L-2-hydroxyglutarate remodeling of the epigenome and epitranscriptome creates a metabolic vulnerability in kidney cancer models.

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule L-2-hdroxyglutarate (L-2HG) is elevated in the most common histology of renal cancer. Similar to other oncometabolites, L-2HG has the potential to profoundly impact gene expression. Here, we demonstrate that L-2HG remodels amino acid metabolism in renal cancer cells through the combined effects on histone methylation and RNA N6-methyladenosine (m6A). The combined effects of L-2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high L-2HG kidney cancers demonstrates reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high L-2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport.

BACKGROUND: Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer's, Parkinson's, and Huntington's disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function. METHODS: We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos. RESULTS: We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide "on-board" ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons. CONCLUSION: NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Auxin exposure disrupts feeding behavior and fatty acid metabolism in adult Drosophila.

The ease of genetic manipulation in Drosophila melanogaster using the Gal4/UAS system has been beneficial in addressing key biological questions. Current modifications of this methodology to temporally induce transgene expression require temperature changes or exposure to exogenous compounds, both of which have been shown to have detrimental effects on physiological processes. The recently described auxin-inducible gene expression system (AGES) utilizes the plant hormone auxin to induce transgene expression and is proposed to be the least toxic compound for genetic manipulation, with no obvious effects on Drosophila development and survival in one wild-type strain. Here, we show that auxin delays larval development in another widely used fly strain, and that short- and long-term auxin exposure in adult Drosophila induces observable changes in physiology and feeding behavior. We further reveal a dosage response to adult survival upon auxin exposure, and that the recommended auxin concentration for AGES alters feeding activity. Furthermore, auxin-fed male and female flies exhibit a significant decrease in triglyceride levels and display altered transcription of fatty acid metabolism genes. Although fatty acid metabolism is disrupted, auxin does not significantly impact adult female fecundity or progeny survival, suggesting AGES may be an ideal methodology for studying limited biological processes. These results emphasize that experiments using temporal binary systems must be carefully designed and controlled to avoid confounding effects and misinterpretation of results.

Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogaster.

Drosophila melanogaster larval development relies on a specialized metabolic state that utilizes carbohydrates and other dietary nutrients to promote rapid growth. One unique feature of the larval metabolic program is that Lactate Dehydrogenase (Ldh) activity is highly elevated during this growth phase when compared to other stages of the fly life cycle, indicating that Ldh serves a key role in promoting juvenile development. Previous studies of larval Ldh activity have largely focused on the function of this enzyme at the whole animal level, however, Ldh expression varies significantly among larval tissues, raising the question of how this enzyme promotes tissue-specific growth programs. Here we characterize two transgene reporters and an antibody that can be used to study Ldh expression in vivo. We find that all three tools produce similar Ldh expression patterns. Moreover, these reagents demonstrate that the larval Ldh expression pattern is complex, suggesting the purpose of this enzyme varies across cell types. Overall, our studies validate a series of genetic and molecular reagents that can be used to study glycolytic metabolism in the fly.

Metabolomic analysis of Drosophila melanogaster larvae lacking pyruvate kinase.

Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster are complicated by the fact that the fly genome encodes 6 Pyk paralogs whose functions remain poorly defined. To address this issue, we used sequence distance and phylogenetic approaches to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs, while the other 5 Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of 2 different Pyk mutant strains revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, our analysis also unexpectedly reveals that pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with our metabolomic findings, a complementary RNA-seq analysis revealed that genes involved in lipid metabolism and protease activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, our study provides both insight into how Drosophila larval metabolism adapts to disruption of glycolytic metabolism as well as immediate clinical relevance, considering that Pyk deficiency is the most common congenital enzymatic defect in humans.

Auxin Exposure Disrupts Feeding Behavior and Fatty Acid Metabolism in Adult Drosophila.

The ease of genetic manipulation in Drosophila melanogaster using the Gal4/UAS system has been beneficial in addressing key biological questions. Current modifications of this methodology to temporally induce transgene expression require temperature changes or exposure to exogenous compounds, both of which have been shown to have detrimental effects on physiological processes. The recently described auxin-inducible gene expression system (AGES) utilizes the plant hormone auxin to induce transgene expression and is proposed to be the least toxic compound for genetic manipulation, with no obvious effects on Drosophila development and survival in one wild-type strain. Here we show that auxin delays larval development in another widely-used fly strain, and that short- and long-term auxin exposure in adult Drosophila induces observable changes in physiology and feeding behavior. We further reveal a dosage response to adult survival upon auxin exposure, and that the recommended auxin concentration for AGES alters feeding activity. Furthermore, auxin fed male and female flies exhibit a significant decrease in triglyceride levels and display altered transcription of fatty acid metabolism genes. Although fatty acid metabolism is disrupted, auxin does not significantly impact adult female fecundity or progeny survival, suggesting AGES may be an ideal methodology for studying limited biological processes. These results emphasize that experiments using temporal binary systems must be carefully designed and controlled to avoid confounding effects and misinterpretation of results.

Perspectives on the Drosophila melanogaster Model for Advances in Toxicological Science.

The use of Drosophila melanogaster for studies of toxicology has grown considerably in the last decade. The Drosophila model has long been appreciated as a versatile and powerful model for developmental biology and genetics because of its ease of handling, short life cycle, low cost of maintenance, molecular genetic accessibility, and availability of a wide range of publicly available strains and data resources. These features, together with recent unique developments in genomics and metabolomics, make the fly model especially relevant and timely for the development of new approach methodologies and movements toward precision toxicology. Here, we offer a perspective on how flies can be leveraged to identify risk factors relevant to environmental exposures and human health. First, we review and discuss fundamental toxicologic principles for experimental design with Drosophila. Next, we describe quantitative and systems genetics approaches to resolve the genetic architecture and candidate pathways controlling susceptibility to toxicants. Finally, we summarize the current state and future promise of the emerging field of Drosophila metabolomics for elaborating toxic mechanisms. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogaster.

Drosophila melanogaster larval development relies on a specialized metabolic state that utilizes carbohydrates and other dietary nutrients to promote rapid growth. One unique feature of the larval metabolic program is that Lactate Dehydrogenase (Ldh) activity is highly elevated during this growth phase when compared to other stages of the fly life cycle, indicating that Ldh serves a key role in promoting juvenile development. Previous studies of larval Ldh activity have largely focused on the function of this enzyme at the whole animal level, however, Ldh expression varies significantly among larval tissues, raising the question of how this enzyme promotes tissue-specific growth programs. Here we characterize two transgene reporters and an antibody that can be used to study Ldh expression in vivo . We find that all three tools produce similar Ldh expression patterns. Moreover, these reagents demonstrate that the larval Ldh expression pattern is complex, suggesting the purpose of this enzyme varies across cell types. Overall, our studies validate a series of genetic and molecular reagents that can be used to study glycolytic metabolism in the fly.

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport.

Background: Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer's, Parkinson's, and Huntington's disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function. Methods: We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos. Results: We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide "on-board" ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons. Conclusion: NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Metabolomic analysis of Drosophila melanogaster larvae lacking Pyruvate kinase.

Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster , however, are complicated by the fact that the fly genome encodes six Pyk paralogs whose functions remain poorly defined. To address this issue, we used sequence distance and phylogenetic approaches to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs, while the other five Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of two different Pyk mutant backgrounds revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, our analysis also unexpectedly reveals that steady state pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with our metabolomic findings, a complementary RNA-seq analysis revealed that genes involved in lipid metabolism and peptidase activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, our study provides both insight into how Drosophila larval metabolism adapts to disruption of glycolytic metabolism as well as immediate clinical relevance, considering that Pyk deficiency is the most common congenital enzymatic defect in humans.

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster.

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human health. This deficit of chemical safety information is exacerbated by current testing methods in mammals that are expensive, labor-intensive, and time-consuming. Recently, scientists and regulators have been working to develop new approach methodologies (NAMs) for chemical safety testing that are cheaper, more rapid, and reduce animal suffering. One of the key NAMs to emerge is the use of invertebrate organisms as replacements for mammalian models to elucidate conserved chemical modes of action across distantly related species, including humans. To advance these efforts, here, we describe a method that uses the fruit fly, Drosophila melanogaster, to assess chemical safety. The protocol describes a simple, rapid, and inexpensive procedure to measure the viability and feeding behavior of exposed adult flies. In addition, the protocol can be easily adapted to generate samples for genomic and metabolomic approaches. Overall, the protocol represents an important step forward in establishing Drosophila as a standard model for use in precision toxicology.

Aging and memory are altered by genetically manipulating lactate dehydrogenase in the neurons or glia of flies.

The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory. Although studies in vertebrates have revealed that lactate shuttling is important for cognitive function, it is uncertain if this form of metabolic coupling is conserved in invertebrates or is influenced by age. Lactate dehydrogenase (Ldh) is a rate limiting enzyme that interconverts lactate and pyruvate. Here we genetically manipulated expression of Drosophila melanogaster lactate dehydrogenase (dLdh) in neurons or glia to assess the impact of altered lactate metabolism on invertebrate aging and long-term courtship memory at different ages. We also assessed survival, negative geotaxis, brain neutral lipids (the core component of lipid droplets) and brain metabolites. Both upregulation and downregulation of dLdh in neurons resulted in decreased survival and memory impairment with age. Glial downregulation of dLdh expression caused age-related memory impairment without altering survival, while upregulated glial dLdh expression lowered survival without disrupting memory. Both neuronal and glial dLdh upregulation increased neutral lipid accumulation. We provide evidence that altered lactate metabolism with age affects the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2HG), and neutral lipid accumulation. Collectively, our findings indicate that the direct alteration of lactate metabolism in either glia or neurons affects memory and survival but only in an age-dependent manner.

Wolbachia is a nutritional symbiont in Drosophila melanogaster.

The intracellular bacterium Wolbachia is a common symbiont of many arthropods and nematodes, well studied for its impacts on host reproductive biology. However, its broad success as a vertically transmitted infection cannot be attributed to manipulations of host reproduction alone. Using the Drosophila melanogaster model and their natively associated Wolbachia strain "wMel", we show that Wolbachia infection supports fly development and buffers against nutritional stress. Wolbachia infection across several fly genotypes and a range of nutrient conditions resulted in reduced pupal mortality, increased adult emergence, and larger size. We determined that the exogenous supplementation of pyrimidines rescued these phenotypes in the Wolbachia-free, flies suggesting that Wolbachia plays a role in providing this metabolite that is normally limiting for fly growth. Additionally, Wolbachia was sensitive to host pyrimidine metabolism: Wolbachia titers increased upon transgenic knockdown of the Drosophila de novo pyrimidine synthesis pathway but not knockdown of the de novo purine synthesis pathway. We propose that Wolbachia acts as a nutritional symbiont to supplement fly development and enhance host fitness.

Renal oncometabolite L-2-hydroxyglutarate imposes a block in kidney tubulogenesis: Evidence for an epigenetic basis for the L-2HG-induced impairment of differentiation.

2-Hydroxyglutarate (2HG) overproducing tumors arise in a number of tissues, including the kidney. The tumorigenesis resulting from overproduced 2HG has been attributed to the ability of 2HG alter gene expression by inhibiting α-ketoglutarate (αKG)-dependent dioxygenases, including Ten-eleven-Translocation (TET) enzymes. Genes that regulate cellular differentiation are reportedly repressed, blocking differentiation of mesenchymal cells into myocytes, and adipocytes. In this report, the expression of the enzyme responsible for L2HG degradation, L-2HG dehydrogenase (L2HGDH), is knocked down, using lentiviral shRNA, as well as siRNA, in primary cultures of normal Renal Proximal Tubule (RPT) cells. The knockdown (KD) results in increased L-2HG levels, decreased demethylation of 5mC in genomic DNA, and increased methylation of H3 Histones. Consequences include reduced tubulogenesis by RPT cells in matrigel, and reduced expression of molecular markers of differentiation, including membrane transporters as well as HNF1α and HNF1ß, which regulate their transcription. These results are consistent with the hypothesis that oncometabolite 2HG blocks RPT differentiation by altering the methylation status of chromatin in a manner that impedes the transcriptional events required for normal differentiation. Presumably, similar alterations are responsible for promoting the expansion of renal cancer stem-cells, increasing their propensity for malignant transformation.

Honey bee symbiont buffers larvae against nutritional stress and supplements lysine.

Honey bees have suffered dramatic losses in recent years, largely due to multiple stressors underpinned by poor nutrition [1]. Nutritional stress especially harms larvae, who mature into workers unable to meet the needs of their colony [2]. In this study, we characterize the metabolic capabilities of a honey bee larvae-associated bacterium, Bombella apis (formerly Parasaccharibacter apium), and its effects on the nutritional resilience of larvae. We found that B. apis is the only bacterium associated with larvae that can withstand the antimicrobial larval diet. Further, we found that B. apis can synthesize all essential amino acids and significantly alters the amino acid content of synthetic larval diet, largely by supplying the essential amino acid lysine. Analyses of gene gain/loss across the phylogeny suggest that four amino acid transporters were gained in recent B. apis ancestors. In addition, the transporter LysE is conserved across all sequenced strains of B. apis. Finally, we tested the impact of B. apis on developing honey bee larvae subjected to nutritional stress and found that larvae supplemented with B. apis are bolstered against mass reduction despite limited nutrition. Together, these data suggest a novel role of B. apis as a nutritional mutualist of honey bee larvae.

The Drosophila melanogaster enzyme glycerol-3-phosphate dehydrogenase 1 is required for oogenesis, embryonic development, and amino acid homeostasis.

As the fruit fly, Drosophila melanogaster, progresses from one life stage to the next, many of the enzymes that compose intermediary metabolism undergo substantial changes in both expression and activity. These predictable shifts in metabolic flux allow the fly meet stage-specific requirements for energy production and biosynthesis. In this regard, the enzyme glycerol-3-phosphate dehydrogenase 1 (GPDH1) has been the focus of biochemical genetics studies for several decades and, as a result, is one of the most well-characterized Drosophila enzymes. Among the findings of these earlier studies is that GPDH1 acts throughout the fly lifecycle to promote mitochondrial energy production and triglyceride accumulation while also serving a key role in maintaining redox balance. Here, we expand upon the known roles of GPDH1 during fly development by examining how depletion of both the maternal and zygotic pools of this enzyme influences development, metabolism, and viability. Our findings not only confirm previous observations that Gpdh1 mutants exhibit defects in larval development, lifespan, and fat storage but also reveal that GPDH1 serves essential roles in oogenesis and embryogenesis. Moreover, metabolomics analysis reveals that a Gpdh1 mutant stock maintained in a homozygous state exhibits larval metabolic defects that significantly differ from those observed in the F1 mutant generation. Overall, our findings highlight unappreciated roles for GPDH1 in early development and uncover previously undescribed metabolic adaptations that could allow flies to survive the loss of this key enzyme.

Ecdysone and 20-hydroxyecdysone are not required to activate glycolytic gene expression in Drosophila melanogaster embryos.

Many of the Drosophila enzymes involved in carbohydrate metabolism are coordinately up-regulated approximately midway through embryogenesis. Previous studies have demonstrated that this metabolic transition is controlled by the Drosophila Estrogen-Related Receptor (dERR), which is stabilized and activated immediately prior to onset of glycolytic gene expression. The mechanisms that promote dERR activity, however, are poorly understood and other transcriptional regulators could control this metabolic transition, independent of dERR. In this regard, the steroid hormone 20-hydroxyecdysone (20E) represents an intriguing candidate for regulating glycolytic gene expression in embryos - not only does the embryonic 20E pulse immediately precede transcriptional up-regulation of glycolytic metabolism, but 20E is also known to promote Lactate dehydrogenase gene expression. Here I test the hypothesis that embryonic 20E signaling is required to activate glycolytic gene expression. Using developmental northern blots, I demonstrate that the transcriptional up-regulation of glycolytic genes during embryogenesis still occurs in shadow mutants, which are unable to synthesize either ecdysone or 20E. My finding indicates that ecdysone and 20E signaling are not required for this mid-embryonic metabolic transition.

miR-125-chinmo pathway regulates dietary restriction-dependent enhancement of lifespan in Drosophila.

Dietary restriction (DR) extends healthy lifespan in diverse species. Age and nutrient-related changes in the abundance of microRNAs (miRNAs) and their processing factors have been linked to organismal longevity. However, the mechanisms by which they modulate lifespan and the tissue-specific role of miRNA-mediated networks in DR-dependent enhancement of lifespan remains largely unexplored. We show that two neuronally enriched and highly conserved microRNAs, miR-125 and let-7 mediate the DR response in Drosophila melanogaster. Functional characterization of miR-125 demonstrates its role in neurons while its target chinmo acts both in neurons and the fat body to modulate fat metabolism and longevity. Proteomic analysis revealed that Chinmo exerts its DR effects by regulating the expression of FATP, CG2017, CG9577, CG17554, CG5009, CG8778, CG9527, and FASN1. Our findings identify miR-125 as a conserved effector of the DR pathway and open the avenue for this small RNA molecule and its downstream effectors to be considered as potential drug candidates for the treatment of late-onset diseases and biomarkers for healthy aging in humans.

Balancing energy expenditure and storage with growth and biosynthesis during Drosophila development.

Sustaining life requires efficient uptake of nutrients and conversion to useable forms. Almost everything about this process is dynamic. Nutrient availability fluctuates and changing environmental conditions impose new demands that can tip the metabolic equilibrium from biosynthesis and macromolecule storage to energy expenditure. At the same time, the organism itself changes, particularly during the rapid growth and differentiation in early development and also later in life as the adult ages. Here we review what has been learned from Drosophila melanogaster as an experimental model about the connections between external signals, signaling pathways, tissues and organs that allow animals to balance energy storage with expenditure in the face of change, both intrinsic and extrinsic.