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Pyruvate occupies a central node in carbohydrate metabolism such that how it is produced and consumed can optimize a cell for energy production or biosynthetic capacity. This has been primarily studied in proliferating cells, but observations from the post-mitotic Drosophila fat body led us to hypothesize that pyruvate fate might dictate the rapid cell growth observed in this organ during development. Indeed, we demonstrate that augmented mitochondrial pyruvate import prevented cell growth in fat body cells in vivo as well as in cultured mammalian hepatocytes and human hepatocyte-derived cells in vitro . This effect on cell size was caused by an increase in the NADH/NAD + ratio, which rewired metabolism toward gluconeogenesis and suppressed the biomass-supporting glycolytic pathway. Amino acid synthesis was decreased, and the resulting loss of protein synthesis prevented cell growth. Surprisingly, this all occurred in the face of activated pro-growth signaling pathways, including mTORC1, Myc, and PI3K/Akt. These observations highlight the evolutionarily conserved role of pyruvate metabolism in setting the balance between energy extraction and biomass production in specialized post-mitotic cells.

Nutrient digestion, absorption, and export must be coordinated in the gut to meet the nutritional needs of the organism. We used the Drosophila intestine to characterize the mechanisms that coordinate the fate of dietary lipids. We identified enterocytes specialized in absorbing and exporting lipids to peripheral organs. Distinct hepatocyte-like cells, called oenocytes, communicate with these enterocytes to adjust intestinal lipid storage and export. A single transcription factor, Drosophila hepatocyte nuclear factor 4 (dHNF4), supports this gut-liver axis. In enterocytes, dHNF4 maximizes dietary lipid export by preventing their sequestration in cytoplasmic lipid droplets. In oenocytes, dHNF4 promotes the expression of the insulin antagonist ImpL2 to activate Foxo and suppress lipid retention in enterocytes. Disruption of this switch between lipid storage and export is associated with intestinal inflammation, suggesting a lipidic origin for inflammatory bowel diseases. These studies establish dHNF4 as a central regulator of intestinal metabolism and inter-organ lipid trafficking., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Drosophila tracheal system consists of a widespread tubular network that provides respiratory functions for the animal. Its development, from ten pairs of placodes in the embryo to the final stereotypical branched structure in the adult, has been extensively studied by many labs as a model system for understanding tubular epithelial morphogenesis. Throughout these studies, a breathless ( btl ) -GAL4 driver has provided an invaluable tool to either mark tracheal cells during development or to manipulate gene expression in this tissue. A distinct shortcoming of this approach, however, is that btl-GAL4 cannot be used to specifically visualize tracheal cells in the presence of other GAL4 drivers or other UAS constructs, restricting its utility. Here we describe a direct-drive btl-nGFP reporter that can be used as a specific marker of tracheal cells throughout development in combination with any GAL4 driver and/or UAS construct. This reporter line should facilitate the use of Drosophila as a model system for studies of tracheal development and tubular morphogenesis.

Pulses of the steroid hormone ecdysone act through transcriptional cascades to direct the major developmental transitions during the Drosophila life cycle. These include the prepupal ecdysone pulse, which occurs 10 ​hours after pupariation and triggers the onset of adult morphogenesis and larval tissue destruction. E93 encodes a transcription factor that is specifically induced by the prepupal pulse of ecdysone, supporting a model proposed by earlier work that it specifies the onset of adult development. Although a number of studies have addressed these functions for E93, little is known about its roles in the salivary gland where the E93 locus was originally identified. Here we show that E93 is required for development through late pupal stages, with mutants displaying defects in adult differentiation and no detectable effect on the destruction of larval salivary glands. RNA-seq analysis demonstrates that E93 regulates genes involved in development and morphogenesis in the salivary glands, but has little effect on cell death gene expression. We also show that E93 is required to direct the proper timing of ecdysone-regulated gene expression in salivary glands, and that it suppresses earlier transcriptional programs that occur during larval and prepupal stages. These studies support the model that the stage-specific induction of E93 in late prepupae provides a critical signal that defines the end of larval development and the onset of adult differentiation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Successful reproduction is dependent on the transfer of male seminal proteins to females upon mating. These proteins arise from secretory tissues in the male reproductive tract, including the prostate and seminal vesicles in mammals and the accessory gland in insects. Although detailed functional studies have provided important insights into the mechanisms by which accessory gland proteins support reproduction, much less is known about the molecular mechanisms that regulate their expression within this tissue. Here we show that the Drosophila HR39 nuclear receptor is required for the proper expression of most genes that encode male accessory gland proteins. Consistent with this role, HR39 mutant males are infertile. In addition, tissue-specific RNAi and genetic rescue experiments indicate that HR39 acts within the accessory glands to regulate gene expression and male fertility. These results provide new directions for characterizing the mammalian orthologs of HR39, the SF-1 and LRH-1 nuclear receptors, both of which are required for glandular secretions and reproduction. In addition, our studies provide a molecular mechanism to explain how the accessory glands can maintain the abundant levels of seminal fluid production required to support fertility., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Lipid levels are maintained by balancing lipid uptake, synthesis, and mobilization. Although many studies have focused on the control of lipid synthesis and mobilization, less is known about the regulation of lipid digestion and uptake., Results: Here we show that the Drosophila E78A nuclear receptor plays a central role in intestinal lipid homeostasis through regulation of the CG17192 digestive lipase. E78A mutant adults fail to maintain proper systemic lipid levels following eclosion, with this effect largely restricted to the intestine. Transcriptional profiling by RNA-seq revealed a candidate gene for mediating this effect, encoding the predicted adult intestinal lipase CG17192. Intestine-specific disruption of CG17192 results in reduced lipid levels similar to that seen in E78A mutants. In addition, dietary supplementation with free fatty acids, or intestine-specific expression of either E78A or CG17192, is sufficient to restore lipid levels in E78A mutant adults., Conclusion: These studies support the model that E78A is a central regulator of adult lipid homeostasis through its effects on CG17192 expression and lipid digestion. This work also provides new insights into the control of intestinal lipid uptake and demonstrate that nuclear receptors can play an important role in these pathways., (© 2020 American Association of Anatomists.)

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. Here we show that the Drosophila estrogen-related receptor ( dERR ) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. dERR 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 dERR, 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, we 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 dERR , leading to the metabolic defects observed in mutants. Our data support the model that dERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly., (© 2020 Beebe et al.; Published by Cold Spring Harbor Laboratory Press.)

Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation., Competing Interests: Declaration of Interests The University of Utah has filed a patent related to the mitochondrial pyruvate carrier, of which J.R. and C.S.T. are listed as co-inventors. All other authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multiple signaling pathways in the adult Drosophila enterocyte sense cellular damage or stress and signal to intestinal stem cells (ISCs) to undergo proliferation and differentiation, thereby maintaining intestinal homeostasis. Here we show that misregulation of mitochondrial pyruvate metabolism in enterocytes can stimulate ISC proliferation and differentiation. Our studies focus on the Mitochondrial Pyruvate Carrier (MPC), which is an evolutionarily-conserved protein complex that resides in the inner mitochondrial membrane and transports cytoplasmic pyruvate into the mitochondrial matrix. Loss of MPC function in enterocytes induces Unpaired cytokine expression, which activates the JAK/STAT pathway in ISCs, promoting their proliferation. Upd3 and JNK signaling are required in enterocytes for ISC proliferation, indicating that this reflects a canonical non-cell autonomous damage response. Disruption of lactate dehydrogenase in enterocytes has no effect on ISC proliferation but it suppresses the proliferative response to a loss of enterocyte MPC function, suggesting that lactate contributes to this pathway. These studies define an important role for cellular pyruvate metabolism in differentiated enterocytes to maintain stem cell proliferation rates., (Copyright © 2019 Wisidagama, Thummel.)

Background: The ADCK proteins are predicted mitochondrial kinases. Most studies of these proteins have focused on the Abc1/Coq8 subfamily, which contributes to Coenzyme Q biosynthesis. In contrast, little is known about ADCK1 despite its evolutionary conservation in yeast, Drosophila, Caenorhabditis elegans and mammals., Results: We show that Drosophila ADCK1 mutants die as second instar larvae with double mouth hooks and tracheal breaks. Tissue-specific genetic rescue and RNAi studies show that ADCK1 is necessary and sufficient in the trachea for larval viability. In addition, tracheal-rescued ADCK1 mutant adults have reduced lifespan, are developmentally delayed, have reduced body size, and normal levels of basic metabolites., Conclusion: The larval lethality and double mouth hooks seen in ADCK1 mutants are often associated with reduced levels of the steroid hormone ecdysone, suggesting that this gene could contribute to controlling ecdysone levels or bioavailability. Similarly, the tracheal defects in these animals could arise from defects in intracellular lipid trafficking. These studies of ADCK1 provide a new context to define the physiological functions of this poorly understood member of the ADCK family of predicted mitochondrial proteins., (© 2019 Wiley Periodicals, Inc.)

Animals must adjust their metabolism as they progress through development in order to meet the needs of each stage in the life cycle. Here, we show that the dHNF4 nuclear receptor acts at the onset of Drosophila adulthood to direct an essential switch in lipid metabolism. Lipid stores are consumed shortly after metamorphosis but contribute little to energy metabolism. Rather, dHNF4 directs their conversion to very long chain fatty acids and hydrocarbons, which waterproof the animal to preserve fluid homeostasis. Similarly, HNF4α is required in mouse hepatocytes for the expression of fatty acid elongases that contribute to a waterproof epidermis, suggesting that this pathway is conserved through evolution. This developmental switch in Drosophila lipid metabolism promotes lifespan and desiccation resistance in adults and suppresses hallmarks of diabetes, including elevated glucose levels and intolerance to dietary sugars. These studies establish dHNF4 as a regulator of the adult metabolic state., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Nutrients play a central role in controlling the form and function of the intestinal epithelium. In this issue of Developmental Cell, Mattila et al. (2018) and Obniski et al. (2018) uncover important mechanisms by which Drosophila intestinal stem cells respond to dietary signals, linking nutrients to tissue homeostasis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The Testicular Receptors 2 and 4 (TR2, TR4) comprise a small subfamily of orphan nuclear receptors. Genetic studies in mouse models have identified roles for TR4 in developmental progression, fertility, brain development, and metabolism, as well as genetic redundancy with TR2. Here we study the adult functions of the single Drosophila member of this subfamily, DHR78, with the goal of defining its ancestral functions in the absence of genetic redundancy., Results: We show that DHR78 mutants have a shortened lifespan, reduced motility, and mated DHR78 mutant females display a reduced feeding rate. Transcriptional profiling reveals a major role for DHR78 in promoting the expression of genes that are expressed in the midgut, suggesting that it contributes to nutrient uptake. We also identify roles for DHR78 in maintaining the expression of genes in the ecdysone and Notch signaling pathways., Conclusions: This study provides a new context for linking the molecular activity of the TR orphan nuclear receptors with their complex roles in adult physiology and lifespan. Developmental Dynamics 247:315-322, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

Metabolomic analysis provides a powerful new tool for studies of Drosophila physiology. This approach allows investigators to detect thousands of chemical compounds in a single sample, representing the combined contributions of gene expression, enzyme activity, and environmental context. Metabolomics has been used for a wide range of studies in Drosophila , often providing new insights into gene function and metabolic state that could not be obtained using any other approach. In this review, we survey the uses of metabolomic analysis since its entry into the field. We also cover the major methods used for metabolomic studies in Drosophila and highlight new directions for future research., (Copyright © 2017 by the Genetics Society of America.)

Many studies have focused on defining the critical transcription factors that specify tissue morphogenesis and differentiation. Our understanding of how these spatial regulators are deployed in the proper temporal order, however, has remained less clear. In this issue of Genes & Development , Uyehara and colleagues (pp. 862-875) provide new insights into the mechanisms by which temporal and spatial regulators are coordinated to control Drosophila wing development during metamorphosis., (© 2017 Praggastis and Thummel; Published by Cold Spring Harbor Laboratory Press.)

Objective: A significant portion of the heritable risk for complex metabolic disorders cannot be attributed to classic Mendelian genetic factors. At least some of this missing heritability is thought to be due to the epigenetic influence of parental and grandparental metabolic state on offspring health. Previous work suggests that this transgenerational phenomenon is evolutionarily conserved in Drosophila . These studies, however, have all depended on dietary paradigms to alter parental metabolic state, which can have inconsistent heritable effects on the metabolism of offspring., Methods: Here we use AKHR null alleles to induce obesity in the parental generation and then score both metabolic parameters and genome-wide transcriptional responses in AKHR heterozygote F1 progeny and genetically wild-type F2 progeny., Results: Unexpectedly, we observe elevated glycogen levels and changes in gene expression in AKHR heterozygotes due to haploinsufficiency at this locus. We also show that genetic manipulation of parental metabolism using AKHR mutations results in significant physiological changes in F2 wild-type offspring of the grandpaternal/maternal lineage., Conclusions: Our results demonstrate that genetic manipulation of parental metabolism in Drosophila can have an effect on the health of F2 progeny, providing a non-dietary paradigm to better understand the mechanisms behind the transgenerational inheritance of metabolic state.

Objective: Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC) is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion., Methods: To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, we used genetically-engineered Drosophila and mice with loss of MPC activity in insulin-producing cells., Results: In both species, MPC deficiency results in elevated blood sugar concentrations and glucose intolerance accompanied by impaired glucose-stimulated insulin secretion. In mouse islets, β-cell MPC-deficiency resulted in decreased respiration with glucose, ATP-sensitive potassium (KATP) channel hyperactivity, and impaired insulin release. Moreover, treatment of pancreas-specific MPC knockout mice with glibenclamide, a sulfonylurea KATP channel inhibitor, improved defects in islet insulin secretion and abnormalities in glucose homeostasis in vivo. Finally, using a recently-developed biosensor for MPC activity, we show that the MPC is rapidly stimulated by glucose treatment in INS-1 insulinoma cells suggesting that glucose sensing is coupled to mitochondrial pyruvate carrier activity., Conclusions: Altogether, these studies suggest that the MPC plays an important and ancestral role in insulin-secreting cells in mediating glucose sensing, regulating insulin secretion, and controlling systemic glycemia.

Although mutations in HNF4A were identified as the cause of Maturity Onset Diabetes of the Young 1 (MODY1) two decades ago, the mechanisms by which this nuclear receptor regulates glucose homeostasis remain unclear. Here we report that loss of Drosophila HNF4 recapitulates hallmark symptoms of MODY1, including adult-onset hyperglycemia, glucose intolerance and impaired glucose-stimulated insulin secretion (GSIS). These defects are linked to a role for dHNF4 in promoting mitochondrial function as well as the expression of Hex-C, a homolog of the MODY2 gene Glucokinase. dHNF4 is required in the fat body and insulin-producing cells to maintain glucose homeostasis by supporting a developmental switch toward oxidative phosphorylation and GSIS at the transition to adulthood. These findings establish an animal model for MODY1 and define a developmental reprogramming of metabolism to support the energetic needs of the mature animal.

SIRT1 is a member of the sirtuin family of NAD+-dependent deacetylases, which couple cellular metabolism to systemic physiology. Although studies in mouse models have defined a central role for SIRT1 in maintaining metabolic health, the molecular mechanisms remain unclear. Here we show that loss of the Drosophila SIRT1 homolog sir2 leads to the age-progressive onset of hyperglycemia, obesity, glucose intolerance, and insulin resistance. Tissue-specific functional studies show that Sir2 is both necessary and sufficient in the fat body (analogous to the mammalian liver) to maintain glucose homeostasis and peripheral insulin sensitivity. Transcriptional profiling of sir2 mutants by RNA-seq revealed a major overlap with genes regulated by the nuclear receptor Hepatocyte Nuclear Factor 4 (HNF4). Consistent with this, Drosophila HNF4 mutants display diabetic phenotypes similar to those of sir2 mutants, and protein levels for dHNF4 are reduced in sir2 mutant animals. We show that Sir2 exerts these effects by deacetylating and stabilizing dHNF4 through protein interactions. Increasing dHNF4 expression in sir2 mutants is sufficient to rescue their insulin signaling defects, defining this nuclear receptor as an important downstream effector of Sir2 signaling. This study demonstrates that the key metabolic activities of SIRT1 have been conserved through evolution, provides a genetic model for functional studies of phenotypes related to type 2 diabetes, and establishes HNF4 as a critical downstream target by which Sir2 maintains metabolic health.

In this issue of Developmental Cell, Okamoto and Nishimura (2015) identify a positive feedback loop between neuronal cells that maintains insulin signaling and growth under restricted nutritional conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Disorders arising from impaired assembly of succinate dehydrogenase (SDH) result in a myriad of pathologies, consistent with its unique role in linking the citric acid cycle and electron transport chain. In spite of this critical function, however, only a few factors are known to be required for SDH assembly and function. We show here that two factors, Sdh6 (SDHAF1) and Sdh7 (SDHAF3), mediate maturation of the FeS cluster SDH subunit (Sdh2/SDHB). Yeast and Drosophila lacking SDHAF3 are impaired in SDH activity with reduced levels of Sdh2. Drosophila lacking the Sdh7 ortholog SDHAF3 are hypersensitive to oxidative stress and exhibit muscular and neuronal dysfunction. Yeast studies revealed that Sdh6 and Sdh7 act together to promote Sdh2 maturation by binding to a Sdh1/Sdh2 intermediate, protecting it from the deleterious effects of oxidants. These studies in yeast and Drosophila raise the possibility that SDHAF3 mutations may be associated with idiopathic SDH-associated diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Succinate dehydrogenase (SDH) occupies a central place in cellular energy production, linking the tricarboxylic cycle with the electron transport chain. As a result, a subset of cancers and neuromuscular disorders result from mutations affecting any of the four SDH structural subunits or either of two known SDH assembly factors. Herein we characterize an evolutionarily conserved SDH assembly factor designated Sdh8/SDHAF4, using yeast, Drosophila, and mammalian cells. Sdh8 interacts specifically with the catalytic Sdh1 subunit in the mitochondrial matrix, facilitating its association with Sdh2 and the subsequent assembly of the SDH holocomplex. These roles for Sdh8 are critical for preventing motility defects and neurodegeneration in Drosophila as well as the excess ROS generated by free Sdh1. These studies provide insights into the mechanisms by which SDH is assembled and raise the possibility that some forms of neuromuscular disease may be associated with mutations that affect this SDH assembly factor., (Copyright © 2014 Elsevier Inc. All rights reserved.)

As the incidence of complex metabolic disease increases in developed countries, so too does the need to understand the causes and risk factors for these disorders. In addition to the well-known contribution of genetics and environment to metabolic dysfunction, many studies have demonstrated that a significant degree of non-genetic heritable risk can be transmitted from parents to offspring over multiple generations. Understanding the mechanisms by which this occurs could change how we study and treat complex metabolic disorders. In this review, we summarize recent advances in this field utilizing Drosophila, mice, and humans, and propose potential molecular mechanisms that underlie the transgenerational inheritance of metabolic state., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Recent research using Drosophila melanogaster has seen a resurgence in studies of metabolism and physiology. This review focuses on major methods used to conduct this work. These include protocols for dietary interventions, measurements of triglycerides, cholesterol, glucose, trehalose, and glycogen, stains for lipid detection, and the use of gas chromatography-mass spectrometry (GC-MS) to detect major polar metabolites. It is our hope that this will provide a useful framework for both new and current researchers in the field., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rapidly proliferating cells such as cancer cells and embryonic stem cells rely on a specialized metabolic program known as aerobic glycolysis, which supports biomass production from carbohydrates. The fruit fly Drosophila melanogaster also utilizes aerobic glycolysis to support the rapid growth that occurs during larval development. Here we use singular value decomposition analysis of modENCODE RNA-seq data combined with GC-MS-based metabolomic analysis to analyze the changes in gene expression and metabolism that occur during Drosophila embryogenesis, spanning the onset of aerobic glycolysis. Unexpectedly, we find that the most common pattern of co-expressed genes in embryos includes the global switch to glycolytic gene expression that occurs midway through embryogenesis. In contrast to the canonical aerobic glycolytic pathway, however, which is accompanied by reduced mitochondrial oxidative metabolism, the expression of genes involved in the tricarboxylic cycle (TCA cycle) and the electron transport chain are also upregulated at this time. Mitochondrial activity, however, appears to be attenuated, as embryos exhibit a block in the TCA cycle that results in elevated levels of citrate, isocitrate, and α-ketoglutarate. We also find that genes involved in lipid breakdown and β-oxidation are upregulated prior to the transcriptional initiation of glycolysis, but are downregulated before the onset of larval development, revealing coordinated use of lipids and carbohydrates during development. These observations demonstrate the efficient use of nutrient stores to support embryonic development, define sequential metabolic transitions during this stage, and demonstrate striking similarities between the metabolic state of late-stage fly embryos and tumor cells., (Copyright © 2014 Tennessen et al.)

Pesticide resistance poses a major challenge for the control of vector-borne human diseases and agricultural crop protection. Although a number of studies have defined how mutations in specific target proteins can lead to insecticide resistance, much less is known about the mechanisms by which constitutive overexpression of detoxifying enzymes contributes to metabolic pesticide resistance. Here we show that the Nrf2/Keap1 pathway is constitutively active in two laboratory-selected DDT-resistant strains of Drosophila, 91R and RDDTR, leading to the overexpression of multiple detoxifying genes. Disruption of the Drosophila Nrf2 ortholog, CncC, or overexpression of Keap1, is sufficient to block this transcriptional response. In addition, a CncC-responsive reporter is highly active in both DDT-resistant strains and this response is dependent on the presence of an intact CncC binding site in the promoter. Microarray analysis revealed that ∼20% of the genes differentially expressed in the 91R strain are known CncC target genes. Finally, we show that CncC is partially active in these strains, consistent with the fitness cost associated with constitutive activation of the pathway. This study demonstrates that the Nrf2/Keap1 pathway contributes to the widespread overexpression of detoxification genes in insecticide-resistant strains and raises the possibility that inhibitors of this pathway could provide effective synergists for insect population control., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier.

Although transintestinal cholesterol efflux has been identified as an important means of clearing excess sterols, the mechanisms that underlie this process remain poorly understood. Here, we show 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recent studies in mammals have demonstrated a central role for the circadian clock in maintaining metabolic homeostasis. In spite of these advances, however, little is known about how these complex pathways are coordinated. Here, we show that fundamental aspects of the circadian control of metabolism are conserved in the fruit fly Drosophila. We assay feeding behavior and basic metabolite levels in individual flies and show that, like mammals, Drosophila display a rapid increase in circulating sugar following a meal, which is subsequently stored in the form of glycogen. These daily rhythms in carbohydrate levels are disrupted in clock mutants, demonstrating a critical role for the circadian clock in the postprandial response to feeding. We also show that basic metabolite levels are coordinated in a clock-dependent manner and that clock function is required to maintain lipid homeostasis. By examining feeding behavior, we show that flies feed primarily during the first 4 hours of the day and that light suppresses a late day feeding bout through the cryptochrome photoreceptor. These studies demonstrate that central aspects of feeding and metabolism are dependent on the circadian clock in Drosophila. Our work also uncovers novel roles for light and cryptochrome on both feeding behavior and metabolism.

Adult body size in higher animals is dependent on the amount of growth that occurs during the juvenile stage. The duration of juvenile development, therefore, must be flexible and responsive to environmental conditions. When immature animals experience environmental stresses such as malnutrition or disease, maturation can be delayed until conditions improve and normal growth can resume. In contrast, when animals are raised under ideal conditions that promote rapid growth, internal checkpoints ensure that maturation does not occur until juvenile development is complete. Although the mechanisms that regulate growth and gate the onset of maturation have been investigated for decades, the emerging links between childhood obesity, early onset puberty, and adult metabolic disease have placed a new emphasis on this field. Remarkably, genetic studies in the fruit fly Drosophila melanogaster have shown that the central regulatory pathways that control growth and the timing of sexual maturation are conserved through evolution, and suggest that this aspect of animal life history is regulated by a common genetic architecture. This review focuses on these conserved mechanisms and highlights recent studies that explore how Drosophila coordinates developmental growth with environmental conditions., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Living organisms, from bacteria to humans, display a coordinated transcriptional response to xenobiotic exposure, inducing enzymes and transporters that facilitate detoxification. Several transcription factors have been identified in vertebrates that contribute to this regulatory response. In contrast, little is known about this pathway in insects. Here we show that the Drosophila Nrf2 (NF-E2-related factor 2) ortholog CncC (cap 'n' collar isoform-C) is a central regulator of xenobiotic detoxification responses. A binding site for CncC and its heterodimer partner Maf (muscle aponeurosis fibromatosis) is sufficient and necessary for robust transcriptional responses to three xenobiotic compounds: phenobarbital (PB), chlorpromazine, and caffeine. Genetic manipulations that alter the levels of CncC or its negative regulator, Keap1 (Kelch-like ECH-associated protein 1), lead to predictable changes in xenobiotic-inducible gene expression. Transcriptional profiling studies reveal that more than half of the genes regulated by PB are also controlled by CncC. Consistent with these effects on detoxification gene expression, activation of the CncC/Keap1 pathway in Drosophila is sufficient to confer resistance to the lethal effects of the pesticide malathion. These studies establish a molecular mechanism for the regulation of xenobiotic detoxification in Drosophila and have implications for controlling insect populations and the spread of insect-borne human diseases.

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. Here, we show that the Drosophila ortholog of the estrogen-related receptor (ERR) family of nuclear receptors directs a critical metabolic transition during development. dERR 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Animals balance nutrient storage and mobilization to maintain metabolic homeostasis, a process that is disrupted in metabolic diseases like obesity and diabetes. Here, we show that DHR38, the single fly ortholog of the mammalian nuclear receptor 4A family of nuclear receptors, regulates glycogen storage during the larval stages of Drosophila melanogaster. DHR38 is expressed and active in the gut and body wall of larvae, and its expression levels change in response to nutritional status. DHR38 null mutants have normal levels of glucose, trehalose (the major circulating form of sugar), and triacylglycerol but display reduced levels of glycogen in the body wall muscles, which constitute the primary storage site for carbohydrates. Microarray analysis reveals that many metabolic genes are mis-regulated in DHR38 mutants. These include phosphoglucomutase, which is required for glycogen synthesis, and the two genes that encode the digestive enzyme amylase, accounting for the reduced amylase enzyme activity seen in DHR38 mutant larvae. These studies demonstrate that a critical role of nuclear receptor 4A receptors in carbohydrate metabolism has been conserved through evolution and that nutritional regulation of DHR38 expression maintains the proper uptake and storage of glycogen during the growing larval stage of development.

The steroid hormone ecdysone triggers the rapid destruction of larval tissues through transcriptional cascades that culminate in rpr and hid expression and caspase activation. Here, we show that mutations in Mdh2 and Med24 block caspase cleavage and larval salivary gland cell death. Mdh2 encodes a predicted malate dehydrogenase that localizes to mitochondria. Consistent with this proposed function, Mdh2 mutants have significantly lower levels of ATP and accumulate late-stage citric acid cycle intermediates, suggesting that the cell death defects arise from a deficit in energy production. Med24 encodes a component of the Mediator transcriptional coactivator complex. Unexpectedly, however, expression of the key death regulator genes is normal in Med24 mutant salivary glands. This study identifies novel mechanisms for controlling the destruction of larval tissues during Drosophila metamorphosis and provides new directions for our understanding of steroid-triggered programmed cell death., (Copyright (c) 2010 Wiley-Liss, Inc.)

Studies of the onset of metamorphosis have identified an ecdysone-triggered transcriptional cascade that consists of the sequential expression of the transcription-factor-encoding genes DHR3, betaFTZ-F1, E74A and E75A. Although the regulatory interactions between these genes have been well characterized by genetic and molecular studies over the past 20 years, their developmental functions have remained more poorly understood. In addition, a transcriptional sequence similar to that observed in prepupae is repeated before each developmental transition in the life cycle, including mid-embryogenesis and the larval molts. Whether the regulatory interactions between DHR3, betaFTZ-F1, E74A and E75A at these earlier stages are similar to those defined at the onset of metamorphosis, however, is unknown. In this study, we turn to embryonic development to address these two issues. We show that mid-embryonic expression of DHR3 and betaFTZ-F1 is part of a 20-hydroxyecdysone (20E)-triggered transcriptional cascade similar to that seen in mid-prepupae, directing maximal expression of E74A and E75A during late embryogenesis. In addition, DHR3 and betaFTZ-F1 exert overlapping developmental functions at the end of embryogenesis. Both genes are required for tracheal air filling, whereas DHR3 is required for ventral nerve cord condensation and betaFTZ-F1 is required for proper maturation of the cuticular denticles. Rescue experiments support these observations, indicating that DHR3 has essential functions independent from those of betaFTZ-F1. DHR3 and betaFTZ-F1 also contribute to overlapping transcriptional responses during embryogenesis. Taken together, these studies define the lethal phenotypes of DHR3 and betaFTZ-F1 mutants, and provide evidence for functional bifurcation in the 20E-responsive transcriptional cascade.

Triacylglycerol (TAG) homeostasis is an integral part of normal physiology and essential for proper energy metabolism. Here we show 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. We show that DHR96 function is required in the midgut for the breakdown of dietary fat and that 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.

Cholesterol homeostasis is required to maintain normal cellular function and avoid the deleterious effects of hypercholesterolemia. Here we show that 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.

Drosophila HNF4 (dHNF4) is the single ancestral ortholog of a highly conserved subfamily of nuclear receptors that includes two mammalian receptors, HNFalpha and HNFgamma, and 269 members in C. elegans. We show here that dHNF4 null mutant larvae are sensitive to starvation. Starved mutant larvae consume glycogen normally but retain lipids in their midgut and fat body and have increased levels of long-chain fatty acids, suggesting that they are unable to efficiently mobilize stored fat for energy. Microarray studies support this model, indicating reduced expression of genes that control lipid catabolism and beta-oxidation. A GAL4-dHNF4;UAS-lacZ ligand sensor can be activated by starvation or exogenous long-chain fatty acids, suggesting that dHNF4 is responsive to dietary signals. Taken together, our results support a feed-forward model for dHNF4, in which fatty acids released from triglycerides activate the receptor, inducing enzymes that drive fatty acid oxidation for energy production.

DHR38 is the only Drosophila member of the NR4A subclass of vertebrate nuclear receptors, which have been implicated in multiple biological pathways, including neuronal function, apoptosis, and metabolism. Although an earlier study identified three point mutations in DHR38, none of these were shown to be a null allele for the locus, leaving it unclear whether a complete loss of DHR38 function might uncover novel roles for the receptor. Here we show that a specific DHR38 null allele, DHR38(Y214), leads to fully penetrant pharate adult lethality, similar to the most severe phenotype associated with the EMS-induced mutations. DHR38(Y214) mutants display minor effects on ecdysone-regulated transcription at the onset of metamorphosis. In contrast, cuticle gene expression is significantly reduced in DHR38(Y214) mutant pupae. These studies define the essential functions of DHR38 and provide a genetic context for further characterization of its roles during development., ((c) 2009 Wiley-Liss, Inc.)

The steroid hormone ecdysone triggers the rapid and massive destruction of larval tissues through transcriptional cascades that culminate in rpr and hid expression and caspase activation. Here we describe the use of genetic screens to further our understanding of this steroid-triggered programmed cell death response. Pupal lethal mutants were screened for specific defects in larval salivary gland destruction. A pilot screen using existing P-element collections resulted in the identification of mutations in known cell death regulators, E74 and hid, as well as multiple alleles in CBP (nejire) and dTrf2. A large-scale EMS mutagenesis screen on the third chromosome resulted in the recovery of 48 mutants. These include seven multiallelic complementation groups, at least five of which do not map to regions or genes previously associated with cell death. Five mutants display defects in the transcriptional induction of rpr and hid, and all display a penetrant block in caspase activation. Three were mapped to specific genes: CG5146, which encodes a protein of unknown function, Med24, which encodes a component of the RNA polymerase II mediator complex, and CG7998, which encodes a putative mitochondrial malate dehydrogenase. These genetic screens provide new directions for understanding the regulation of programmed cell death during development.

Expression of the heterochronic microRNA let-7 is tightly correlated with the onset of adult development in many animals, suggesting that it functions as an evolutionarily conserved developmental timer. This hypothesis has now been confirmed by studies in Drosophila.

Neuroendocrine signaling pathways play a central role in modulating animal body size in response to environmental signals. Little is known, however, regarding how these neuroendocrine circuits are controlled. An important advance in this area is reported in this issue of Genes & Development by Kaplan and colleagues (pp. 1877-1893), who show that serotonergic neurons regulate the growth of peripheral tissues in Drosophila through the insulin/IGF pathway.

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval.

The TATA box-binding protein (TBP) related factor 2 (TRF2) has been well characterized at a biochemical level and in cultured cells. Relatively little, however, is known about how TRF2 functions in specific biological pathways during development. Here, we show that Drosophila TRF2 (dTRF2) plays an essential role in responses to the steroid hormone ecdysone during the onset of metamorphosis. Hypomorphic dTrf2 mutations lead to developmental arrest during prepupal and early pupal stages with defects in major ecdysone-triggered biological responses, including puparium formation, anterior spiracle eversion, gas bubble translocation, adult head eversion, and larval salivary gland cell death. The transcription of key ecdysone-regulated target genes is delayed and reduced in dTrf2 mutants. dTrf2 appears to be required for the proper timing and levels of ecdysone-regulated gene expression required for entry into metamorphosis., (Copyright 2007 Wiley-Liss, Inc.)

The past few years have seen a shift in the use of Drosophila, from studies of growth and development toward genetic characterization of carbohydrate, sterol, and lipid metabolism. This research, reviewed below, establishes a new foundation for using this simple genetic model system to define the basic regulatory mechanisms that underlie metabolic homeostasis and holds the promise of providing new insights into the causes and treatments of critical human disorders such as diabetes and obesity.

A pulse of the steroid hormone ecdysone triggers the destruction of larval salivary glands during Drosophila metamorphosis through a transcriptional cascade that converges on reaper (rpr) and head involution defective (hid) induction, resulting in caspase activation and cell death. We identify the CREB binding protein (CBP) transcriptional cofactor as essential for salivary gland cell death. We show that CBP acts 1 d before the onset of metamorphosis in apparent response to a mid-third instar ecdysone pulse, when CBP is necessary and sufficient for down-regulation of the Drosophila inhibitor of apoptosis 1 (DIAP1). It is only after DIAP1 levels are reduced that salivary glands become competent to die through rpr/hid-mediated cell death. Before this time, high levels of DIAP1 block salivary gland cell death, even in the presence of ectopic rpr expression. This study shows that naturally occurring changes in inhibitor of apoptosis levels can be critical for regulating cell death during development. It also provides a molecular mechanism for the acquisition of competence in steroid signaling pathways.

Previous studies have shown that ecdysone (E), and its immediate downstream product 20-hydroxyecdysone (20E), can have different biological functions in insects, suggesting that E acts as a distinct hormone. Here, we use Drosophila larval organ culture in combination with microarray technology to identify genes that are transcriptionally regulated by E, but which show little or no response to 20E. These genes are coordinately expressed for a brief temporal interval at the onset of metamorphosis, suggesting that E acts together with 20E to direct puparium formation. We also show that E74B, pepck, and CG14949 can be induced by juvenile hormone III (JH III) in organ culture, and that CG14949 can be induced by JH independently of protein synthesis. In contrast, E74A and E75A show no response to JH in this system. These studies demonstrate that larval organ culture can be used to identify Drosophila genes that are regulated by hormones other than 20E, and provide a basis for studying crosstalk between multiple hormone signaling pathways.