Hongde Li

Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth

Significance Oncometabolites are small molecules that promote tumor formation and growth. L-2-hydroxyglutarate (L-2HG) is a putative oncometabolite that is associated with gliomas and renal cell carcinomas, as well as a severe neurometabolic disorder known as L-2-hydroxyglutaric aciduria. However, despite that L-2HG is commonly considered a metabolic waste product, this compound was recently discovered to control immune cell fate, thereby demonstrating that it has endogenous functions in healthy animal cells. Here, we find that the fruit fly, Drosophila melanogaster, also synthesizes high concentrations of L-2HG during normal larval growth. Our discovery establishes the fly as a genetic model for studying this putative oncometabolite in healthy animal tissues. 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. Here, we demonstrate 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). We also show that 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, we use a genetic approach to demonstrate that dLDH and L-2HG influence position effect variegation and DNA methylation, suggesting that this compound serves to coordinate glycolytic flux with epigenetic modifications. Overall, our studies 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.

Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium

Corneal endothelium (CE) is among the most metabolically active tissues in the body. This elevated metabolic rate helps the CE maintain corneal transparency by its ion and fluid transport properties, which when disrupted, leads to visual impairment. Here we demonstrate that glutamine catabolism (glutaminolysis) through TCA cycle generates a large fraction of the ATP needed to maintain CE function, and this glutaminolysis is severely disrupted in cells deficient in NH3:H+ cotransporter Solute Carrier Family 4 Member 11 (SLC4A11). Considering SLC4A11 mutations leads to corneal endothelial dystrophy and sensorineural deafness, our results indicate that SLC4A11-associated developmental and degenerative disorders result from altered glutamine catabolism. Overall, our results describe an important metabolic mechanism that provides CE cells with the energy required to maintain high level transport activity, reveal a direct link between glutamine metabolism and developmental and degenerative neuronal diseases, and suggest an approach for protecting the CE during ophthalmic surgeries.

Methods for studying the metabolic basis of Drosophila development

The field of metabolic research has experienced an unexpected renaissance. While this renewed interest in metabolism largely originated in response to the global increase in diabetes and obesity, studies of metabolic regulation now represent the frontier of many biomedical fields. This trend is especially apparent in developmental biology, where metabolism influences processes ranging from stem cell differentiation and tissue growth to sexual maturation and reproduction. In this regard, the fruit fly Drosophila melanogaster has emerged as a powerful tool for dissecting conserved mechanisms that underlie developmental metabolism, often with a level of detail that is simply not possible in other animals. Here we describe why the fly is an ideal system for exploring the relationship between metabolism and development, and outline a basic experimental strategy for conducting these studies. WIREs Dev Biol 2017, 6:e280. doi: 10.1002/wdev.280

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Metabolomic Analysis Reveals That the Drosophila Gene lysine Influences Diverse Aspects of Metabolism

The fruit fly Drosophila melanogaster has emerged as a powerful model for investigating the molecular mechanisms that regulate animal metabolism. However, a major limitation of these studies is that many metabolic assays are tedious, dedicated to analyzing a single molecule, and rely on indirect measurements. As a result, Drosophila geneticists commonly use candidate gene approaches, which, while important, bias studies toward known metabolic regulators. In an effort to expand the scope of Drosophila metabolic studies, we used the classic mutant lysine (lys) to demonstrate how a modern metabolomics approach can be used to conduct forward genetic studies. Using an inexpensive and well-established gas chromatography-mass spectrometry-based method, we genetically mapped and molecularly characterized lys by using free lysine levels as a phenotypic readout. Our efforts revealed that lys encodes the Drosophila homolog of Lysine Ketoglutarate Reductase/Saccharopine Dehydrogenase, which is required for the enzymatic degradation of lysine. Furthermore, this approach also allowed us to simultaneously survey a large swathe of intermediate metabolism, thus demonstrating that Drosophila lysine catabolism is complex and capable of influencing seemingly unrelated metabolic pathways. Overall, our study highlights how a combination of Drosophila forward genetics and metabolomics can be used for unbiased studies of animal metabolism, and demonstrates that a single enzymatic step is intricately connected to diverse aspects of metabolism.

The Drosophila mitochondrial citrate carrier regulates L-2-hydroxyglutarate accumulation by coupling the tricarboxylic acid cycle with glycolysis

The oncometabolites D- and L-2-hydroxyglutarate (2HG) broadly interfere with cellular metabolism, physiology, and gene expression. A key regulator of 2HG metabolism is the mitochondrial citrate carrier (CIC), which, when mutated, promotes excess D-/L-2HG accumulation. The mechanism by which CIC influences 2HG levels, however, remains unknown. Here we studied the Drosophila gene scheggia (sea), which encodes the fly CIC homolog, to explore the mechanisms linking mitochondrial citrate efflux to L-2HG metabolism. Our findings demonstrate that decreased Drosophila CIC activity results in elevated glucose catabolism and increased lactate production, thereby creating a metabolic environment that inhibits L-2HG degradation.

A Drosophila model of combined D-2- and L-2-hydroxyglutaric aciduria reveals a mechanism linking mitochondrial citrate export with oncometabolite accumulation

ABSTRACT The enantiomers of 2-hydroxyglutarate (2HG) are potent regulators of metabolism, chromatin modifications and cell fate decisions. Although these compounds are associated with tumor metabolism and commonly referred to as oncometabolites, both D- and L-2HG are also synthesized by healthy cells and likely serve endogenous functions. The metabolic mechanisms that control 2HG metabolism in vivo are poorly understood. One clue towards how cells regulate 2HG levels has emerged from an inborn error of metabolism known as combined D- and L-2HG aciduria (D-/L-2HGA), which results in elevated D- and L-2HG accumulation. Because this disorder is caused by mutations in the mitochondrial citrate transporter (CIC), citrate must somehow govern 2HG metabolism in healthy cells. The mechanism linking citrate and 2HG, however, remains unknown. Here, we use the fruit fly Drosophila melanogaster to elucidate a metabolic link between citrate transport and L-2HG accumulation. Our study reveals that the Drosophila gene scheggia (sea), which encodes the fly CIC homolog, dampens glycolytic flux and restricts L-2HG accumulation. Moreover, we find that sea mutants accumulate excess L-2HG owing to elevated lactate production, which inhibits L-2HG degradation by interfering with L-2HG dehydrogenase activity. This unexpected result demonstrates that citrate indirectly regulates L-2HG stability and reveals a feedback mechanism that coordinates L-2HG metabolism with glycolysis and the tricarboxylic acid cycle. Finally, our study also suggests a potential strategy for preventing L-2HG accumulation in human patients with CIC deficiency. This article has an associated First Person interview with the first author of the paper. Summary: This study reveals a mechanism that links export of mitochondrial citrate to accumulation of the oncometabolite L-2-hydroxyglutarate, suggesting a potential treatment for individuals with combined D-2- and L-2-hydroxyglutaric aciduria, a rare inborn error of metabolism.

Inflammation-induced DNA methylation of DNA polymerase gamma alters the metabolic profile of colon tumors

BackgroundInflammation, metabolism, and epigenetic modulation are highly interconnected processes that can be altered during tumorigenesis. However, because of the complexity of these interactions, direct cause and effect during tumorigenesis have been difficult to prove. Previously, using a murine model of inflammation-induced colon tumorigenesis, we determined that the promoter of the catalytic subunit of DNA polymerase gamma (Polg) is DNA hypermethylated and silenced in inflammation-induced tumors, but not in non-inflammation-induced (mock) tumors, suggesting that inflammation can induce silencing of Polg through promoting DNA methylation during tumorigenesis. Polg is the only mitochondrial DNA polymerase and mutations in Polg cause mitochondrial diseases in humans. Because of the role of mitochondria in metabolism, we hypothesized that silencing of Polg in inflammation-induced tumors would result in these tumors having altered metabolism in comparison to mock tumors.MethodsInflammation-induced and mock colon tumors and colon epithelium from a mouse model of inflammation-induced colon tumorigenesis were assayed for alterations in Polg expression, mitochondria, and metabolism. Organoids derived from these tissues were used to study the direct effect of loss of Polg on mitochondria and metabolism.ResultsWe demonstrate that inflammation-induced tumors with reduced Polg expression have decreased mitochondrial DNA content and numbers of mitochondria compared to normal epithelium or mock tumors. Tumoroids derived from mock and inflammation-induced tumors retained key characteristics of the original tumors. Inflammation-induced tumoroids had increased glucose uptake and lactate secretion relative to mock tumoroids. shRNA-mediated knockdown of Polg in mock tumoroids reduced mtDNA content, increased glucose uptake and lactate secretion, and made the tumoroids more resistant to oxidative stress.ConclusionsThese results suggest that inflammation-induced DNA methylation and silencing of Polg plays an important role in the tumorigenesis process by resulting in reduced mitochondria levels and altered metabolism. An enhanced understanding of how metabolism is altered in and drives inflammation-induced tumorigenesis will provide potential therapeutic targets.