InteractiveFly: GeneBrief

estrogen-related receptor: Biological Overview | References


Gene name - estrogen-related receptor

Synonyms - dERR

Cytological map position - 66B11-66B11

Function - nuclear receptor

Keywords - A coordinate switch in gene expression that drives a metabolic program normally associated with proliferating cells - supports growth during larval development

Symbol - ERR

FlyBase ID: FBgn0035849

Genetic map position - chr3L:8176477-8179766

Classification - NR_LBD: The ligand binding domain of nuclear receptors; zf-C4: Zinc finger

Cellular location - nuclear



NCBI link: EntrezGene

ERR orthologs: Biolitmine
Recent literature
Li, H., Chawla, G., Hurlburt, A.J., Sterrett, M.C., Zaslaver, O., Cox, J., Karty, J.A., Rosebrock, A.P., Caudy, A.A. and Tennessen, J.M. (2017). Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth. Proc Natl Acad Sci U S A 114: 1353-1358. PubMed ID: 28115720
Summary:
L-2-hydroxyglutarate (L-2HG) has emerged as a putative oncometabolite that is capable of inhibiting enzymes involved in metabolism, chromatin modification, and cell differentiation. However, despite the ability of L-2HG to interfere with a broad range of cellular processes, this molecule is often characterized as a metabolic waste product. This study demonstrates that Drosophila larvae use the metabolic conditions established by aerobic glycolysis to both synthesize and accumulate high concentrations of L-2HG during normal developmental growth. A majority of the larval L-2HG pool is derived from glucose and dependent on the Drosophila estrogen-related receptor (dERR), which promotes L-2HG synthesis by up-regulating expression of the Drosophila homolog of lactate dehydrogenase (dLdh). dLDH is both necessary and sufficient for directly synthesizing L-2HG and the Drosophila homolog of L-2-hydroxyglutarate dehydrogenase (dL2HGDH), which encodes the enzyme that breaks down L-2HG, is required for stage-specific degradation of the L-2HG pool. In addition, dLDH also indirectly promotes L-2HG accumulation via synthesis of lactate, which activates a metabolic feed-forward mechanism that inhibits dL2HGDH activity and stabilizes L-2HG levels. Finally, dLDH and L-2HG influence position effect variegation and DNA methylation, suggesting that this compound serves to coordinate glycolytic flux with epigenetic modifications. Overall, these data demonstrate that growing animal tissues synthesize L-2HG in a controlled manner, reveal a mechanism that coordinates glucose catabolism with L-2HG synthesis, and establish the fly as a unique model system for studying the endogenous functions of L-2HG during cell growth and proliferation.

Kovalenko, E. V., Mazina, M. Y., Krasnov, A. N. and Vorobyeva, N. E. (2019). The Drosophila nuclear receptors EcR and ERR jointly regulate the expression of genes involved in carbohydrate metabolism. Insect Biochem Mol Biol 112: 103184. PubMed ID: 31295549
Summary:
The rate of carbohydrate metabolism is tightly coordinated with developmental transitions in Drosophila, and fluctuates depending on the requirements of a particular developmental stage. These successive metabolic switches result from changes in the expression levels of genes encoding glycolytic, tricarboxylic acid cycle (TCA), and oxidative phosphorylation enzymes. This report describes a repressive action of ecdysone signaling on the expression of glycolytic genes and enzymes of glycogen metabolism in Drosophila development. The basis of this effect is an interaction between the ecdysone receptor (EcR) and the estrogen-related receptor (ERR), a specific regulator of the Drosophila glycolysis. An overlapping DNA-binding pattern was found for the EcR and ERR in the Drosophila S2 cells. EcR was detected at a subset of the ERR target genes responsible for carbohydrate metabolism. The 20-hydroxyecdysone treatment of both the Drosophila larvae and the S2 cells decreased transcriptional levels of ERR targets. A joint action mode is proposed for both the EcR and ERR, for at least a subset of the glycolytic genes. Both receptors bind to the same regulatory regions and may form or be part of a joint transcriptional regulatory complex in the Drosophila S2 cells.
Bartolo, G., Gonzalez, L. O., Alameh, S., Valencia, C. A. and Martchenko Shilman, M. (2020). Identification of glucocorticoid receptor in Drosophila melanogaster. BMC Microbiol 20(1): 161. PubMed ID: 32539689
Summary:
Vertebrate glucocorticoid receptor (GR) is an evolutionary-conserved cortisol-regulated nuclear receptor that controls key metabolic and developmental pathways. Upon binding to cortisol, GR acts as an immunosuppressive transcription factor. Drosophila melanogaster, a model organism to study innate immunity, can also be immunosuppressed by glucocorticoids. However, while the genome of fruit fly harbors 18 nuclear receptor genes, the functional homolog of vertebrate GR has not been identified. This study demonstrated that while D. melanogaster is susceptible to Saccharomyces cerevisiae oral infection, the oral exposure to cortisol analogs, cortisone acetate or estrogen, increases fly sensitivity to yeast challenge. To understand the mechanism of this steroid-induced immunosuppression, the closest genetic GR homolog was identified as D. melanogaster Estrogen Related Receptor (ERR) gene. Drosophila ERR is necessary for cortisone acetate- and estrogen-mediated increase in sensitivity to fungal infection: while ERR mutant flies are as sensitive to the fungal challenge as the wildtype flies, the yeast-sensitivity of ERR mutants is not increased by these steroids. Interestingly, the fungal cortisone analog, ergosterol, did not increase the susceptibility of Drosophila to yeast infection. The immunosuppressive effect of steroids on the sensitivity of flies to fungi is evolutionary conserved in insects, as we show that estrogen significantly increases the yeast-sensitivity of Culex quinquefasciatus mosquitoes, whose genome contains a close ortholog of the fly ERR gene. This study identifies a D. melanogaster gene that structurally resembles vertebrate GR and is functionally necessary for the steroid-mediated immunosuppression to fungal infections.
BIOLOGICAL OVERVIEW

Metabolism must be coordinated with development to provide the appropriate energetic needs for each stage in the life cycle. Little is known, however, about how this temporal control is achieved. This study shows that the Drosophila ortholog of the estrogen-related receptor (ERR) family of nuclear receptors directs a critical metabolic transition during development. Drosophila ERR mutants die as larvae with low ATP levels and elevated levels of circulating sugars. The expression of active dERR protein in mid-embryogenesis triggers a coordinate switch in gene expression that drives a metabolic program normally associated with proliferating cells, supporting the dramatic growth that occurs during larval development. This study shows that dERR plays a central role in carbohydrate metabolism, demonstrates that a proliferative metabolic program is used in normal developmental growth, and provides a molecular context to understand the close association between mammalian ERR family members and cancer (Tennessen, 2011).

Metabolism must be tightly coupled with developmental progression, with distinct metabolic programs supporting the nutritional and energetic requirements of each stage in the life cycle. Relatively few studies, however, have addressed the molecular mechanisms that provide this temporal coordination. One of the best-characterized metabolic transitions occurs in the developing mammalian heart and is represented by a switch in substrate utilization from glucose and lactate metabolism during fetal stages to postnatal dependence on fatty acid oxidation (Lehman, 2002). This switch is accompanied by the coordinate induction of genes involved in mitochondrial β oxidation and oxidative metabolism and mediated by the nuclear receptors PPARα and ERRγ (Lehman, 2002; Alaynick, 2007). Interestingly, this process reverts in pathological forms of cardiac hypertrophy, demonstrating that metabolic transitions can be associated with disease. In addition, the nutritional status of early developmental stages can have a profound effect on later metabolic health. One of the most dramatic manifestations of this interplay between nutrition and development is the impact of childhood obesity on the incidence of type 2 diabetes and obesity in adults. Obesity also impacts the timing of sexual maturation, linking a metabolic state to a key developmental transition. In spite of this important interplay between nutrition, metabolism, and development, little is understood about how these pathways are integrated (Tennessen, 2011).

Nuclear receptors are a specialized family of ligand-regulated transcription factors that play central roles in controlling development, growth, and metabolism. They are defined by a conserved zinc-finger DNA-binding domain and a C-terminal ligand-binding domain (LBD) that can impart multiple regulatory functions. One subfamily of these receptors are the estrogen-related receptors (ERRs), represented by three paralogs in mammals, ERRα, ERRβ, and ERRγ. Although some synthetic estrogen analogs can suppress the constitutive transcriptional activity of these receptors, they have no known naturally occurring ligands. Genetic studies in mice have demonstrated roles for ERR family members in mitochondrial biogenesis, oxidative phosphorylation, and lipid metabolism (for a review, see Tremblay, 2007). Consistent with these functions, ERRα mutant mice are lean and resistant to diet-induced obesity (Luo, 2003). In addition, ERRα and ERRγ are essential for proper cardiac metabolism. ERRα is required for energy production in response to cardiac stress (Huss, 2007), whereas ERRĪ³ directs a metabolic switch that allows the postnatal heart to metabolize fatty acids (Alaynick, 2007). Recent studies, however, suggest that ERRs play a broader role in metabolic homeostasis (Ao, 2008; Charest-Marcotte, 2010; Eichner, 2010). Moreover, all three mammalian ERRs are associated with cancer progression. ERRα is necessary for the normal growth of estrogen receptor-negative breast cancer tumor grafts, and elevated ERRα expression is associated with aggressive forms of breast cancer. In contrast, ERRβ inhibits the progression of prostate cancer, and increased expression of ERRγ is correlated with a favorable clinical prognosis for breast cance. These observations suggest that ERR family members coordinate cell growth and proliferation with metabolism. The molecular basis for this relationship, however, remains unclear (Tennessen, 2011).

This study presents a functional study of the Drosophila ERR ortholog, dERR. The presence of only a single ERR gene in flies eliminates the potential genetic redundancy between multiple members of the ERR subfamily, allowing determination of its key ancestral functions. dERR is shown to be an essential regulator of carbohydrate metabolism during larval stages. dERR mutants die during the second larval instar with abnormally high levels of circulating sugar and diminished concentrations of ATP and triacylglycerides (TAG). These metabolic defects result from decreased expression of genes involved in glycolysis, the pentose phosphate pathway, and other aspects of carbohydrate metabolism. These genes are coordinately induced midway through embryogenesis, in apparent direct response to the expression of activated dERR protein. Interestingly, this dERR-regulated metabolic state is ideally suited to promote larval growth by converting dietary carbohydrates into biomass and is strikingly reminiscent of the Warburg effect, the observation that cancer cells exhibit glycolysis with lactate secretion and mitochondrial respiration even in the presence of oxygen. These studies reveal that an ERR family member coordinates metabolism with growth, indicate that a proliferative metabolic program is used in the context of normal development, and suggest that mammalian ERRs are associated with cancer through their ability to promote the Warburg effect (Tennessen, 2011).

Drosophila larval development is characterized by an ∼200-fold increase in body mass. This period of dramatic growth requires the efficient conversion of dietary nutrients into cellular building blocks, such as amino acids, fatty acids, and nucleotides. The metabolic basis that supports juvenile growth, however, has not been defined. The studies described here support the model that Drosophila larval growth depends on a form of aerobic glycolysis that is similar to the Warburg effect, and that this metabolic program is established as a mid-embryonic transcriptional switch in response to activated dERR protein (Tennessen, 2011).

Microarray study of dERR mutant larvae revealed a profound and widespread effect on genes that control carbohydrate metabolism, including highly reduced expression of almost all genes in glycolysis and the pentose phosphate pathway (PPP). This is consistent with the ∼2-fold increase in trehalose seen in dERR mutants, along with highly reduced levels of ATP. Mutation of the rate-limiting step in glycolysis, Phosphofructokinase (Pfk), results in elevated trehalose levels, similar to the dERR mutant, and ectopic expression of Phosphoglucose isomerase (Pgi) and Pfk in dERR mutants is sufficient to rescue this phenotype. These studies demonstrate an essential role for dERR in carbohydrate catabolism during larval stages (Tennessen, 2011).

Metabolic profiling confirms these observations, showing significant accumulation of a number of sugars, including glucose-6-phosphate, the first intermediate in the glycolytic pathway. This study, however, also reveals changes in the levels of a number of other key metabolites, providing a broader understanding of dERR function. Lactate is almost completely absent in dERR mutants, consistent with lactate dehydrogenase being one of the most highly downregulated genes in these animals. Levels of α-ketoglutarate, malate, and other late-stage TCA cycle intermediates are also significantly reduced in dERR mutant larvae, along with depletion of several amino acids, of which the most significant and reproducible is proline (Tennessen, 2011).

Interestingly, when taken together with the widespread effects of dERR on glycolysis and the PPP, these changes in metabolite levels are consistent with a form of aerobic glycolysis that is normally associated with cell proliferation. In the context of cancer, this metabolic signature is referred to as the Warburg effect. The increase in carbohydrate metabolism is not designed to produce ATP, but rather promotes the synthesis of amino acids, lipids, and nucleotides, thereby supporting cellular proliferation. Both highly proliferative cells and Drosophila larvae shunt large quantities of glucose through the PPP, allowing them to generate ribose-5-phosphate for nucleotide synthesis and NADPH for fatty acid synthesis and other biosynthetic reactions. Inadequate acetyl-CoA production from glycolysis in dERR mutants and reduced NADPH generation via the PPP likely results in decreased fatty acid synthesis, accounting for the reduced TAG levels in these animals). In support of this hypothesis, TAG levels are reduced by 32% in animals that carry a loss-of-function mutation in glucose-6-phosphate dehydrogenase, which encodes a rate-limiting step in the PPP. Moreover, mutations in PPP enzymes significantly decrease the rate of carbohydrate-dependent fatty acid synthesis in larvae. Similarly, elevated Ldh activity, which is a hallmark of cancer metabolism, is present in normal Drosophila larvae, which display elevated expression of the Drosophila ortholog of Ldh and high levels of Ldh enzyme activity. This enzyme converts pyruvate into lactate, preventing pyruvate from entering the mitochondria and generating NAD+ to promote maximal glycolytic flux. As a result of this diversion of pyruvate away from energy production, the TCA cycle in proliferating cells becomes dependent on amino acids derived from glutamic acid. Large amounts of glutamine are consumed by these cells to anaplerotically maintain the concentration of TCA intermediates. In an analogous manner, Drosophila larval metabolism appears to rely heavily on proline, which is significantly reduced in dERR mutants. Many insects use proline to generate energy and anaplerotically replenish the TCA cycle in the same way that proliferating yeast and cancer cells rely on glutamine. Taken together, these observations support the model that dERR establishes a metabolic state that is related to cellular proliferation, and that this function is essential for larval viability and growth (Tennessen, 2011).

These studies also provide initial insights into the tissue-specific metabolic programs regulated by dERR during larval stages. When dERR is expressed in only the muscle or epidermis, it promotes transcription of the core glycolytic pathway. In contrast, specific expression of dERR in the fat body upregulates Phosphogluconate dehydrogenase (Pgd), which encodes an essential enzyme in the PPP that is induced by sugar consumption. These results are consistent with the metabolic requirements of these different tissues. The dramatic expansion of the epidermis during larval growth, along with its production of cuticle, requires efficient glucose catabolism, as does the muscle to provide larval movement. In contrast, the fat body is one of the principle sites where sugar is processed by the PPP, and efficient lipid storage requires PPP activity. These observations indicate that dERR promotes appropriate tissue-specific metabolic programs during larval development (Tennessen, 2011).

A role for dERR in directing a metabolic state normally associated with cell proliferation provides a new context to understand how Drosophila larvae undergo their remarkable 200-fold increase in mass during the 4 days of larval development. It has long been known that many glycolytic enzymes are induced at the onset of larval development and that the PPP and Ldh are highly active at this stage. No evidence, however, has tied these pathways together. In addition, although yeast use a similar mechanism to support their proliferation, no studies have addressed the metabolic state that accompanies juvenile development in higher organisms. These results suggest that dERR directs a coordinated metabolic program that supports the unusual growth that occurs during this stage. These studies of the dERR-regulated transcriptional program also reveal that this metabolic state is established by a coordinate switch in gene expression during mid-embryogenesis. Accumulation of active dERR protein in 12-18 hr embryos directly induces Pfk transcription and, probably, other key dERR target genes. This transcriptional switch in turn establishes the metabolic requirements for the next stage in development. The timing of the accumulation of active dERR protein could be regulated at a number of levels including posttranslational modifications, ligand binding, and/or cofactor recruitment. Further studies are needed to define the mechanisms that regulate this response. It is interesting to note that the use of aerobic glycolysis to support developmental growth may not be restricted to Drosophila larval stages. Many animals undergo exponential growth during embryonic and fetal stages of development. It will be interesting to determine whether similar metabolic states are associated with early growth in other organisms (Tennessen, 2011).

Studies of mammalian ERR family members have largely focused on their roles in mitochondrial biogenesis and oxidative phosphorylation (for a review, see Tremblay and Giguère, 2007). The microarray study of RNA isolated from dERR mutant larvae, however, identified very few genes associated with β oxidation, the TCA cycle, or the electron transport chain. Although genes involved in oxidoreductase activity are upregulated in dERR mutants, most encode cytochrome P450s with unknown functions. Similarly, a number of genes encoding mitochondrial ribosomal proteins are expressed at reduced levels in dERR mutants, although these effects are relatively minor (≤1.5-fold). dERR mutant larvae have normal mitochondrial genome number and mitochondrial morphology, demonstrating no detectable effect on mitochondrial biogenesis. These observations are consistent with a primary function for dERR in biomass production during larval stages, rather than energy generation and oxidative phosphorylation. Rather, it is speculated that dERR may play a more central role in mitochondrial function during adult stages, when the fly is highly dependent on oxidative metabolism to support its increased mobility (Tennessen, 2011).

Conversely, several recent studies have expanded understanding of the metabolic functions of mammalian ERR family members to include those controlled by dERR. ERRγ regulates glycolytic gene expression in the heart, ERRα is bound to the extended promoters of genes involved in glycolysis and the TCA cycle, and ERRγ regulates several genes in these pathways (Alaynick, 2007; Charest-Marcotte, 2010; Eichner, 2010). Similarly, ERRα is required for the upregulation of genes encoding glycolytic enzymes when cells are raised under hypoxic conditions (Ao, 2008). These studies of dERR define carbohydrate metabolism as a key ancestral function for this nuclear receptor subclass, aspects of which have been conserved through evolution to mammals (Tennessen, 2011).

This work also provides a new context to understand roles for mammalian ERR family members in cancer progression. Many studies have demonstrated a close association between ERR receptors and cancer. The molecular basis for this association, however, remains unclear, likely because of the functional redundancy and crosstalk between mammalian ERR paralogs. A recent paper has provided an initial step in this direction, showing that the miR-378 microRNA, which promotes the Warburg effect in BT-474 cancer cells, downregulates ERRγ expression in this context (Eichner, 2010). These studies of the single Drosophila ERR family member raise the possibility that mammalian ERRs control the dramatic cellular proliferation associated with cancer through their ability to promote the Warburg effect (Tennessen, 2011).

Drosophila estrogen-related receptor directs a transcriptional switch that supports adult glycolysis and lipogenesis

Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. This study shows that the Drosophila Estrogen-related receptor (ERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. ERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by ERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, it was found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on ERR, leading to the metabolic defects observed in mutants. These data support the model that ERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly (Beebe, 2020).

This study focuses on the transcriptional regulation of metabolism by ERR in Drosophila. The ERRs comprise a subfamily of orphan nuclear receptors that act as transcriptional activators. There are three mammalian ERR paralogs: ERRα, ERRβ, and ERRγ. ERRβ functions as a pluripotency factor in stem cells and is required for the growth of extraembryonic tissues as well as neuronal regulation of feeding behavior and systemic energy balance (Huss, 2015). In contrast, extensive studies have defined metabolic functions for ERRα and ERRγ in multiple tissues, including the liver, muscle, and adipose tissue (Huss, 2015; Misra, 2017; Xia, 2019). These have revealed central roles for these receptors in oxidative energy metabolism and mitochondrial function, in part through their close association with the PGC-1α coactivator. Genetic studies of ERR family members, however, have been complicated by their functional redundancy. ERRα and ERRγ are expressed widely in overlapping patterns in the mouse and ERRγ expression is elevated in the absence of ERRα, indicating compensatory control. A recent study in brown adipose tissue demonstrated the majority of the adaptive response to adrenergic stimulation is dependent on the combined functions of these two ERR paralogs (Brown, 2018). Similarly, ERRβ and ERRγ are expressed in overlapping patterns in the mouse hindbrain and ERRγ levels are elevated in ERRβ mutant neurons with downstream effects on target gene expression (Byerly, 2013). A current goal for the field is to dissect these redundant ERR activities through the use of double mutants (Beebe, 2020).

These studies are focused on functional analysis of the single ERR ortholog in Drosophila with the goal of better understanding its ancestral functions in the absence of genetic redundancy. Initial work showed that ERR null mutants go through early development normally but die as mid-second instar larvae with hyperglycemia and hypolipidemia (Tennessen, 2011). GC/MS metabolomics revealed that wild-type larvae normally display aerobic glycolytic metabolism similar to cancer cells, with abundant lactate and 2-hydroxyglutarate, and this progrowth metabolic profile is dependent on ERR function (Tennessen, 2011; Li, 2017). Interestingly, dERR establishes this metabolic state midway through embryogenesis when it coordinately induces genes that encode enzymes in glycolysis, the pentose phosphate pathway (PPP), and lactate dehydrogenase (Ldh) (Tennessen, 2011; Tennessen, 2014). These observations support the model that ERR links metabolism with developmental progression by establishing a metabolic state in late embryos that is optimized for the rapid and massive growth that occurs during larval development (Beebe, 2020).

This study extenda functional analysis of ERR to the adult fly when there is no developmental growth. Using a conditional allele, it was shown that, similar to ERR mutant larvae, mutant adults display reduced expression of genes in the glycolytic and pentose phosphate pathways, many of which appear to be directly induced by the receptor. In contrast to larvae, however, this results in reduced levels of citrate and downstream lipogenesis, leading to an almost complete absence of stored triglycerides in the adult fat body. Transcriptional profiling of staged control and ERR mutant pupae and adults revealed that this metabolic state is normally induced during mid-pupal development and the glycolytic transcriptional switch is disrupted in ERR mutants. It is concluded that ERR acts during pupal stages to contribute to the physiological state of the adult fly by up-regulating metabolic pathways that support glucose oxidation and lipogenesis. This study provides insights into the molecular mechanisms that allow the animal to transition from an immobile pupa into a highly motile and reproductively active adult fly (Beebe, 2020).

Metabolism changes dramatically as animals progress through development. How these systemic metabolic states are coordinated with the life cycle, however, remains unclear. Although some transitions can occur slowly and thus could involve complex compensatory and feedback regulation, other metabolic transitions occur within a period of a few hours or days and require a more rapid and direct form of control. This study shows that the onset of adulthood in Drosophila is accompanied by a widespread switch in gene expression that occurs midway through metamorphosis. This transition involves the central pathways of energy metabolism, glycolysis, the TCA cycle, and electron transport chain, and thus is ideally designed to meet the increased energy demands of adult motility. This study shows that the ERR nuclear receptor is required for part of this transcriptional response, inducing the expression of key genes in glycolysis, the PPP, and lipogenesis. Below wthis regulatory response is described in more detail and a model is proposedfor how ERR contributes to the pupal-to-adult metabolic transition (Beebe, 2020).

CRISPR/Cas9 was used to generate a conditional ERR mutant allele in order to facilitate functional studies during later stages of development. Although widely used in mice, conditional alleles are rarely implemented in Drosophila largely due to the ease of RNAi studies and clonal analysis. Conditional alleles, however, provide an ideal way to characterize organism-wide responses at any stage of development as well as a genetic alternative to cell type and tissue-specific RNAi, which can be complicated by partial knockdown and off-target effects (Beebe, 2020).

ERR mutant adults eclose at a normal rate and with normal metabolites. This implies that metamorphosis can proceed with basal levels of glycolysis. This is perhaps not surprising given that insects do not feed during pupal stages but instead depend on stored energy and nutrients recycled from the autophagic death of larval tissues. In addition, the time course analysis of metabolic gene expression reveals an unexpected role for ERR in enhancing glycolysis at puparium formation. ERR mutants show a modest but significant reduction in glycolytic transcripts during early stages of metamorphosis, which in control animals are already at relatively low levels. Interestingly, this correlates with the transcriptional induction of ERR mRNA in apparent response to the late larval pulse of 20-hydroxyecdysone. Further studies are required to determine the possible significance of this regulatory connection with ecdysone signaling and roles for glycolysis during early metamorphosis (Beebe, 2020).

The accumulation of lipid in the newly formed adult fat body appears to be entirely dependent on de novo lipogenesis, as reflected by the absence of triglycerides and neutral lipids in adults starved at eclosion (Storelli, 2019). It is proposed that this efficient conversion of dietary nutrients into stored fat is regulated by ERR at the onset of adulthood. Several lines of evidence support this model. First, ERR mutants rapidly become hypolipidemic, leading to an almost complete absence of lipids by 1 wk of age, resembling the hypolipidemia seen in controls starved at eclosion (Storelli, 2019). In addition, unlike controls, a high sugar diet has no effect on the low triglyceride levels in ERR mutant adults. This is consistent with the reduced expression of genes that encode glycolytic enzymes, many of which appear to be directly regulated by ERR. This reduced rate of glycolysis leads to an accumulation of glucose along with reduced downstream TCA cycle intermediates, including citrate, which is the key precursor for lipogenesis. In addition, ATP citrate lyase (ATPCL) is expressed at reduced levels in ERR mutants. This gene encodes the enzyme that converts cytoplasmic citrate into the critical precursor for lipid synthesis, acetyl CoA. Lipogenesis is also dependent on NADPH, which is largely derived from the PPP, another pathway that appears to be under direct control by ERR. In addition, Gpdh, which encodes a key enzyme that supports triglyceride biosynthesis, is expressed at reduced levels in ERR mutants. Taken together, these effects of ERR on glycolysis, the PPP, citrate levels, Gpdh expression, and ATPCL expression, can account for the reduced levels of fatty acids seen in dERR mutants along with the reduced levels of stored fat (Beebe, 2020).

In addition to hypolipidemia, ERR mutant adults rapidly develop hyperglycemia and hypoproteinemia. As mentioned above, the hyperglycemia is likely due, at least in part, to reduced glycolysis, which leads to an accumulation of glucose along with fructose and mannitol. Similarly, the reduced protein levels seen in ERR mutants are consistent with the fundamental block in energy metabolism in these animals. It is likely that ERR mutants are breaking down protein in order to compensate for the reduced entry of pyruvate into the TCA cycle. This possibility is supported by the major reductions in alanine and proline in ERR mutants seen by metabolomic analysis. In addition, a shift from glucose oxidation to protein catabolism is a hallmark of the starved state and thus could contribute to the shortened life span and reduced motility seen in ERR mutant adults. It is also interesting to note that ERR mutant larvae do not display a reproducible effect on total protein levels. Consistent with this, proline is only reduced about twofold in mutant larvae and alanine levels are only slightly affected (Tennessen, 2011). Similarly, citrate, isocitrate, and succinate levels are all unaffected in dERR mutant larvae and significantly reduced in ERR mutant adults. These stage-specific differences appear to reflect a more significant reduction in TCA cycle activity in mutant adults and thus could explain why triglycerides are more severely affected at this stage (Beebe, 2020).

Previous studies have demonstrated that the adult fat body is a critical tissue for de novo lipogenesis. The current results support this conclusion and show that ERR and glycolysis are both required in this tissue for proper lipid accumulation. Disruption of either ERR or Pfk in the adult fat body leads to reduced levels of triglycerides and reduced neutral lipids in adult fat tissue. These phenotypes are similar to the hypolipidemia seen in ERR mutants, supporting the proposal that efficient glycolysis in the adult fat body is required to maintain appropriate levels of stored lipid. However, it is important to note that ERR also has effects on multiple metabolic pathways that act in tissues outside the fat body. Further studies are required to dissect these activities and determine how they might contribute to adult physiology and health (Beebe, 2020).

Interestingly, roles for ERR in maintaining stored lipid reserves appear to be conserved through evolution. ERRα mutant mice appear normal and are fully viable but display reduced body weight, reduced peripheral adipose mass, and are resistant to diet-induced obesity. Recent studies of ERRα mutant mice have confirmed and extended these original observations and also shown that pharmacological inhibition of ERRα leads to reduced body weight as well as reduced hepatic lipid accumulation and reduced gonadal and inguinal fat mass (Kim, 2013; B'Chir, 2018). To date, however, no studies have demonstrated a direct role for ERRα in supporting normal glycolytic oxidation or lipogenesis and thus it remains unclear whether the molecular mechanisms that are described in this study for ERR are conserved through evolution. However, it is important to note that glycolytic genes are expressed at reduced levels in ERRγ mutant hearts and ERRα is bound to almost every gene in the glycolytic pathway in the mouse liver. In addition, ERR has been shown to support glycolytic Warburg metabolism in cancer cells. Further studies are required to address possible roles for mammalian ERR family members in supporting glucose oxidation for energy production and lipogenesis (Beebe, 2020).

Although these studies demonstrate that ERR is required to establish adult lipid reserves, its central role in glycolysis is also consistent with the high energetic demands of adult motility. Indeed, at peak capacity, Drosophila flight sustains remarkable mechanical power driven by highly efficient energy production. Adult flies must therefore maintain high levels of pyruvate to fuel ATP synthesis through mitochondrial oxidative phosphorylation. Unlike larvae, which efficiently convert pyruvate into lactate to support aerobic glycolysis, Ldh expression is relatively low during pupal and adult development. In addition, previous studies have shown that the dHNF4 nuclear receptor triggers a transcriptional switch in newly emerged adults that supports mitochondrial function and maximal ATP production (Barry, 2016). It is proposed that it is the coordinated activities of ERR and HNF4 that establish the efficient energetic state that characterizes adult life. ERR is required for a transcriptional switch during mid-pupal stages that upregulates glycolysis along with other pathways. This is followed by the induction of dHNF4 at the end of metamorphosis, activating downstream target genes that promote mitochondrial oxidative metabolism (Barry and Thummel 2016). Taken together, these two nuclear receptors can direct the efficient conversion of glucose into ATP, promoting oxidative energy production. In addition, this nuclear receptor mediated metabolic switch provides a molecular context to understand the so-called 'U-shaped curve of oxygen consumption that occurs during insect metamorphosis. Studies in multiple insect species have shown that their overall metabolic rate drops at the onset of metamorphosis and increases prior to adult eclosion, as represented by a Ushaped curve of oxygen consumption. This profile correlates with the relatively low levels of metabolic gene expression that were observed during metamorphosis. In addition, the results support the proposal that ERR and HNF4, acting together at the onset of adulthood, end the U-shaped curve of low metabolic rate during metamorphosis and establish the efficient physiological state that sustains adult energetic needs (Beebe, 2020).

Interestingly, previous studies have shown that nuclear receptors can cooperate to link developmental progression with metabolism. Both ERRγ and PPARα contribute to a postnatal change in fuel utilization in the mouse heart, from primarily glucose during fetal stages to fatty acids following birth. This switch in energy substrate preference allows the newborn pup to effectively utilize dietary lipids from maternal milk. Taken together with these studies of ERR and HNF4 in Drosophila, these studies in mammals highlight the importance of nuclear receptors in coupling developmental progression with metabolism and raise the possibility that other metabolic switches may be discovered through future studies of these transcriptional regulators (Beebe, 2020).

Transcriptional profiling of staged pupae and young adults revealed that the central glycolytic genes are coordinately induced during mid-pupal stages in a ERR-dependent manner. Unexpectedly, it was also found that most genes in the TCA cycle and ETC are globally up-regulated in synchrony with this switch. The transcription factors that mediate this regulation, however, remain unknown. Only a few genes in the TCA cycle and ETC display a small change in their expression in ERR and HNF4 mutants. Similarly, although HNF4 plays an important role in supporting adult mitochondrial activity, the global transcriptional induction observed in mid-pupae precedes the HNF4-regulated switch at the onset of adulthood (Barry, 2016; Storelli, 2019). These observations highlight the importance of transcriptional switches as a molecular mechanism that links developmental progression with changes in metabolic state, and raise the question of what other regulators feed into these pathways. A number of transcription factors have been identified that play central roles in metabolism; however, most of these, such as Myc, DHR38, Mondo, Sugarbabe, and Cabut, regulate carbohydrate metabolism, with no clear effect on the TCA cycle or ETC. Further studies are required to identify the key regulator(s) that establish adult mitochondrial oxidative metabolism. It will also be interesting to see whether studies in vertebrates will continue to generalize the observations reported in this study and provide further insights into the molecular mechanisms that couple switches in metabolic state with the changing demands of developmental progression through the life cycle (Beebe, 2020).

HIF- and non-HIF-regulated hypoxic responses require the estrogen-related receptor in Drosophila melanogaster

Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. This study investigated how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. Hypoxic-induced transcriptional changes were found to be comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. Normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and these animals are unable to mobilize glycogen in hypoxia. Furthermore, it was found that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs (Li, 2013).

A triple mutant of the Drosophila ERR confers ligand-induced suppression of activity

The steroid hormone (NR3) subfamily of nuclear receptors was until recently believed to be restricted to deuterostomes. However, a novel nuclear receptor belonging to the NR3 subfamily was recently identified in the Drosophila genome, indicating the existence of an ancestor before the evolutionary split of deuterostomes and protostomes. This receptor, termed the Drosophila estrogen-related receptor (dERR), most closely resembles the human and mouse estrogen-related receptors (ERRs) in both the DNA binding domain (DBD) (approximately 85% identical) and the ligand binding domain (LBD) (approximately 35% identical). This study describe the functional analysis and rational design of ligand responsive dERR mutants created by protein engineering of the LBD. On the basis of homology modeling, three amino acid residues in the LBD were identified and mutated to enable ligand-dependent suppression of transcriptional activity. The results show that the Y295A/T333I/Y365L triple mutant is significantly suppressed by the known ERR inverse agonists 4-hydroxytamoxifen (OHT) and diethylstilbestrol (DES), in comparison to the wild-type dERR receptor, which was inefficiently suppressed by these substances. The coactivator mGRIP-1 (mouse glucocorticoid receptor interacting protein 1) was shown to significantly increase the activity of the triple mutant in transfection experiments, and the addition of OHT resulted in an efficient suppression of the activity. Accordingly, the ability to functionally interact with a coactivator is still maintained by the Y295A/T333I/Y365L mutant. These findings demonstrate the potential of using rational design and engineering of the LBD to study the function of a nuclear receptor lacking identified ligands (Ostberg, 2003).


Functions of ERR orthologs in other species

Estrogen-related receptors mediate the adaptive response of brown adipose tissue to adrenergic stimulation

Adrenergic stimulation of brown adipose tissue (BAT) induces acute and long-term responses. The acute adrenergic response activates thermogenesis by uncoupling oxidative phosphorylation and enabling increased substrate oxidation. Long-term, adrenergic signaling remodels BAT, inducing adaptive transcriptional changes that expand thermogenic capacity. This study shows that the estrogen-related receptors alpha and gamma (ERRalpha, ERRgamma) are collectively critical effectors of adrenergically stimulated transcriptional reprogramming of BAT. Mice lacking adipose ERRs (ERRalphagamma(Ad-/-)) have reduced oxidative and thermogenic capacity and rapidly become hypothermic when exposed to cold. ERRalphagamma(Ad-/-) mice treated long term with a beta3-adrenergic agonist fail to expand oxidative or thermogenic capacity and do not increase energy expenditure in response to norepinephrine (NE). Furthermore, ERRalphagamma(Ad-/-) mice fed a high-fat diet do not lose weight or show improved glucose tolerance when dosed with beta3-adrenergic agonists. The molecular basis of these defects is the finding that ERRs mediate the bulk of the transcriptional response to adrenergic stimulation (Brown, 2018).


REFERENCES

Search PubMed for articles about Drosophila ERR

Alaynick, W. A. et al. (2007). ERRgamma directs and maintains the transition to oxidative metabolism in the postnatal heart. Cell Metab. 6: 13-24. PubMed ID: 17618853

Ao, A., Wang, H., Kamarajugadda, S. and Lu, J. (2000). Involvement of estrogen-related receptors in transcriptional response to hypoxia and growth of solid tumors Proc. Natl. Acad. Sci. 105: 7821-7826. PubMed ID: 18509053

Barry, W. E. and Thummel, C. S. (2016). The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults. Elife 5. PubMed ID: 27185732

B'Chir, W., Dufour, C. R., Ouellet, C., Yan, M., Tam, I. S., Andrzejewski, S., Xia, H., Nabata, K., St-Pierre, J. and Giguere, V. (2018). Divergent role of estrogen-related receptor alpha in lipid- and fasting-induced hepatic steatosis in mice. Endocrinology 159(5): 2153-2164. PubMed ID: 29635284

Beebe, K., Robins, M. M., Hernandez, E. J., Lam, G., Horner, M. A. and Thummel, C. S. (2020). Drosophila estrogen-related receptor directs a transcriptional switch that supports adult glycolysis and lipogenesis. Genes Dev. PubMed ID: 32165409

Brown, E. L., Hazen, B. C., Eury, E., Wattez, J. S., Gantner, M. L., Albert, V., Chau, S., Sanchez-Alavez, M., Conti, B. and Kralli, A. (2018). Estrogen-related receptors mediate the adaptive response of brown adipose tissue to adrenergic stimulation. iScience 2: 221-237. PubMed ID: 29888756

Byerly, M. S., Al Salayta, M., Swanson, R. D., Kwon, K., Peterson, J. M., Wei, Z., Aja, S., Moran, T. H., Blackshaw, S. and Wong, G. W. (2013). Estrogen-related receptor beta deletion modulates whole-body energy balance via estrogen-related receptor gamma and attenuates neuropeptide Y gene expression. Eur J Neurosci 37(7): 1033-1047. PubMed ID: 23360481

Charest-Marcotte, A. et al. (2010). The homeobox protein Prox1 is a negative modulator of ERRalpha/PGC-1alpha bioenergetic functions Genes Dev. 24: 537-542. PubMed ID: 20194433

Eichner, L. J. et al. (2010). miR-378∗ mediates metabolic shift in breast cancer cells via the PGC-1β/ERRγ transcriptional pathway. Cell Metab. 12: 352-361. PubMed ID: 20889127

Huss, J. M. et al. (2007). The nuclear receptor ERRalpha is required for the bioenergetic and functional adaptation to cardiac pressure overload. Cell Metab. 6: 25-37. PubMed ID: 17618854

Huss, J. M., Garbacz, W. G. and Xie, W. (2015). Constitutive activities of estrogen-related receptors: Transcriptional regulation of metabolism by the ERR pathways in health and disease. Biochim Biophys Acta 1852(9): 1912-1927. PubMed ID: 26115970

Kim, D. K., Gang, G. T., Ryu, D., Koh, M., Kim, Y. N., Kim, S. S., Park, J., Kim, Y. H., Sim, T., Lee, I. K., Choi, C. S., Park, S. B., Lee, C. H., Koo, S. H. and Choi, H. S. (2013). Inverse agonist of nuclear receptor ERRgamma mediates antidiabetic effect through inhibition of hepatic gluconeogenesis. Diabetes 62(9): 3093-3102. PubMed ID: 23775767

Lehman, J. J. and Kelly, D. P. (2002). Transcriptional activation of energy metabolic switches in the developing and hypertrophied heart. Clin. Exp. Pharmacol. Physiol. 29: 339-345. PubMed ID: 11985547

Li, B. and Dewey, C. N. (2011). RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12: 323. PubMed ID: 21816040

Li, H., Chawla, G., Hurlburt, A. J., Sterrett, M. C., Zaslaver, O., Cox, J., Karty, J. A., Rosebrock, A. P., Caudy, A. A. and Tennessen, J. M. (2017). Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth. Proc Natl Acad Sci U S A 114(6): 1353-1358. PubMed ID: 28115720

Li, Y., Padmanabha, D., Gentile, L. B., Dumur, C. I., Beckstead, R. B. and Baker, K. D. (2013). HIF- and non-HIF-regulated hypoxic responses require the estrogen-related receptor in Drosophila melanogaster. PLoS Genet 9: e1003230. PubMed ID: 23382692

Luo, J. et al. (2003). Reduced fat mass in mice lacking orphan nuclear receptor estrogen-related receptor alpha. Mol. Cell. Biol. 23: 7947-7956. PubMed ID: 14585956

Misra, J., Kim, D. K. and Choi, H. S. (2017). ERRgamma: a junior orphan with a senior role in metabolism. Trends Endocrinol Metab 28(4): 261-272. PubMed ID: 28209382

Ostberg, T. et al. (2003). A triple mutant of the Drosophila ERR confers ligand-induced suppression of activity. Biochemistry 42(21): 6427-35. PubMed ID: 12767224

Storelli, G., Nam, H. J., Simcox, J., Villanueva, C. J. and Thummel, C. S. (2018). Drosophila HNF4 directs a switch in lipid metabolism that supports the transition to adulthood. Dev Cell. PubMed ID: 30554999

Tennessen, J. M., Baker, K. D., Lam, G., Evans, J. and Thummel, C. S. (2011). The Drosophila estrogen-related receptor directs a metabolic switch that supports developmental growth. Cell Metab. 13(2): 139-48. PubMed ID: 21284981

Tennessen, J. M., Bertagnolli, N. M., Evans, J., Sieber, M. H., Cox, J. and Thummel, C. S. (2014). Coordinated metabolic transitions during Drosophila embryogenesis and the onset of aerobic glycolysis. G3 (Bethesda) 4(5): 839-850. PubMed ID: 24622332

Tremblay, A. M. and Giguère, V. (2007). The NR3B subgroup: an overview. Nucl. Recept. Signal. 5: e009. PubMed ID: 18174917

Xia, H., Dufour, C. R. and Giguere, V. (2019). ERRalpha as a bridge between transcription and function: role in liver metabolism and disease. Front Endocrinol (Lausanne) 10: 206. PubMed ID: 31024446


Biological Overview

date revised: 15 April 2020

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