::gfp,  a 4-kb genomic fragment, including the full-length  acs-2  gene plus 2 kb of upstream sequence, was cloned into the vector L3691. Germ-line transformation was performed using 50 ng/μl of  acs:2::gfp  and 50 ng/μl pRF4  rol-6(su1006).  For N2/ acs-2::gfp  and  nhr-49(nr2041)/acs-2::gfp,  over ten independent transmitting lines were obtained and analyzed by GFP microscopy. GFP reporter expression was observed using a Zeiss Axiovert S100 fluorescence microscope (Carl Zeiss, Thornwood, New York, United States) under a 40X objective. For rescue experiments, 50–100 ng/μl of  acs-2::gfp  was injected into  nhr-49(nr2041)  and WT animals along with 50 ng/μl of pRF4 rol-6(su1006). Transgenic adults, selected for rolling behavior, were placed on Nile Red plates and assayed for fat content as described above. pRF4  rol-6(su1006)  was injected into  nhr-49(nr2041)  in combination with unrelated plasmids to demonstrate that the lowered fat content was not a result of hosting a transgenic array or caused by expression of the  rol-6(su1006)  marker gene.

RNAi constructs All of the RNAi constructs used in the NHR screen were created by cloning full-length NHR cDNAs into the vector L4440 (Andy Fire, Stanford University). The  fat-7  RNAi vector was a gift from Jennifer Watts (Washington State University). All of the other RNAi constructs were obtained from the Ahringer RNAi library [ 32 ].
RNAi constructs
All of the RNAi constructs used in the NHR screen were created by cloning full-length NHR cDNAs into the vector L4440 (Andy Fire, Stanford University). The  fat-7  RNAi vector was a gift from Jennifer Watts (Washington State University). All of the other RNAi constructs were obtained from the Ahringer RNAi library [ 32 ].fat-7Life span assays Approximately 30–40 starved L1 worms were transferred to RNAi bacteria (HT115 transformed with RNAi clone) and life-span assays were carried out at 23 °C as described previously [ 33 ].  nhr-49(nr2041)  and WT life-span assays were carried out at 23 °C on OP50 bacteria.
Life span assays
Approximately 30–40 starved L1 worms were transferred to RNAi bacteria (HT115 transformed with RNAi clone) and life-span assays were carried out at 23 °C as described previously [ 33 ].  nhr-49(nr2041)  and WT life-span assays were carried out at 23 °C on OP50 bacteria.nhr-49(nr2041)Nile Red assays Nile Red fat-staining assays were carried out as described previously [ 12 ].
Nile Red assays
Nile Red fat-staining assays were carried out as described previously [ 12 ].Selection of fatty acid and glucose metabolism genes Most of the fatty acid and glucose metabolism candidate genes examined in this study were initially identified using the KEGG pathway database ( http://www.genome.ad.jp/kegg/pathway.html ). Other genes were identified using BLAST searches designed to find  C. elegans  open reading frames that were highly related to known mammalian glucose and fat metabolism enzymes, or to plant glyoxylate cycle genes. For prediction of β-oxidation enzyme subcellular localization, we used the following online prediction tools. TargetP server version 1.01 ( http://www.cbs.dtu.dk/services/TargetP/ ) [ 34 ] and PSORT ( http://psort.nibb.ac.jp/form.html ).
Selection of fatty acid and glucose metabolism genes
Most of the fatty acid and glucose metabolism candidate genes examined in this study were initially identified using the KEGG pathway database ( http://www.genome.ad.jp/kegg/pathway.html ). Other genes were identified using BLAST searches designed to find  C. elegans  open reading frames that were highly related to known mammalian glucose and fat metabolism enzymes, or to plant glyoxylate cycle genes.For prediction of β-oxidation enzyme subcellular localization, we used the following online prediction tools. TargetP server version 1.01 ( http://www.cbs.dtu.dk/services/TargetP/ ) [ 34 ] and PSORT ( http://psort.nibb.ac.jp/form.html ).Preparation of nematode total RNA Both  C. elegans  N2-Bristol (WT) and  nhr-49(nr2041)  were grown at 23 °C on high-growth plates seeded with OP50 bacteria. Gravid adults from 30 10-cm plates were bleached, and embryos were dispersed onto 15-cm nematode growth media (NGM)-lite plates seeded with OP50. The worms were distributed as follows: for L1 harvest, 80,000 embryos/plate; for L2 harvest, 40,000 embryos/plate; for L3 harvest, 20,000 embryos/plate; and for L4 harvest, 10,000 embryos/plate. The remaining embryos were saved for embryonic RNA preparation. Worms were harvested at the appropriate stage, washed twice with M9, and frozen in liquid nitrogen. For RNAi experiments, 10,000 embryos were placed onto NGM-lite plates containing 8 mM IPTG and 100 μg/ml carbenicillin seeded with control bacteria (HT115 transformed with empty L4440 vector) or  nhr-49  RNAi bacteria (HT115 transformed with L4440- nhr-49 ). Worms were harvested at the L4 stage of development, washed twice with M9, and frozen in liquid nitrogen. For RNA preparation, worms were thawed at 65 °C for 10 min, and RNA was isolated using the Tri-Reagent Kit (Molecular Research Center, Cincinnati, Ohio, United States). Isolated total RNA was subjected to DNAase treatment and further purification using RNAeasy (Qiagen, Valencia, California, United States).
Preparation of nematode total RNA
Both  C. elegans  N2-Bristol (WT) and  nhr-49(nr2041)  were grown at 23 °C on high-growth plates seeded with OP50 bacteria. Gravid adults from 30 10-cm plates were bleached, and embryos were dispersed onto 15-cm nematode growth media (NGM)-lite plates seeded with OP50. The worms were distributed as follows: for L1 harvest, 80,000 embryos/plate; for L2 harvest, 40,000 embryos/plate; for L3 harvest, 20,000 embryos/plate; and for L4 harvest, 10,000 embryos/plate. The remaining embryos were saved for embryonic RNA preparation. Worms were harvested at the appropriate stage, washed twice with M9, and frozen in liquid nitrogen. For RNAi experiments, 10,000 embryos were placed onto NGM-lite plates containing 8 mM IPTG and 100 μg/ml carbenicillin seeded with control bacteria (HT115 transformed with empty L4440 vector) or  nhr-49  RNAi bacteria (HT115 transformed with L4440- nhr-49 ). Worms were harvested at the L4 stage of development, washed twice with M9, and frozen in liquid nitrogen. For RNA preparation, worms were thawed at 65 °C for 10 min, and RNA was isolated using the Tri-Reagent Kit (Molecular Research Center, Cincinnati, Ohio, United States). Isolated total RNA was subjected to DNAase treatment and further purification using RNAeasy (Qiagen, Valencia, California, United States).nhr-49(nr2041)nhr-49nhr-49QRT-PCR cDNA was prepared from 5 μg of total RNA in a 100-μl reaction using the Protoscript cDNA preparation kit (New England Biolabs, Beverly, Massachusetts, United States). Primer pairs (primer sequences available upon request) were diluted into 96-well cell culture plates at a concentration of 3 μM. Next, 30-μl PCR reactions were prepared in 96-well plates. Each PCR reaction was carried out with  Taq DNA Polymerase (Invitrogen, Carlsbad, California, United States) and consisted of the following reaction mixture: 0.3 μM primers, 1/500th of the cDNA reaction (corresponds to cDNA derived from 10 ng of total RNA), 125 μM dNTPs, 1.5 mM MgCl 2 , and 1X reaction buffer (20 mM Tris pH 8.4, 50 mM KCl). 0.15 μl (0.75 units) of  Taq DNA Polymerase was used for each reaction. Formation of double-stranded DNA product was monitored using SYBR-Green (Molecular Probes, Eugene, Oregon, United States). All QRT-PCR reactions were carried out and analyzed on a DNA Engine-Opticon 2 (MJ Research, Waltham, Massachusetts, United States). Data were collected using RNA from at least three independent  C. elegans  growths. To determine the relationship between mRNA abundance and PCR cycle number, all primer sets were calibrated using serial dilutions of cDNA preparations. Primer sets were also calibrated by performing QRT-PCR reactions on serial dilutions of  C. elegans  genomic DNA. Relative abundance is reported as the mRNA abundance of each gene relative to the mRNA abundance of several control genes, which are expressed at constant levels throughout development.
QRT-PCR
cDNA was prepared from 5 μg of total RNA in a 100-μl reaction using the Protoscript cDNA preparation kit (New England Biolabs, Beverly, Massachusetts, United States). Primer pairs (primer sequences available upon request) were diluted into 96-well cell culture plates at a concentration of 3 μM. Next, 30-μl PCR reactions were prepared in 96-well plates. Each PCR reaction was carried out with  Taq DNA Polymerase (Invitrogen, Carlsbad, California, United States) and consisted of the following reaction mixture: 0.3 μM primers, 1/500th of the cDNA reaction (corresponds to cDNA derived from 10 ng of total RNA), 125 μM dNTPs, 1.5 mM MgCl 2 , and 1X reaction buffer (20 mM Tris pH 8.4, 50 mM KCl). 0.15 μl (0.75 units) of  Taq DNA Polymerase was used for each reaction. Formation of double-stranded DNA product was monitored using SYBR-Green (Molecular Probes, Eugene, Oregon, United States). All QRT-PCR reactions were carried out and analyzed on a DNA Engine-Opticon 2 (MJ Research, Waltham, Massachusetts, United States). Data were collected using RNA from at least three independent  C. elegans  growths. To determine the relationship between mRNA abundance and PCR cycle number, all primer sets were calibrated using serial dilutions of cDNA preparations. Primer sets were also calibrated by performing QRT-PCR reactions on serial dilutions of  C. elegans  genomic DNA. Relative abundance is reported as the mRNA abundance of each gene relative to the mRNA abundance of several control genes, which are expressed at constant levels throughout development.TaqTaqGC/MS Fatty acids were isolated from 10,000 L4 animals grown on a single 15-cm NGM-Lite plate. For WT and  nhr-49(nr2041)  experiments, worms were grown at 23 °C on a lawn of OP50. For RNAi, 10,000 embryos were placed on control bacteria, or on  fat-5, fat-7, ech-1, acs-2,  or  nhr-49  RNAi bacteria. Fatty acid extract was prepared as described previously [ 17 ]. GC/MS spectra were collected on an HP 6890 gas chromatograph outfitted with a J&W DB-XLB column. The mass spectrometer was an HP MSD 5973 (Agilent, Palo Alto, California, United States) and data were analyzed using Chemstation version A.03.00 software (Agilent). Peaks were assigned using fatty acid standards.
GC/MS
Fatty acids were isolated from 10,000 L4 animals grown on a single 15-cm NGM-Lite plate. For WT and  nhr-49(nr2041)  experiments, worms were grown at 23 °C on a lawn of OP50. For RNAi, 10,000 embryos were placed on control bacteria, or on  fat-5, fat-7, ech-1, acs-2,  or  nhr-49  RNAi bacteria. Fatty acid extract was prepared as described previously [ 17 ]. GC/MS spectra were collected on an HP 6890 gas chromatograph outfitted with a J&W DB-XLB column. The mass spectrometer was an HP MSD 5973 (Agilent, Palo Alto, California, United States) and data were analyzed using Chemstation version A.03.00 software (Agilent). Peaks were assigned using fatty acid standards.nhr-49(nr2041)fat-5, fat-7, ech-1, acs-2,nhr-49GFP reporter analysis and ectopic expression of ACS-2::GFP To make the P nhr-49 GFP plasmid, a genomic fragment including 3.5 kb of the  nhr-49  upstream sequence and 88 bp of the first exon of  nhr-49  was ligated into the L3691 GFP expression vector (a gift of A. Fire). Germ-line transformation was performed following standard procedures [ 35 ]. P nhr-49 GFP was injected at the following concentrations: 1 ng/μl, 5 ng/μl and 20 ng/μl along with the co-injection marker pRF4  rol-6(su1006)  injected at 60 ng/μl. At all three P nhr-49 GFP concentrations, we were unable to obtain stably transmitting lines. Animals used for GFP imaging were derived from the F1 generation and were mosaic, hosting the P nhr-49 GFP transgene in only a subset of tissues. To construct  acs-2::gfp,  a 4-kb genomic fragment, including the full-length  acs-2  gene plus 2 kb of upstream sequence, was cloned into the vector L3691. Germ-line transformation was performed using 50 ng/μl of  acs:2::gfp  and 50 ng/μl pRF4  rol-6(su1006).  For N2/ acs-2::gfp  and  nhr-49(nr2041)/acs-2::gfp,  over ten independent transmitting lines were obtained and analyzed by GFP microscopy. GFP reporter expression was observed using a Zeiss Axiovert S100 fluorescence microscope (Carl Zeiss, Thornwood, New York, United States) under a 40X objective. For rescue experiments, 50–100 ng/μl of  acs-2::gfp  was injected into  nhr-49(nr2041)  and WT animals along with 50 ng/μl of pRF4 rol-6(su1006). Transgenic adults, selected for rolling behavior, were placed on Nile Red plates and assayed for fat content as described above. pRF4  rol-6(su1006)  was injected into  nhr-49(nr2041)  in combination with unrelated plasmids to demonstrate that the lowered fat content was not a result of hosting a transgenic array or caused by expression of the  rol-6(su1006)  marker gene.
GFP reporter analysis and ectopic expression of ACS-2::GFP
To make the P nhr-49 GFP plasmid, a genomic fragment including 3.5 kb of the  nhr-49  upstream sequence and 88 bp of the first exon of  nhr-49  was ligated into the L3691 GFP expression vector (a gift of A. Fire). Germ-line transformation was performed following standard procedures [ 35 ]. P nhr-49 GFP was injected at the following concentrations: 1 ng/μl, 5 ng/μl and 20 ng/μl along with the co-injection marker pRF4  rol-6(su1006)  injected at 60 ng/μl. At all three P nhr-49 GFP concentrations, we were unable to obtain stably transmitting lines. Animals used for GFP imaging were derived from the F1 generation and were mosaic, hosting the P nhr-49 GFP transgene in only a subset of tissues.nhr-49nhr-49nhr-49nhr-49rol-6(su1006)nhr-49nhr-49To construct  acs-2::gfp,  a 4-kb genomic fragment, including the full-length  acs-2  gene plus 2 kb of upstream sequence, was cloned into the vector L3691. Germ-line transformation was performed using 50 ng/μl of  acs:2::gfp  and 50 ng/μl pRF4  rol-6(su1006).  For N2/ acs-2::gfp  and  nhr-49(nr2041)/acs-2::gfp,  over ten independent transmitting lines were obtained and analyzed by GFP microscopy. GFP reporter expression was observed using a Zeiss Axiovert S100 fluorescence microscope (Carl Zeiss, Thornwood, New York, United States) under a 40X objective.acs-2::gfp,acs-2acs:2::gfprol-6(su1006).acs-2::gfpnhr-49(nr2041)/acs-2::gfp,For rescue experiments, 50–100 ng/μl of  acs-2::gfp  was injected into  nhr-49(nr2041)  and WT animals along with 50 ng/μl of pRF4 rol-6(su1006). Transgenic adults, selected for rolling behavior, were placed on Nile Red plates and assayed for fat content as described above. pRF4  rol-6(su1006)  was injected into  nhr-49(nr2041)  in combination with unrelated plasmids to demonstrate that the lowered fat content was not a result of hosting a transgenic array or caused by expression of the  rol-6(su1006)  marker gene.acs-2::gfpnhr-49(nr2041)rol-6(su1006)nhr-49(nr2041)rol-6(su1006)Supporting Information Table S1 Raw Data for QRT-PCR Screen Data are shown as C t , the number of cycles required for amplification of a particular target gene from a cDNA preparation. Changes in gene expression in the  nhr-49(nr2041)  mutant were measured as ΔC t . ΔC t  are obtained by subtracting  nhr-49 (C t ) from N2(C t ) and adjusting for RNA concentration differences with a correction factor (CF). The CF was determined by aligning the data to a set of standard genes. Thus the equation for ΔC t  is as follows:

 Genes were designated as  nhr-49  dependent if the C t  was >1.0 in multiple developmental stages (and was greater than the standard error).  nhr-49  targets are marked with an X. The QRT-PCR results of the major  nhr-49  targets,  acs-2, ech-1, fat-5,  and  fat-7,  were confirmed with a second primer pair. (100 KB XLS). Click here for additional data file.
Supporting Information
Raw Data for QRT-PCR Screen
Data are shown as C t , the number of cycles required for amplification of a particular target gene from a cDNA preparation. Changes in gene expression in the  nhr-49(nr2041)  mutant were measured as ΔC t . ΔC t  are obtained by subtracting  nhr-49 (C t ) from N2(C t ) and adjusting for RNA concentration differences with a correction factor (CF). The CF was determined by aligning the data to a set of standard genes. Thus the equation for ΔC t  is as follows:nhr-49(nr2041)nhr-49Genes were designated as  nhr-49  dependent if the C t  was >1.0 in multiple developmental stages (and was greater than the standard error).  nhr-49  targets are marked with an X. The QRT-PCR results of the major  nhr-49  targets,  acs-2, ech-1, fat-5,  and  fat-7,  were confirmed with a second primer pair.nhr-49nhr-49nhr-49acs-2, ech-1, fat-5,fat-7,(100 KB XLS).Click here for additional data file.