preparation-drosophila-larval-samples-for-gas-chromatography-mass

Preparation of <em>Drosophila</em> 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 ...

Indiana University

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.

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

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 NH: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.

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

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 For further resources related to this article, please visit the WIREs website.

Methods for studying the metabolic basis of Drosophila development.

To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11+/+ and Slc4a11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy.

Conditionally Immortal Slc4a11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11-/- Mouse Model.

The fruit fly  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,  geneticists commonly use candidate gene approaches, which, while important, bias studies toward known metabolic regulators. In an effort to expand the scope of  metabolic studies, we used the classic mutant  () 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  by using free lysine levels as a phenotypic readout. Our efforts revealed that  encodes the  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  lysine catabolism is complex and capable of influencing seemingly unrelated metabolic pathways. Overall, our study highlights how a combination of  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.

Metabolomic Analysis Reveals That the  Gene  Influences Diverse Aspects of Metabolism.

In the beverage fermentation industry, especially at the craft or micro level, there is a movement to incorporate as many local ingredients as possible to both capture terroir and stimulate local economies. In the case of craft beer, this has traditionally only encompassed locally sourced barley, hops, and other agricultural adjuncts. The identification and use of novel yeasts in brewing lags behind. We sought to bridge this gap by bio-prospecting for wild yeasts, with a focus on the American Midwest. We isolated 284 different strains from 54 species of yeast and have begun to determine their fermentation characteristics. During this work, we found several isolates of five species that produce lactic acid and ethanol during wort fermentation: Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus, and Wickerhamomyces anomalus. Tested representatives of these species yielded excellent attenuation, lactic acid production, and sensory characteristics, positioning them as viable alternatives to lactic acid bacteria (LAB) for the production of sour beers. Indeed, we suggest a new LAB-free paradigm for sour beer production that we term "primary souring" because the lactic acid production and resultant pH decrease occurs during primary fermentation, as opposed to kettle souring or souring via mixed culture fermentation.

Hongde Li

Department of Biology

Indiana University

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

Hongde Li

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

Indiana University

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

Department of Biology