InteractiveFly: GeneBrief

Hormone receptor-like in 96: Biological Overview | References

BIOLOGICAL OVERVIEW

Cholesterol homeostasis is required to maintain normal cellular function and avoid the deleterious effects of hypercholesterolemia. The Drosophila DHR96 nuclear receptor binds cholesterol and is required for the coordinate transcriptional response of genes that are regulated by cholesterol and involved in cholesterol uptake, trafficking, and storage. DHR96 mutants die when grown on low levels of cholesterol and accumulate excess cholesterol when maintained on a high-cholesterol diet. The cholesterol accumulation phenotype can be attributed to misregulation of npc1b, an ortholog of the mammalian Niemann-Pick C1-like 1 gene NPC1L1, which is essential for dietary cholesterol uptake. These studies define DHR96 as a central regulator of cholesterol homeostasis (Horner, 2010).

Cholesterol is an essential component of cell membranes that influences the permeability and fluidity of the lipid bilayer. Cholesterol also acts as a precursor for steroid hormone biosynthesis and contributes to cell-cell signaling pathways. These critical cellular functions are supported by regulatory mechanisms that maintain normal cholesterol levels and prevent hypercholesterolemia, which is a major risk factor for cardiovascular disease in humans. Cholesterol homeostasis in vertebrates is achieved primarily through de novo synthesis and dietary uptake. Although extensive studies have defined a central role for the sterol regulatory element-binding protein (SREBP) family of transcription factors in controlling cholesterol synthesis (Brown, 1997), the mechanisms that regulate dietary cholesterol absorption remain more poorly understood. One central component of this pathway is the Niemann-Pick C1-like 1 gene NPC1L1 (see Drosophila NPC1), which encodes a plasma membrane protein that mediates the uptake of dietary cholesterol by the intestine. Mouse mutants for NPC1L1 display significantly reduced levels of cholesterol absorption and are insensitive to treatment with the anti-hypercholesterolemia drug ezetimibe, which acts as a specific NPC1L1 inhibitor. Another major regulator of cholesterol homeostasis is the liver X receptor α (LXRα) nuclear receptor, which binds cholesterol metabolites and regulates the transcription of genes that control cholesterol transport and metabolism, including NPC1L1 (Duval, 2006; Kalaany, 2006; Valasek, 2007; Horner, 2010 and references therein).

Drosophila was used as a model system to study the regulation of cholesterol homeostasis. Unlike vertebrates, insects are cholesterol auxotrophs that are unable to synthesize this essential compound. Little is known, however, about the mechanisms that regulate the uptake of dietary cholesterol in Drosophila. A recent study showed that the fly ortholog of NPC1L1, npc1b, is expressed specifically in the midgut and is essential for dietary cholesterol absorption (Voght, 2007). Other NPC disease gene homologs in Drosophila also contribute to cholesterol homeostasis. The Drosophila ortholog of vertebrate NPC1, npc1a, and two of the eight fly NPC2 homologs, npc2a and npc2b, play important roles in intracellular cholesterol trafficking and synthesis of the steroid hormone 20-hydroxyecdysone (20E) (Huang, 2005; Huang, 2007; Fluegel, 2006). Other predicted regulators of cholesterol metabolism in Drosophila, however, remain unstudied, and upstream factors that might sense cholesterol levels and control cholesterol homeostasis are undefined (Horner, 2010).

This study shows that the Drosophila DHR96 nuclear receptor binds cholesterol, is essential for survival on a low-cholesterol diet, and is required to maintain cholesterol homeostasis when animals are grown on a high-cholesterol diet. Dietary cholesterol regulates the transcription of many genes that are expressed in the midgut and that act in lipid metabolism, and this transcriptional response fails to occur in DHR96 mutants. Misregulation of one of these genes, npc1b, is sufficient to explain the cholesterol accumulation defect seen in DHR96 mutants, defining npc1b as a critical functional target of the receptor. This study provides a new framework for understanding the molecular mechanisms that regulate cholesterol homeostasis (Horner, 2010).

Mass spectrometry (MS) was used to identify potential ligands for DHR96. The DHR96 ligand-binding domain (LBD) was overexpressed, purified from insect cells, and subjected to electrospray ionization (ESI) MS, under both denaturing and nondenaturing conditions. The full mass range spectrum of the sample from the denaturing condition had a series of peaks corresponding to the DHR96 LBD, with a measured molecular weight (MW) of 31,831.92 Da, close to the predicted theoretical mass (31,830.84 Da for His₆-DHR96_S471-H723). Under nondenaturing conditions, the full mass range scan detected an additional series of peaks corresponding to a MW of 32,218.44 Da. This mass shift indicates that the DHR96 LBD copurifies with a molecule of 386.52 Da in a stoichiometry of 1:1, indicative of specific ligand binding (Horner, 2010).

One of the presumptive DHR96/ligand complex peaks (2685.87 m/z) was then selected for collision-induced dissociation (CID) with a collision voltage of 10 V, resulting in the generation of two ions in the 12⁺ charge state. One ion represents the mass of the intact DHR96 LBD/ligand complex (2685.87 m/z; 32218.44 Da), while the other represents the unbound DHR96 LBD (2653.66 m/z; 31831.92 Da). Using a higher collision voltage of 50 V completely disrupted the DHR96 LBD/ligand complex to generate a CID spectrum with only an unbound receptor (2653.60 m/z; 31831.2 Da). The charge state of the receptor ion did not change upon loss of the ligand and no peak was observed in the lower mass range, indicating that the ligand is a neutral molecule. These observations, along with the mass shift of 386.52 Da, suggest that the bound molecule may be cholesterol (MW, 386.6 Da) (Horner, 2010).

To further verify the identity of the molecule bound to the DHR96 LBD, purified receptor was extracted with chloroform-methanol and a portion of the extract was derivatized and analyzed by gas chromatography/MS (GC/MS) with electron ionization (EI). A single major peak at 19 min was observed in the GC/MS chromatogram. The retention time of this peak was identical to that of a derivatized cholesterol standard. Moreover, the major peak of the DHR96 sample on GC/MS produced an EI spectrum that matched a previously described spectrum for derivatized cholesterol. Based on these two independent modes of MS - ESI/MS and GC/MS - it is concluded that the DHR96 LBD is capable of binding a single molecule of cholesterol. GC/MS analysis of several other Drosophila nuclear receptors indicated that this interaction is specific to DHR96. In addition, although this interaction is stable through three rounds of protein chromatography, the partial dissociation of ligands at 10 V and complete dissociation at 50 V suggests that the bound cholesterol is exchangeable (Horner, 2010).

The observation that DHR96 binds cholesterol raises the possibility that it may mediate transcriptional responses to this compound. To test this possibility, control and DHR96 mutants were grown on low-cholesterol medium in the absence or presence of 0.03% cholesterol, and were subjected to microarray analysis. From this analysis, 117 genes were identified that are up-regulated at least 1.4-fold in response to cholesterol in wild-type larvae, along with 270 genes that are down-regulated. This response appears to be rapid, occurring within 1-2 h of cholesterol treatment, and displays similar cholesterol dose response profiles. A response to sitosterol treatment was also seen, indicating that it is not specific to cholesterol. Interestingly, a number of the cholesterol-regulated genes are predicted to play central roles in cholesterol metabolism and transport. These include CG32186, which encodes a predicted ABCA3-like transporter; CG6472, which encodes a lipoprotein lipase (LPL) homolog; and CG8112, which encodes acyl-CoA:cholesterol acetyltransferase (ACAT). This enzyme plays a critical role in esterifying cholesterol, which is the primary stored form of intracellular cholesterol. Four Drosophila homologs of NPC genes are also regulated by cholesterol, including npc1b and three NPC2 homologs: npc2b, npc2c, and npc2d (Huang, 2007; Horner, 2010).

Importantly, this transcriptional response is almost entirely dependent on DHR96 function. Only 13% of the genes that are up-regulated by cholesterol in wild-type larvae (15 genes), and 10% of the genes that are down-regulated (27 genes), display a similar profile of expression in DHR96 mutants treated with cholesterol. Moreover, many additional genes become responsive to cholesterol in DHR96 mutants, with 355 genes up-regulated and 446 genes down-regulated at least 1.4-fold, indicating that DHR96 normally plays a key role in suppressing this transcriptional program. These DHR96-regulated genes include four of the remaining five NPC2 family members: npc2e, npc2f, npc2g, and npc2h. DHR96 also regulates Lip3 (CG8823), which encodes a predicted cholesterol ester hydrolase; CG9663, which encodes an ABCG1 homolog; CG11162, which encodes a sterol-C4-methyl oxidase; and several genes that encode predicted stearoyl-CoA-desaturases. Many genes involved in other aspects of lipid metabolism are also misregulated in DHR96 mutants. In addition, DHR96 itself is down-regulated approximately twofold by cholesterol in wild-type larvae, and this response fails to occur in DHR96 mutants, suggesting that there is autoregulation. Further examination of the cholesterol-regulated genes in both wild-type and DHR96 mutant larvae revealed that many of these genes are expressed in the midgut, consistent with the critical role of dietary cholesterol uptake in a cholesterol auxotroph such as Drosophila (Horner, 2010).

Northern blot hybridizations confirmed that genes such as npc2c and CG14745 (which encodes a predicted peptidoglycan recognition protein) are induced by cholesterol in wild-type larvae, while genes such as npc1b, CG5932 (which encodes a gastric lipase), and CG31148 (which encodes a predicted enzyme in sphingolipid metabolism) are repressed by cholesterol. Other genes, such as npc2e, are not responsive to cholesterol in wild-type larvae. All of these genes, however, are misregulated in DHR96 mutants (Horner, 2010).

The central role of DHR96 in mediating transcriptional responses to cholesterol suggests that it may contribute to the regulation of cholesterol homeostasis. As an initial test of this possibility, how control and DHR96¹ mutants respond to growth on a low-cholesterol diet was examined. Whereas most wild-type larvae grown on this medium develop through to adulthood, DHR96 mutants arrest their development primarily as second instar larvae and die within several days. Supplementation with a complete nutrient source, yeast, was sufficient to rescue this lethality. Efficient rescue was also achieved by supplementing with cholesterol, demonstrating that the lack of this essential nutrient is a cause of the lethality. Widespread heat-induced expression of a wild-type DHR96 transgene, or specific expression of DHR96 in the midgut, is also sufficient to rescue the lethality of DHR96 mutants grown on the low-cholesterol diet. Specific expression of DHR96 in the fat body of DHR96 mutants had no effect. Taken together, these observations indicate that DHR96 mutants are unable to survive under limiting cholesterol conditions, and that this phenotype is due to a specific loss of DHR96 function in the midgut. Similar results were obtained when control and DHR96 mutants were raised on a chloroform-extracted medium that is deprived of all sterols. A dose response study on the low-cholesterol diet revealed that 0.03% cholesterol is the ideal concentration for rescue. This amount is identical to the optimal amount required for Drosophila larval survival on a minimal defined medium. A number of other sterols are able to substitute for cholesterol in these experiments, including 7-dehydrocholesterol, ergosterol, dehydroergosterol, sitosterol, and stigmasterol, consistent with their ability to support normal growth of wild-type Drosophila. Supplementing the low-cholesterol medium with other lipids, however, including triacylglycerol (TAG) and oleic acid, had no effect, indicating that the rescue is specific to sterols (Horner, 2010).

Several lines of evidence indicate that proper cholesterol metabolism is essential for larval development. Drosophila larvae grown in the absence of sterols arrest development at the first or second instar, similar to the lethal phase of DHR96 mutants grown under low-cholesterol conditions. Similarly, mutants for npc1a die as first instar larvae due to defects in intracellular cholesterol trafficking and reduced production of the molting hormone 20E (Huang, 2005; Fluegel, 2006). Double mutants for npc2a and npc2b also fail to progress through development and can be rescued by feeding 20E (Huang, 2007). DHR96 mutants grown on a high-cholesterol diet fail to show any clear defects in intracellular cholesterol localization, as detected by filipin staining. In addition, feeding 20E to DHR96 mutants maintained on the low-cholesterol diet does not rescue their lethality. These observations do not support the hypothesis that the lethality in DHR96 mutants is due to a defect in cholesterol trafficking that affects 20E production. Rather, this phenotype is likely to arise from other defects, such as inefficient cholesterol utilization, changes in cholesterol storage, or disruption of other lipid metabolic pathways (Horner, 2010).

To determine whether DHR96 mutants display defects in cholesterol homeostasis, total cholesterol levels were measured in control and DHR96¹ mutant larvae grown on the low-cholesterol medium, in either the absence or presence of 0.03% cholesterol. DHR96 mutants have the same level of cholesterol as wild-type larvae when propagated without cholesterol supplementation. In the presence of added cholesterol, however, DHR96 mutants display cholesterol levels that are significantly higher than the 10%-20% increase seen in control larvae. This cholesterol accumulation defect can be rescued by either a wild-type genomic DHR96 transgene or expression of wild-type DHR96 in the midgut of mutant larvae, indicating that it arises from a specific loss of DHR96 function in this tissue (Horner, 2010).

Expression of npc1b, which is required for dietary cholesterol absorption, is down-regulated when wild-type larvae are treated with cholesterol. The observation that this switch fails to occur in DHR96 mutants could explain why these animals accumulate excess cholesterol when grown on a high-cholesterol diet. Consistent with this, it was found that DHR96 is expressed in the region of the larval midgut where npc1b exerts its functions. This overlap in DHR96 and npc1b expression also appears to be functionally significant because specific expression of wild-type DHR96 in the midgut of DHR96 mutants is sufficient to restore appropriate npc1b transcriptional repression in response to cholesterol. Importantly, the accumulation of cholesterol in DHR96 mutants is dependent on npc1b function because npc1b;DHR96 double mutants have wild-type levels of cholesterol. This is consistent with the normal cholesterol levels seen in npc1b mutants at this stage of development, which likely derive from maternal loading of cholesterol during oogenesis (Horner, 2010).

Whether DHR96 mutants require npc1b function for their ability to absorb dietary cholesterol was tested. Control larvae, DHR96¹ mutants, npc1b¹ mutants, and npc1b¹;DHR96¹ double mutants were grown on the low-cholesterol medium in the presence of ³H-cholesterol. Radioactive glucose was also added to the food in order to normalize the levels of cholesterol absorption and to control for different feeding rates. Under these conditions, DHR96 mutants display a similar level of cholesterol uptake as wild-type larvae. This result is consistent with the wild-type levels of total cholesterol seen in DHR96 mutants grown on a low-cholesterol diet. In contrast, both npc1b mutants and npc1b;DHR96 double mutants display a dramatic reduction in cholesterol absorption. This result agrees with the original study of npc1b mutants, and indicates that DHR96 mutants require NPC1b to take up dietary cholesterol. A similar result was seen with a key plant sterol, ³H-sitosterol, suggesting that this pathway mediates general sterol absorption. Moreover, the npc1b mutation has no effect on the ability of DHR96 mutants to absorb oleic acid, consistent with its specific role in sterol uptake. Taken together, these observations indicate that the misregulation of npc1b expression in DHR96 mutants is sufficient to explain their inability to maintain proper cholesterol homeostasis when grown on a high-cholesterol diet (Horner, 2010).

It is important to note, however, that the cholesterol accumulation defect seen in DHR96 mutants does not appear to be related to the lethality that is observed when the mutant is grown on a low-cholesterol diet. DHR96 mutants that arrest their development when maintained on a low-cholesterol diet display normal levels of cholesterol. In addition, the npc1b;DHR96 double mutants are small and die earlier than npc1b mutants alone when raised in either the presence or absence of cholesterol, in spite of their wild-type cholesterol levels. Thus, it is likely that the DHR96 mutants suffer from additional metabolic defects beyond their inability to properly regulate npc1b transcription. This conclusion is supported by the widespread effects of the DHR96 mutation on the expression of genes involved in lipid metabolism and midgut physiology, as well as the expression of DHR96 in tissues outside the midgut. Further studies of DHR96 mutants should help to uncover its other key metabolic activities (Horner, 2010).

These reported here define essential functions for DHR96 in maintaining cholesterol homeostasis during larval development. A parallel set of studies has also identified nonessential functions for DHR96 in lipid metabolism during adult stages due to their reduced levels of stored energy in the form of TAG (Sieber, 2009). Interestingly, microarray analysis of DHR96 mutant adults raised on normal growth medium revealed widespread effects on the transcription of genes expressed in the midgut, many of which are identical to the DHR96 regulatory targets described in this study. Among this core set of DHR96 target genes is CG5932, which encodes a gastric lipase that is required for the breakdown of dietary fat. Misregulation of CG5932 in the midgut is sufficient to explain the defects observed in TAG homeostasis in DHR96 mutants, analogous to the important role of DHR96 in regulating npc1b expression to maintain proper cholesterol homeostasis. Interestingly, CG5932 transcription is also regulated by cholesterol, raising the possibility that this dietary sterol, acting through DHR96, may coordinate dietary TAG breakdown with cholesterol absorption. This convergence of results points to a central role for DHR96 in midgut physiology and provides a strong foundation to refine understanding of its critical regulatory functions in maintaining lipid homeostasis (Horner, 2010).

DHR96 is a nuclear protein, in the presence or absence of cholesterol, consistent with its predicted role as a direct regulator of transcription. It has a unique P-box sequence within its DNA-binding domain (DBD), which determines its DNA-binding specificity. This sequence is shared by only three Caenorhabditis elegans nuclear receptors: DAF-12, NHR-48, and NHR-8. Although a preferred binding site has been identified for DAF-12, it not known whether DAF-12 contacts this sequence as a homodimer or a heterodimer with another nuclear receptor (Shostak, 2004). Similarly, it is unclear whether DHR96 binds DNA as a monomer, homodimer, or heterodimer with the Drosophila retinoid X receptor (RXR) ortholog, like its mammalian homologs. In addition, the observation that DHR96 target genes display all possible combinations of regulatory responses to cholesterol and the DHR96 mutation indicates that there is further complexity in this pathway. Studies are under way to define the molecular mechanisms by which DHR96 regulates target gene transcription (Horner, 2010).

Ligands have been identified for only two of the 18 canonical nuclear receptors in Drosophila: ecdysone receptor (EcR) and E75. This study adds a third such adopted orphan receptor to the list: DHR96. The identification of cholesterol as a DHR96 ligand fits with its membership in the NR1I subfamily of nuclear receptors. The closest mammalian and C. elegans homologs of DHR96 -- pregnane X receptor (PXR), constitutive androstane receptor (CAR), vitamin D3 receptor (VDR), and DAF-12 -- are all transcriptionally responsive to cholesterol derivatives. Similar responses to sterol derivatives are seen for members of the next most closely related group of receptors, the NR1H group, which includes LXR, farnesoid X receptor (FXR), and EcR. These observations support the proposal that the NR1H and NR1I groups arose from an ancestral progenitor that acted as a sterol receptor in primitive organisms (Horner, 2010).

It is important, however, to note that although cholesterol copurifies with the DHR96 LBD, it may not be the natural ligand for this receptor. In efforts to address this issue, no changes in DHR96 activity were detected in response to exogenous cholesterol, dietary factors, or genetic backgrounds that disrupt cholesterol absorption or intracellular trafficking. Further studies are required to determine whether cholesterol or a related metabolite acts as a regulatory ligand to modulate the activation status of DHR96 (Horner, 2010).

Nuclear receptor DHR96 acts as a sentinel for low cholesterol concentrations in Drosophila melanogaster

All eukaryotic cells have to maintain cholesterol concentrations within defined margins in order to function normally. Perturbing cholesterol homeostasis can result in a wide range of cellular and systemic defects, including cardiovascular diseases, as well as Niemann-Pick disease and Tangier disease. This study shows that DHR96 is indispensable for mediating the transcriptional response to dietary cholesterol and that it acts as a key regulator of the Niemann-Pick type C gene family, as well as of other genes involved in cholesterol uptake, metabolism, and transport. DHR96 mutants are viable and phenotypically normal on a standard medium but fail to survive on diets that are low in cholesterol. DHR96 mutants have aberrant cholesterol levels, demonstrating a defect in maintaining cholesterol homeostasis. Remarkably, it was found that a high-cholesterol diet phenocopies the genomic profile of the DHR96 mutation, indicating that DHR96 resides at the top of a genetic hierarchy controlling cholesterol homeostasis in insects. The study proposes a model whereby DHR96 is activated when cellular cholesterol concentrations drop below a critical threshold in order to protect cells from severe cholesterol deprivation (Bujold, 2010).

This study demonstrates that DHR96 is a critical regulator of cholesterol homeostasis and that it mediates transcriptional changes in response to dietary cholesterol. A central question is why DHR96 mutants fail to survive on a low-cholesterol diet. The most likely answer is that DHR96 mutants are unable to recognize that they are ingesting a low-cholesterol diet and therefore fail to implement the necessary transcriptional programs that are required to adapt to conditions of severe cholesterol paucity. For instance, genes that function to increase cellular cholesterol levels, such as NPC1b and Lip3, are transcriptionally upregulated in wild-type larvae when cholesterol concentrations decline. In DHR96 mutants, however, the upregulation of NPC1b and Lip3 never reaches wild-type expression levels. NPC1b is specifically expressed in the gut and is required for cholesterol absorption, while the predicted function of the Lip3 protein is the hydrolyzing of cholesteryl esters, resulting in the liberation of free cholesterol. This suggests that at least two genes required for increasing cellular cholesterol levels are submaximally expressed in DHR96 mutants (Bujold, 2010).

It appears likely that insufficient cholesterol absorption in DHR96 mutants merely contributes to the failure to survive on low cholesterol, because only a 20% reduction of total cholesterol and cholesteryl ester levels was found in mutant animals compared to controls. With this in mind, it is interesting that genes with roles in reducing cellular cholesterol concentrations are substantially overexpressed in DHR96 mutants, suggesting that cellular cholesterol efflux is increased in mutant animals compared to the wild type. The ACAT and ABCA1 genes fall into this category, since ACAT reduces the pool of free cellular cholesterol through an esterification reaction that adds a fatty acid to the molecule, while ABCA1 encodes an ATP transporter that is involved in the active removal of cholesterol by pumping it across the cell membrane. Wild-type ACAT and ABCA1 are downregulated in response to declining cholesterol concentrations, which is in agreement with the idea that under conditions of dangerously low cholesterol levels, genes that increase cholesterol efflux must be turned off. On lipid-depleted medium, ACAT and ABCA1 mRNA levels are substantially higher in DHR96 mutants than in controls, suggesting that mutant cells actively reduce cellular cholesterol concentrations under conditions of low dietary cholesterol. At the same time, cholesterol uptake is reduced as well, thus aggravating the situation (Bujold, 2010).

The fact that treatment with 1% cholesterol largely phenocopies the genome-wide effects of the DHR96 mutation strongly suggests that DHR96 functions at the top of a gene network controlling the systemic response to varying levels of dietary sterols. It has been shown earlier that DHR96 can bind cholesterol in vivo, suggesting that this nuclear receptor is a cellular sensor for varying sterol levels. This would suggest that cholesterol or a very similar metabolite acts as a direct ligand for DHR96; however, a key question is whether such a ligand would act as an agonist or an antagonist. The “constitutive androstane receptor” (CAR), which is one of the three vertebrate nuclear receptor orthologs of DHR96, displays the unusual feature that it acts as a constitutively active transcription factor in the absence of a ligand. Androstane metabolites, however, act as inverse agonists and deactivate murine CAR upon ligand binding. Prior to this finding, all nuclear receptors were believed to be activated by ligand binding; however, it remains unclear how widespread this mode of nuclear receptor inactivation is (Bujold, 2010).

Observations of this study are best explained by an inverse agonist mechanism similar to what has been described for CAR. Given the fact that DHR96 is only required for survival when the animal is feeding on a low-cholesterol diet, it follows that cholesterol metabolites that might act as ligands for DHR96 are scarce under these conditions. This suggests that DHR96 is active in the absence of a ligand. Conversely, the DHR96 gene is not required for survival when cholesterol concentrations are sufficiently high, suggesting that the DHR96 protein is inactive when potential ligands are abundant, again favoring the view of an inverse agonist mechanism. Perhaps the strongest argument for a mechanism through an inverse agonist derives from the fact that it provides the simplest explanation for the fact that a high-cholesterol diet is able to phenocopy the DHR96 mutation. In accordance with the finding that DHR96 binds to cholesterol, this model predicts that a high-cholesterol diet would result in a widespread deactivation of DHR96 receptor molecules, essentially turning off DHR96 activity. Consequently, one would expect that the molecular consequences of deactivated DHR96 protein (via a high-cholesterol treatment) or removing functional protein altogether (via a null mutation) are very similar indeed. The observation that DHR96 mRNA levels decline in response to increasing cholesterol levels is also compatible with the idea of inverse agonism. Since DHR96 mRNA is possibly regulated by its own protein, one would predict that increasing cholesterol levels reduce DHR96 activity, which in turn results in reduced transcription of the DHR96 gene itself (Bujold, 2010).

Nutrigenomics is a powerful strategy for identifying genes that act in nutrient-dependent pathways, and this study represents a first step toward the identification of genes with hitherto unknown roles in cholesterol homeostasis. It was shown that the expression of four Niemann-Pick genes—NPC1b and NPC2c, -d, and -e—is strongly dependent on the concentration of dietary cholesterol. Other identified genes with predicted roles in sterol biology are ACAT, ABCA1, Lip3, and Cyp12d1. Cholesterol-responsive genes with no known links to cholesterol homeostasis were also found. For instance, CG5932, encoding a gastric lipase; FANCL, encoding a ubiquitin E3 ligase; and CG31148, encoding a glucosylceramidase, are all downregulated in response to increasing cholesterol concentrations. An earlier study shows that DHR96 mutants are resistant to diet-induced obesity, which is at least in part due to the role of DHR96 in regulating CG5932, confirming that this nuclear receptor also has important roles in controlling lipid metabolism. In addition, a study of mice demonstrates that the “Idol” ubiquitin E3 ligase is transcriptionally induced by LXR and triggers proteolytic degradation of the LDL receptor via ubiquitination, thereby downregulating cellular cholesterol uptake. Future work may provide insight into whether FANCL has comparable roles in regulating cholesterol homeostasis in Drosophila (Bujold, 2010).

Although the sequence of DHR96 is most closely related to mammalian PXR and CAR, it shares significant functional characteristics with its more distant cousin, LXRα. LXRα binds oxysterols and, similar to DHR96 mutants, LXRα mutant mice fail to respond properly to dietary cholesterol and accumulate hepatic cholesterol when maintained on a high-cholesterol diet (Peet, 1998). LXRs also play a central role in the transcriptional response to dietary cholesterol in mice, and directly or indirectly control a number of genes that are related to DHR96-regulated genes, including ABCG1, LPL, steroyl-CoA desaturase, NPC1, and NPC2. Finally, like DHR96, LXRs repress NPC1L1 expression, although it is not clear whether this regulation is direct. Taken together, these results indicate that DHR96 and mammalian LXRs act through similar regulatory pathways to control cholesterol homeostasis. This work establishes a new framework for understanding how cholesterol levels are sensed in Drosophila, and the molecular mechanisms by which cholesterol homeostasis is maintained (Horner, 2010).

Coordination of triacylglycerol and cholesterol homeostasis by DHR96 and the Drosophila LipA homolog magro.

Although transintestinal cholesterol efflux has been identified as an important means of clearing excess sterols, the mechanisms that underlie this process remain poorly understood. This study shows that magro, a direct target of the Drosophila DHR96 nuclear receptor, is required in the intestine to maintain cholesterol homeostasis. magro encodes a LipA homolog that is secreted from the anterior gut into the intestinal lumen to digest dietary triacylglycerol. Expression of magro in intestinal cells is required to hydrolyze cholesterol esters and promote cholesterol clearance. Restoring magro expression in the intestine of DHR96 mutants rescues their defects in triacylglycerol and cholesterol metabolism. These studies show that the central role of the intestine in cholesterol efflux has been conserved through evolution, that the ancestral function of LipA is to coordinate triacylglycerol and cholesterol metabolism, and that the region-specific activities of magro correspond to the metabolic functions of its upstream regulator, DHR96 (Sieber, 2012).

Relatively little is known about the mechanisms that regulate cholesterol metabolism in Drosophila. Most studies have focused on DHR96 and the Niemann-Pick (NPC) disease gene homologs, which play important roles in dietary cholesterol absorption and intracellular cholesterol trafficking. This paper identifies the intestine as a key tissue for maintaining cholesterol homeostasis, and a central role is defined for the LipA homolog, Magro, in mediating this function. Like LipA, Magro has dual enzymatic activities, cleaving both TAG and cholesterol esters, consistent with their common fatty acid ester bonds. Mouse LipA mutants display a lack of stored fat in the form of white adipose tissue along with excess cholesterol esters, reflecting the major defects in magro RNAi animals. Similar phenotypes are seen in human LipA mutants suffering from CESD and Wolman's disease. Patients with Wolman’s disease also have digestive dysfunction, which may be related to the defects in lipid uptake that was observed in magro RNAi animals. In addition to these shared phenotypes, however, mammalian LipA mutants display massive accumulations of lipid in the liver, spleen, and intestine—defects that are not apparent in magro RNAi animals. Nonetheless, the parallels between Magro and LipA function in flies and humans establish Drosophila as a system to further understanding of CESD and Wolman's disease and define the ancestral function for this class of acid lipases, demonstrating their central role in the intestine to coordinate TAG and cholesterol homeostasis (Sieber, 2012).

Interestingly, the dual enzymatic functions of Magro appear to arise from distinct regions of the intestine. Disruption of magro function specifically in the proventriculus blocks its TAG lipolytic activity but does not affect cholesterol levels in these animals. In contrast, magro RNAi throughout the intestine affects both TAG and cholesterol homeostasis. These region-specific activities are consistent with dietary supplementation studies with pancreatin and Orlistat. They are also consistent with the expression pattern of Magro-EGFP protein, providing insights into how the dual functions of this enzyme are manifested. Magro-EGFP is expressed abundantly in the large outer columnar cells in the anterior half of the proventriculus. A stream of large acidic vesicles that originate is seen from this region, and it moves in a posterior direction toward the lumen of the intestine. This apparent vesicular trafficking of Magro is consistent with the cells at the anterior end of the proventriculus having secretory functions, depositing peritrophic matrix components into the lumen that lies between the outer and inner cell layers of the proventriculus. The peritrophic matrix is a meshwork of chitin and glycoproteins that provides a protective lining within the gut, much like the mucosal layer of the mammalian intestine. The observation that the Magro-EGFP vesicles reside in the same region of the proventriculus as the developing peritrophic matrix suggests that they are synthesized and exported into the lumen of the gut in a similar manner. This also raises the possibility that the digestive enzymes that are embedded in the peritrophic matrix may originate from vesicular trafficking in the proventriculus. Like magro, many genes with predicted digestive functions, including glucosidases, mannosidases, and endopeptidases, are regulated by DHR96 and expressed in the intestine. Several genes that contribute to the peritrophic matrix are also regulated by DHR96. It would be interesting to determine whether the proventriculus synthesizes and secretes these proteins in a coordinated manner (Sieber, 2012).

In addition to its abundant expression in the proventriculus, Magro-EGFP is also present at lower levels throughout the intestine, visible as punctate cytoplasmic staining in the enterocytes. This expression pattern provides a possible mechanism to explain the role of Magro in maintaining cholesterol homeostasis. It is proposed that Magro acts as a cholesterol esterase in the enterocytes, breaking down stored cholesterol to facilitate its elimination from the intestine. This model is consistent with the neutral lipid stores that are known to reside in the Drosophila intestine, second only to the fat body. It is also consistent with recent evidence that LipA acts as cholesterol esterase in macrophage foam cells to promote ABCA1-mediated cholesterol efflux. These data suggest that LipA and Magro may break down stored cholesterol esters upstream of reverse cholesterol transport and transintestinal cholesterol efflux to promote the clearance of excess sterols. Tissue-specific studies of LipA function in the pancreas and intestine may provide a clearer understanding of its relationship to the apparent exocrine role of Magro in the proventriculus and its ability to promote cholesterol clearance in the intestine (Sieber, 2012).

Finally, these data provide new directions in understanding the roles of LXR family members in lipid metabolism. Both DHR96 mutants and magro RNAi animals display reduced levels of TAG and elevated levels of cholesterol, and restoring magro expression in the intestines of DHR96 mutant animals largely rescues these defects, establishing magro as a key target for DHR96 regulation. These functions for DHR96 parallel those of its mammalian homolog, LXR. LXR activation, specifically in the intestine, results in a dramatic increase in fecal sterol excretion that correlates with increased expression of the ABCG5/ABCG8 sterol transporter. This observation suggests that LXR promotes reverse cholesterol transport in this tissue, which represents the best characterized mechanism for eliminating excess cholesterol from the body. Reverse cholesterol transport involves HDL-mediated transport of cholesterol from peripheral tissues to the hepatobiliary tract, leading to the removal of excess sterol by biliary excretion from the body. However, genetic studies of key components in biliary cholesterol excretion, such as abcb4 mutants and abcg5/abcg8 double mutants, have challenged the importance of reverse cholesterol transport for cholesterol excretion and have led to the proposal that the intestine plays a more direct role in this process (van der Velde, 2010). These studies shift the focus of cholesterol efflux toward the intestine and implicate a central role for LXR in regulating intestinal cholesterol clearance, not only through regulation of reverse cholesterol transport but also through novel targets involved in transintestinal cholesterol efflux. In addition, evidence that intestinal cholesterol esterase activity is critical for clearing excess sterol from the body suggests that acid lipases such as LipA may function downstream from LXR to maintain cholesterol homeostasis. Although there is no direct evidence that LXR regulates LipA expression, a recent study showed that elevated levels of oxidized LDL can repress LipA expression in endothelial cells, an effect that can be reversed by treatment with LXR agonists (Heltianu, 2011). Further studies are required to determine whether the regulatory links between LXR, LipA, and cholesterol homeostasis have been conserved through evolution and whether Drosophila can be used as a simple model system to better define the mechanisms of transintestinal cholesterol efflux (Sieber, 2012).

The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila

Triacylglycerol (TAG) homeostasis is an integral part of normal physiology and essential for proper energy metabolism. This study shows that the single Drosophila ortholog of the PXR and CAR nuclear receptors, DHR96, plays an essential role in TAG homeostasis. DHR96 mutants are sensitive to starvation, have reduced levels of TAG in the fat body and midgut, and are resistant to diet-induced obesity, while DHR96 overexpression leads to starvation resistance and increased TAG levels. DHR96 function is required in the midgut for the breakdown of dietary fat, and it exerts this effect through the CG5932 gastric lipase, which is essential for TAG homeostasis. This study provides insights into the regulation of dietary fat metabolism in Drosophila and demonstrates that the regulation of lipid metabolism is an ancestral function of the PXR/CAR/DHR96 nuclear receptor subfamily (Sieber, 2009).

Fat metabolism is central to the process of energy homeostasis. When nutrients are abundant, dietary fat in the form of triacylglycerol (TAG) is broken down by gastric TAG lipases to release fatty acids. These fatty acids are absorbed by the intestine and used to resynthesize TAG in peripheral tissues. These TAG reserves can be accessed upon nutrient deprivation through the action of specific lipid droplet-associated lipases that release the fatty acids for energy production through mitochondrial fatty acid β-oxidation. Defects in these processes can lead to dramatic changes in TAG levels and a range of physiological disorders, including obesity, diabetes, and cardiovascular disease. The alarming rise in the prevalence of these disorders in human populations has focused attention on understanding the molecular mechanisms that coordinate dietary nutrient uptake with TAG homeostasis. As a result, many regulators of TAG metabolism have been identified, including SREBP, PPAR, and adiponectin. In spite of these advances, however, the molecular mechanisms that coordinate dietary fat uptake, synthesis, storage, and utilization remain poorly understood (Sieber, 2009).

Nuclear receptors (NRs) are ligand-regulated transcription factors that play a central role in metabolic control. They are defined by a conserved zinc-finger DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LBD) that can impart multiple functions, including hormone binding, receptor dimerization, and transactivation. Many NRs are regulated by small lipophilic compounds that include dietary signals and metabolic intermediates, and exert their effects by directing global changes in gene expression that act to maintain metabolic homeostasis. This is exemplified by members of the mammalian PPAR, LXR, and FXR subfamilies, which play critical roles in adipogenesis, lipid metabolism, and cholesterol and bile acid homeostasis, respectively (Chawla, 2001: Sonoda, 2008; Sieber, 2009 and references therein).

This study used Drosophila as a model system to characterize the NR subfamily represented by the Pregnane X Receptor (PXR, NR1I2), Constitutive Androstane Receptor (CAR, NR1I3), and Vitamin D Receptor (VDR, NR1I1) in mammals. Previous studies have defined central roles for these receptors in sensing xenobiotic compounds and directly regulating genes involved in detoxification (Timsit, 2007; Willson, 2002). Initial studies showed that the single ancestral Drosophila ortholog of this NR subclass, DHR96, has similar functions (King-Jones, 2006). A DHR96 null mutant displays increased sensitivity to the sedative effects of phenobarbital and the pesticide DDT as well as defects in the expression of phenobarbital-regulated genes. These studies, however, revealed other potential roles for the receptor -- in particular, unexpected effects on the expression of genes that are predicted to regulate lipid and carbohydrate metabolism (King-Jones, 2006). This observation is in line with recent studies that have implicated roles for the mammalian PXR and CAR NRs in metabolic control (Moreau, 2008). The molecular mechanism by which they exert this effect, however, remains undefined (Sieber, 2009).

This study shows that DHR96 null mutants are sensitive to starvation and have reduced levels of TAG, while DHR96 overexpression leads to starvation resistance and elevated TAG levels. A series of studies using metabolic assays, diets, and the drug Orlistat revealed that DHR96 mutants are defective in their ability to break down dietary lipid. This model was supported by microarray studies, which showed that many genes expressed in the midgut are misregulated in DHR96 mutants, including highly reduced expression of the gastric lipase gene CG5932. CG5932 is required for proper whole-animal TAG levels, and selective expression of CG5932 in the midgut of DHR96 mutants is sufficient to rescue their lean phenotype. Taken together, these data support a role for the PXR/CAR/DHR96 NR subclass in lipid metabolism and define DHR96 as a key regulator of dietary TAG breakdown in the Drosophila midgut (Sieber, 2009).

Although DHR96 null mutants are viable and fertile, with no morphological defects, they die significantly more rapidly than genetically matched control flies under starvation conditions, while DHR96 overexpression leads to starvation resistance. The effects of these genotypes on the major forms of stored energy in the animal, glycogen and TAG, are consistent with their effects on the starvation response. DHR96 mutants have reduced levels of TAG under both fed and starved conditions, while DHR96 overexpression leads to increased TAG levels. Although no effects are seen on whole-animal glycogen levels in fed animals that either lack or overexpress DHR96, the mutants consume significantly more glycogen upon starvation than do controls. This rapid utilization of glycogen stores is most likely due to the decreased energy contribution from TAG. Taken together, these observations suggest that the starvation sensitivity of DHR96 mutants can be attributed to their lean phenotype, while the starvation resistance of the DHR96^2X strain is due to their excess energy in the form of TAG. This proposal is supported by the observation that genetically elevating the levels of TAG in DHR96 mutants by introducing mutations in brummer or AKHR, which control distinct aspects of fat body TAG lipolysis (Grönke, 2007), effectively rescues their starvation sensitivity. In addition, the opposite effects of DHR96 loss of function and gain of function on both the starvation response and TAG levels argue that this receptor plays a central role in maintaining whole-animal TAG homeostasis (Sieber, 2009)

Several lines of evidence support the conclusion that DHR96 exerts its primary metabolic functions through the midgut. These include the initial observation that DHR96 mutants are resistant to treatment with the gastric lipase inhibitor Orlistat and display reduced levels of midgut lipolytic activity. In addition, dietary supplementation with free fatty acids, but not TAG, is sufficient to rescue the lean phenotype of DHR96 mutants, as is midgut-specific expression of wild-type DHR96 in a DHR96 mutant background. A dramatic effect is seen on lipid levels in the midgut, where almost no neutral lipids are detectable in DHR96 mutants and enlarged lipid droplets are evident in DHR96^2X flies maintained on a normal diet. Interestingly, while the lumen of the midgut is not evident in control animals, material was clearly seen in the lumen of DHR96 mutant midguts or in control flies that are treated with Orlistat. In some cases, this material is stained by oil red O, suggesting that it may represent an increased level of undigested fat in these animals. This phenotype would be similar to that seen in humans who have defects in intestinal lipase activity. Likewise, an increase in the passage of undigested dietary fat is a complication associated with Orlistat treatment in patients. Taken together, these observations support the conclusion that DHR96 acts in the midgut to regulate the breakdown of dietary fat (Sieber, 2009).

An essential role for DHR96 in the midgut is further supported by the microarray study, which revealed that many DHR96-regulated genes are primarily expressed in this tissue. Interestingly, many of these genes have predicted roles related to the breakdown of dietary nutrients. These include downregulation of multiple genes with predicted α-mannosidase activity, which is involved in the breakdown of the complex sugars found on glycoproteins. Many genes that encode trypsins and endopeptidases are also expressed at reduced levels in DHR96 mutants as well as a few genes that encode predicted α-glucosidases, which are involved in the breakdown of dietary carbohydrates. In addition, a number of genes involved in the formation of the peritrophic matrix are more abundantly expressed in DHR96 mutants. This matrix is comprised of chitin and peritrophic proteins and acts as a protective layer for the epithelial surface of the midgut (Hegedus, 2009). The peritrophic matrix also has critical roles in facilitating digestion. Only smaller molecules that arise from the initial digestion of complex nutrients, including peptides, sugars, and lipids, can move through the peritrophic matrix for final digestion and absorption by the midgut epithelium. These events are controlled by the selective partitioning of digestive enzymes to different sides of the peritrophic matrix as well as within the matrix itself. Thus, while midgut morphology appears normal in DHR96 mutants, the effect of the mutation on the peritrophic matrix could impact nutrient digestion and absorption (Sieber, 2009).

In addition to genes that regulate different aspects of lipid metabolism, microarray study of DHR96 mutants identified widespread effects on the expression of Drosophila homologs of NPC disease genes. The npc1b gene, which encodes an essential cholesterol transporter and the ortholog of mammalian NPC1L1, is the tenth most highly upregulated gene in DHR96 mutants. In addition, five of the eight Drosophila NPC2 genes are misregulated in DHR96 mutants: npc2c, npc2d, npc2e, npc2g, and npc2h. These genes encode homologs of mammalian NPC2, which is involved in intracellular cholesterol trafficking. Remarkably, two of these genes are the most highly up- and downregulated genes identified in the mutant (npc2e and npc2d, respectively). Moreover, many of these npc genes are located in clusters, suggesting that they are coregulated by the receptor. Although the function of these npc2 genes is unknown, their disproportionate representation within the list of DHR96-regulated genes implies a critical role for the receptor in regulating cholesterol trafficking. Indeed, dietary cholesterol triggers a widespread transcriptional response in Drosophila that is dependent on DHR96 function (Horner, 2010). Moreover, this study showed that DHR96 mutants display defects in their ability to maintain cholesterol homeostasis when grown on a high-cholesterol diet. Taken together with the results presented here, these studies suggest that DHR96 plays an essential role in the midgut to coordinate the processes of TAG and cholesterol breakdown, absorption, and trafficking (Sieber, 2009)

Two genes with predicted TAG lipase activity are expressed at lower levels in DHR96 mutants. One of these genes, CG5932, is abundantly expressed in the larval and adult midgut, encodes a protein that is highly related to human gastric lipase (LIPF, 37% identity over 358 amino acids), and is essential for maintaining whole-animal TAG levels. In addition, restoring CG5932 expression in the midguts of DHR96 mutants is sufficient to rescue their lean phenotype, defining this gene as a critical functional target of the receptor. Interestingly, CG5932 expression is also regulated by starvation, with reduced expression in the absence of food and increased expression upon refeeding (Gershman, 2006). This regulation is consistent with an essential role for CG5932 in the breakdown of dietary fat, where its expression is upregulated when food is present. This response, however, is unaffected in DHR96 mutants, indicating that it is under independent control, possibly by known regulators of the starvation response such as dFOXO (Sieber, 2009).

The identification of CG5932 as a key functional target of DHR96 raises the question of how that regulation is achieved. A range of dietary parameters and candidate ligands were tested using the GAL4-DHR96 ligand sensor but conditions have not been identified that activate the DHR96 LBD. In addition, the DHR96-binding site remains undefined. DHR96 has a unique P box sequence within its DBD, which determines its DNA-binding specificity. This sequence is only shared by three C. elegans NRs, DAF-12, NHR-48, and NHR-8. Consistent with this observation, it was found that DHR96 protein fails to bind to most canonical NR-binding sites, except for weak binding to a palindromic EcR response element. One study has shown that DAF-12 displays preferential binding to a direct repeat of two distinct hexanucleotide sequences (AGGACA and AGTGCA), separated by five nucleotides (DR5) (Shostak, 2004). Whether DAF-12 contacts these sequences as a homodimer or a heterodimer with another NR, however, remains to be determined. The observation that many DHR96-regulated genes are arranged in clusters and DHR96 binds directly to a region upstream from the CG5932 start site provides an ideal context for defining its DNA-binding specificity as well as determining the molecular mechanisms by which DHR96 coordinates target gene transcription (Sieber, 2009).

These studies of DHR96 raise the interesting possibility that the defects in xenobiotic detoxification seen in DHR96 mutants may arise, at least in part, from its role in regulating midgut metabolic activity. As noted in the original study, most of the genes that are regulated by phenobarbital in wild-type flies do so independently of DHR96 (King-Jones, 2006). These genes include representatives of the major classes associated with xenobiotic detoxification: cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), carboxylesterases, and UDP-glucuronosyl transferases (UGTs). Moreover, some phenobarbital-regulated genes that are misregulated in DHR96 mutants still show a transcriptional response to the drug, although that response is muted. These observations indicate that one or more other factors contribute to the transcriptional response to xenobiotic challenge in Drosophila. Interestingly, similar results have been observed in vertebrates, where less then half of the genes that are regulated by xenobiotics are affected in PXR and CAR mutant mice (Maglich, 2002; Ueda, 2002]). It remains unclear, however, whether this lack of regulation might be due to functional redundancy between these mammalian receptors (Sieber, 2009).

There are several possible mechanisms by which the metabolic functions of DHR96 could impact xenobiotic responses. First, a recent study of the expression patterns of Drosophila P450 genes showed that 34 of 60 genes that could be detected in third-instar larvae are expressed in the midgut or hindgut (Chung, 2009). A similar overrepresentation of P450 genes is evident in a microarray study of midgut expressed genes (Li, 2008). These observations indicate that, contrary to previous assumptions, the gut, and not the fat body, may be a critical site for xenobiotic detoxification (Chung, 2009). If this is true, then the sensitivity of DHR96 mutants to phenobarbital or DDT treatment may be affected by the effects of this mutation on midgut physiology. This could occur through defects in the peritrophic matrix or through changes in the ability of the midgut to absorb lipophilic xenobiotic compounds such as DDT. An alternative possibility is that the sensitivity of DHR96 mutants to xenobiotics might be due to their decreased energy stores. Detoxification requires energy expenditure. For example, P450s consume NADPH or NADH for their oxidation of xenobiotics, UGTs consume glucose, and GSTs consume glutathione. Thus, the reduced levels of stored energy in DHR96 mutants might compromise their ability to properly inactivate toxic compounds. In addition, the reduced lipid stores in DHR96 mutants might exert an indirect effect on xenobiotic responses by lowering the ability of the animal to sequester toxins in the fat reserves of the animal. Thus, there are multiple pathways by which the midgut-specific metabolic defects associated with the DHR96 mutation might indirectly affect xenobiotic responses in these animals. Further studies are required to test this possibility and clarify the functional overlaps between the roles of DHR96 in lipid metabolism and xenobiotic detoxification (Sieber, 2009).

Several recent studies have demonstrated roles for both PXR and CAR in lipid metabolism (Moreau, 2008). CAR can repress the transcription of genes encoding carnitine palmitoyltransferase and enoyl-CoA isomerase, key steps in lipid β-oxidation (Ueda, 2002). CAR mutant mice are also sensitive to starvation, much as was observed for DHR96 mutants, and lose weight more rapidly than wild-type mice when maintained on a low-calorie diet (Maglich, 2004). Transgenic expression of a constitutively active form of PXR in the mouse liver leads to hepatic steatosis along with reduced expression of lipid catabolic genes and increased expression of genes involved in lipid synthesis (Zhou, 2006). Importantly, similar effects were observed upon pharmacological activation of PXR using a specific agonist, indicating that the endogenous receptor can contribute to lipid homeostasis (Hoekstra, 2009; Nakamura, 2007). Most recently, a mutation in CAR has been shown to normalize the elevated serum TAG levels seen in leptin-deficient mice or in wild-type mice maintained on a high-fat diet (Maglich, 2009). Conversely, treatment of wild-type mice with a selective CAR agonist leads to increased serum TAG levels, and this response fails to occur in a CAR mutant background. Taken together, these studies indicate that activation of PXR/CAR receptors leads to lipid accumulation while a loss of PXR/CAR activity leads to reduced lipid levels, defining a central role for these NRs in lipid homeostasis. Their role in normal lipid metabolism, however, remains unknown, although it may be masked by functional redundancy between the two receptors. Similarly, the molecular mechanisms by which PXR and CAR can modulate lipid levels remain to be defined (Sieber, 2009)

These genetic studies of DHR96 have demonstrated that metabolic activities of PXR and CAR have been conserved through evolution, and represent an essential ancestral function for this NR subfamily. Moreover, the observation that DHR96 overexpression leads to lipid accumulation and DHR96 mutants are lean suggests that the molecular mechanisms that underlie these effects are also conserved across species. This conclusion is supported by genetic studies of the C. elegans member of this subfamily, DAF-12, which indicate that this receptor is also required for proper levels of stored fat (Gerisch, 2001). This study provides further evidence of a role for this NR subfamily in normal lipid homeostasis and defines the control of dietary fat breakdown as a key step at which this regulation is achieved. In addition, this work provides a foundation for understanding how dietary lipid uptake can impact normal lipid metabolism in Drosophila and provides a genetic model for characterizing how dietary factors can lead to lipid metabolic disorders such as obesity (Sieber, 2009)

The DHR96 nuclear receptor regulates xenobiotic responses in Drosophila

Exposure to xenobiotics such as plant toxins, pollutants, or prescription drugs triggers a defense response, inducing genes that encode key detoxification enzymes. Although xenobiotic responses have been studied in vertebrates, little effort has been made to exploit a simple genetic system for characterizing the molecular basis of this coordinated transcriptional response. ~1000 transcripts are significantly affected by phenobarbital treatment in Drosophila. The Drosophila ortholog of the human SXR and CAR xenobiotic receptors, DHR96, plays a role in this response. A DHR96 null mutant displays increased sensitivity to the sedative effects of phenobarbital (PB) and the pesticide DDT as well as defects in the expression of many phenobarbital-regulated genes. Metabolic and stress-response genes are also controlled by DHR96, implicating its role in coordinating multiple response pathways. This work establishes a new model system for defining the genetic control of xenobiotic stress responses (King-Jones, 2006).

Although studies have used genome-wide microarrays to define the transcriptional profile of insecticide-resistant strains of Drosophila none have characterized the inducible transcriptional response to a toxic compound in wild-type insects. The data presented in this study, examining the effects of PB on transcription in wild-type flies, provide insights into how insects mount a defense response to toxic compounds. Approximately 1000 transcripts are affected by PB treatment, with many showing high-fold changes in expression. The vast majority of these genes encode enzymes, and many of these correspond to known detoxification pathways, including multiple P450s, GSTs, carboxylesterases, and UGTs. Although relatively few genes have been linked to specific xenobiotic functions in insects, the second most highly PB-induced gene (677-fold) Cyp6a8, can metabolize organophosphates, cyclodiene insecticides, and promutagens. Similarly, the PB-inducible Cyp6g1 and Cyp12a4 genes are sufficient to confer resistance to the pesticide Lufenuron. The overall types of enzymes regulated by PB in Drosophila resemble those seen in insecticide-resistant strains of Anopheles and Drosophila, as well as the enzymes affected by xenobiotics in mammalian studies, providing further evidence that the core detoxification machinery has been conserved through evolution, from insects to humans (King-Jones, 2006).

In addition to the main classes of detoxification genes, several gene families emerge from searches for overrepresented InterPro or gene ontology terms. These include 13 PB-inducible genes that encode proteins with a DUF227 domain. This apparently insect-specific domain is proposed to act as a choline/ethanolamine kinase (InterPro IPR004119). A recent study reported that a specific mutation in a DUF227 domain-encoding gene (CG10618) confers resistance to organophosphates in Drosophila. Two acyl-CoA synthetase genes are also among the ten most abundantly induced PB-responsive genes (CG4500 and CG6300/CG11659). Acyl-CoA synthetases have been implicated in xenobiotic metabolism in mammals, and an acyl-CoA synthetase is upregulated in an insecticide-resistant strain of Anopheles (King-Jones, 2006).

Remarkably, many PB-regulated genes not associated with overrepresented gene families may also contribute to general detoxification responses. For example, Jheh1, which is induced 16-fold by PB, encodes an epoxide hydrolase. These enzymes can detoxify epoxides by increasing their solubility and aiding in their excretion. Similarly, the glycine N-methyltransferase encoded by CG6188, which is induced 77-fold by PB, can bind polyaromatic hydrocarbons and contribute to mammalian Cyp1A1 induction. Thus, the genome-wide transcriptional response to PB has revealed not only new members of the known classes of detoxification enzymes but also a number of other pathways that are likely to contribute to xenobiotic responses, providing a new basis for understanding how insects defend themselves against environmental toxins. Recent studies have also shown that many of these genes are regulated in an identical manner by a different drug, the dopamine antagonist chlorpromazine, suggesting that this transcriptional pattern reflects a general defense response to xenobiotics (King-Jones, 2006).

DHR96 protein is ideally positioned to monitor the entry and exit of dietary nutrients and foreign compounds, coordinating xenobiotic stress and metabolic responses within the animal. DHR96 displays a highly restricted pattern of expression, limited primarily to organs that are involved in nutrient and xenobiotic absorption (gastric caeca), metabolism (fat body), and waste elimination (Malpighian tubules). This pattern is similar to that of PXR and CAR, which are most highly expressed in the liver and intestine. This expression pattern also reflects that of the two PB-inducible P450 transcripts that have been spatially localized. Cyp6a2 is expressed in the midgut, fat bodies, and Malpighian tubules of adult flies while Cyp12a4 is expressed primarily in the midgut and Malpighian tubules of third instar larvae. Unlike its mammalian orthologs, however, which reside in the cytoplasm and translocate to the nucleus upon xenobiotic challenge, DHR96 protein appears to be restricted to the nucleus (King-Jones, 2006).

DHR96 mutants display a significant increase in their sensitivity to the sedative effects of PB. This observation is similar to the prolonged sleep phenotype of PB-treated CAR mutant mice and the effects of PB treatment on resistance to the paralytic effects of the muscle relaxant zoxazolamine in CAR mutants. Of 144 genes that change their expression upon intraperitoneal injection of PB in mice, about half are dependent upon CAR for their proper response to the drug. This is similar to the effect seen in Drosophila, where 102 PB-regulated genes are affected by either the DHR96¹ loss-of-function mutation or ectopic DHR96 gain-of-function. In addition, many of these genes encode members of the four classic detoxification enzyme families, demonstrating that the insect xenobiotic response depends upon DHR96 for its proper implementation. Similar effects on xenobiotic gene regulation were seen in PXR-VP16 gain-of-function studies and with PXR mutant mice (King-Jones, 2006).

Interestingly, some genes are only PB-inducible in a DHR96 mutant background. The most strongly affected genes in this group are members of the Juvenile Hormone Binding Protein family (JHBPs. JHBPs are hemolymph carrier proteins that are capable of binding lipophilic compounds such as juvenile hormone, facilitating their transport within the animal and protecting them against nonspecific esterases. Whereas JHBP genes are not responsive to PB in wild-type animals, they are induced by PB in DHR96 mutants, indicating that DHR96 normally acts to block their expression. This could reflect a protective function for DHR96 in that JHBPs may normally protect lipophilic compounds from degradation, thus interfering with drug clearance. Several other genes are also induced by PB only in a DHR96 mutant background, including Glutathione Synthetase and the putative transcription factor CG14965. It remains unclear, however, how these genes might contribute to the detoxification response. Remarkably, a similar effect has been reported for CAR function in mice, where a subset of genes is only responsive to PB in a CAR mutant background. These genes include two P450 genes and genes encoding a calcium binding protein and glucosamine phosphate N-acetyl transferase. This so-called CAR-dependent blocking of gene expression may reflect an evolutionarily conserved aspect of the xenobiotic response pathway (King-Jones, 2006).

Further evidence of a role for DHR96 in xenobiotic response pathways arises from the finding that many potential detoxifying enzymes change their expression in untreated DHR96 mutants. Among this set, 83 genes were found that were downregulated in both untreated DHR96 mutants and PB-treated wild-type flies, including genes that encode transcription factors and numerous proteases and peptidases. Curiously, the relative fold changes of these genes are almost identical, in spite of their responding to two very different conditions, suggesting that untreated DHR96 mutants display some aspects of a toxin response. There are two possible models to explain this observation. First, DHR96 may regulate a specific branch of the xenobiotic network and the loss of DHR96 may result in the accumulation of toxic metabolites (endobiotics) that activate DHR96-independent xenobiotic pathways. Although at first glance, this 'endobiotic model' is attractive, relatively few PB-inducible genes are upregulated in untreated DHR96 mutants (18 genes). Thus, the mutants do not appear to display a xenobiotic response, but rather misregulate a set of genes that are normally repressed by PB treatment. As a result, an alternative model is favored, which proposes that DHR96 is required for normal expression of a subset of genes involved in the xenobiotic response pathway (exemplified by the 83 genes described in this study). PB then acts to inhibit this function of DHR96, resulting in the reduced expression of these DHR96 target genes. In addition, other xenobiotic receptors are likely to provide input to this pathway (King-Jones, 2006).

Given that treatment with a toxic compound is likely to impose stress on the animal, it is not surprising that a strong correlation was observed between genes up- and downregulated by PB and genes regulated in the same manner by stress. Studies with loss-of-function and gain-of-function DHR96 mutations also indicate a role for this receptor in regulating stress responses. Stress-response genes are upregulated in insecticide-resistant strains of Anopheles and some insecticide-resistant strains of insects are more resistant to oxidative stress, providing a functional link between these two pathways. The results suggest that this link is conferred at the level of specific stress-response gene regulation. In addition, PXR has been recently implicated in mediating oxidative stress responses, suggesting that at least some aspects of this regulation have been conserved through evolution (King-Jones, 2006).

PB treatment is also associated with a significant upregulation of genes involved in energy and sugar metabolism. The first two steps in gluconeogenesis are catalyzed by pyruvate carboxylase (CG1516) and phosphoenolpyruvate carboxykinase (PEPCK), enzymes encoded by genes that are upregulated by PB. Other glucose-generating processes show a similar response to PB, including six α-amylase genes that are induced more than 3-fold by the drug. Amylases are secreted by the salivary glands and midgut epithelia to break down dietary starch and glycogen into dextrins. Two genes encoding glucosidases, which can further degrade dextrins into monosaccharides, are also upregulated upon PB treatment. This effect is the opposite of that seen in mammals, where gluconeogenesis is downregulated by PB. It makes sense, however, that this pathway would be upregulated as part of the detoxification response. Toxin metabolism is energetically costly. P450s consume NADPH or NADH for their oxidation of xenobiotics, while UGTs consume glucose and GSTs consume glutathione. Thus, part of the metabolic response to xenobiotics appears to provide the appropriate energetic requirements for detoxification (King-Jones, 2006).

Although loss-of-function and gain-of-function genetic data indicate a role for DHR96 in mediating xenobiotic responses, it is interesting to note that the majority of PB-regulated genes are unaffected by DHR96 mutations. This is similar to studies in mice, where at least half of the detoxification gene network is unaffected by PXR or CAR null mutations. Given the massive coordinate regulation of the PB response, it seems likely that one or more additional transcriptional regulators feed into this pathway. These could include PAS-bHLH family members, analogous to the role of the mammalian aryl hydrocarbon receptor in regulating xenobiotic responses. Functional studies of PB-regulated promoters should provide insights into how their activity is controlled by the drug and identify additional players in their regulation. Similarly, the identification and characterization of direct targets for DHR96 transcriptional control will allow definition of how this receptor exerts its regulatory functions. Finally, DHR96 provides a potential target for the rational design of novel pesticides. By developing compounds that alter DHR96 activity it may be possible to increase the effectiveness of pesticide treatment for insect population control. Taken together, these studies provide a basis for using Drosophila as a genetic model for dissecting the regulation of xenobiotic responses, with implications for better understanding how these pathways are controlled in all higher organisms (King-Jones, 2006).

Search PubMed for articles about Drosophila DHR96

Brown, M. S. and Goldstein, J. L. (1997). The SREBP pathway: Regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89: 331-340. PubMed ID: 9150132

Bujold, M., Gopalakrishnan, A., Nally, E. and King-Jones, K. (2010). Nuclear receptor DHR96 acts as a sentinel for low cholesterol concentrations in Drosophila melanogaster. Mol Cell Biol 30: 793-805. PubMed ID: 19933845

Chawla, A., et al. (2001). Mangelsdorf, Nuclear receptors and lipid physiology: opening the X-files. Science 294: 1866-1870. PubMed ID: 11729302

Chung, H., et al. (2009). Characterization of Drosophila melanogaster cytochrome P450 genes. Proc. Natl. Acad. Sci. 106: 5731-5736. PubMed ID: 19289821

Duval, C., et al. (2006). Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine. Biochem. Biophys. Res. Commun. 340: 1259-1263. PubMed ID: 16414355

Fluegel, M. L., Parker, T. J. and Pallanck, L. J. (2006). Mutations of a Drosophila NPC1 gene confer sterol and ecdysone metabolic defects. Genetics 172(1): 185-96. 16079224

Gerisch, B., et al. (2001). A hormonal signaling pathway influencing C. elegans metabolism, reproductive development, and life span. Dev. Cell 1: 841-851. PubMed ID: 11740945

Gershman, B., et al. (2006). High resolution dynamics of the transcriptional response to nutrition in Drosophila: a key role for dFOXO. Physiol. Genomics 29: 24-34. PubMed ID: 17090700

Grönke, S., Müller, G., Hirsch, J., Fellert, S., Andreou, A., Haase, T., Jäckle, H. and Kühnlein, R. P. (2007). Dual lipolytic control of body fat storage and mobilization in Drosophila. PLoS Biol. 5(6): e137. PubMed ID: 17488184

Hegedus, D., et al. (2009). New insights into peritrophic matrix synthesis, architecture, and function. Annu. Rev. Entomol. 54: 285-302. PubMed ID: 19067633

Heltianu, C., Robciuc, A., Botez, G., Musina, C., Stancu, C., Sima, A. V., and Simionescu, M. (2011). Modified low density lipoproteins decrease the activity and expression of lysosomal acid lipase in human endothelial and smooth muscle cells. Cell Biochem. Biophys. 61: 209-216. PubMed ID: 21499898

Hoekstra, M., et al. (2009). Activation of the nuclear receptor PXR decreases plasma LDL-cholesterol levels and induces hepatic steatosis in LDL receptor knockout mice. Mol. Pharm. 6: 182-189. PubMed ID: 19183106

Horner, M. A., et al. (2009). The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis. Genes Dev. 23(23): 2711-2716. PubMed ID: 19952106

Huang X., Suyama, K., Buchanan, J., Zhu, A. J. and Scott, M. P. (2005). A Drosophila model of the Niemann-Pick type C lysosome storage disease: dnpc1a is required for molting and sterol homeostasis. Development 132(22): 5115-24. PubMed ID: 16221727

Huang, X., Warren, J. T., Buchanan, J., Gilbert, L. I. and Scott, M. P. (2007). Drosophila Niemann-Pick type C-2 genes control sterol homeostasis and steroid biosynthesis: a model of human neurodegenerative disease. Development 134(20): 3733-42. PubMed ID: 17804599

Kalaany, N., and Mangelsdorf, D. (2006). LXRS and FXR: The yin and yang of cholesterol and fat metabolism. Annu. Rev. Physiol. 68: 159-191. PubMed ID: 16460270

King-Jones, K., Horner, M. A., Lam, G., Thummel, C. S. (2006). The DHR96 nuclear receptor regulates xenobiotic responses in Drosophila. Cell Metab. 4(1): 37-48. PubMed ID: 16814731

Li, H. M., et al. (2008). Transcriptomic profiles of Drosophila melanogaster third instar larval midgut and responses to oxidative stress. Insect Mol. Biol. 17: 325-339. PubMed ID: 18651915

Maglich, J. M., et al. (2002). Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Mol. Pharmacol. 62: 638-646. PubMed ID: 12181440

Maglich, J. M., et al. (2004). The nuclear receptor CAR is a regulator of thyroid hormone metabolism during caloric restriction. J. Biol. Chem. 279: 19832-19838. PubMed ID: 15004031

Maglich, J. M., Lobe, D. C. and Moore, J. T. (2009). The nuclear receptor CAR (NR1I3) regulates serum triglyceride levels under conditions of metabolic stress. J. Lipid Res. 50: 439-445. PubMed ID: 18941143

Moreau, A., et al. (2008). Xenoreceptors CAR and PXR activation and consequences on lipid metabolism, glucose homeostasis, and inflammatory response. Mol. Pharm. 5: 35-41. PubMed ID: 18159929

Nakamura, L., et al. (2007). R. Moore, M. Negishi and T. Sueyoshi, Nuclear pregnane X receptor cross-talk with FoxA2 to mediate drug-induced regulation of lipid metabolism in fasting mouse liver. J. Biol. Chem. 282: 9768-9776. PubMed ID: 17267396

Peet, D. J., et al. (1998). Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR α. Cell 93: 693-704. PubMed ID: 9630215

Shostak, Y., et al. (2004). Identification of C. elegans DAF-12-binding sites, response elements, and target genes. Genes Dev. 18: 2529-2544. PubMed ID: 15489294

Sieber, M. H. and Thummel, C. S. (2009). The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila. Cell Metab. 10(6): 481-90. PubMed ID: 19945405

Sieber, M. H. and Thummel, C. S. (2012). Coordination of triacylglycerol and cholesterol homeostasis by DHR96 and the Drosophila LipA homolog magro. Cell Metab. 15(1): 122-7. PubMed ID: 22197324

Sonoda, J., Pei, L. and Evans, R. M. (2008). Nuclear receptors: decoding metabolic disease. FEBS Lett. 582: 2-9. PubMed ID: 1802328

Timsit, Y. E. and Negishi, M. (2007). CAR and PXR: the xenobiotic-sensing receptors. Steroids 72: 231-246. PubMed ID: 17284330

Valasek, M. A., Clarke, S. L. and Repa, J. J. (2007). Fenofibrate reduces intestinal cholesterol absorption via PPARα-dependent modulation of NPC1L1 expression in mouse. J. Lipid Res. 48: 2725-2735. PubMed ID: 17726195

van der Velde, A. E., Brufau, G., and Groen, A. K. (2010). Transintestinal cholesterol efflux. Curr. Opin. Lipidol. 21: 167-171. PubMed ID: 20410820

Voght, S. P., Fluegel, M. L., Andrews, L. A., and Pallanck, L. J. (2007). Drosophila NPC1b promotes an early step in sterol absorption from the midgut epithelium. Cell Metab. 5: 195-205. PubMed ID: 17339027

Willson, T. M. and Kliewer, S. A. (2002). PXR, CAR and drug metabolism, Nat. Rev. Drug Discov. 1: 259-266. PubMed ID: 12120277

Ueda, A., et al. (2002). Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital. Mol. Pharmacol. 61: 1-6. PubMed ID: 11752199

Zhou, J., et al. (2006). A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway. J. Biol. Chem. 281: 15013-15020. PubMed ID: 16556603

date revised: 10 October 2012

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