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WIREs Dev Biol
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Methods for studying the metabolic basis of Drosophila development

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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

A schematic representation of changes in metabolite abundance during Drosophila development. Trehalose, glycogen, and triglycerides undergo dramatic but predictable changes during the Drosophila life cycle. Any metabolic experiment must be interpreted in the context of these developmental trends.
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A schematic diagram illustrating the basic principles of stable‐isotope tracer analysis. U‐13C‐labeled glucose is six atomic mass units (amu) heavier than unlabeled 12C glucose. The catabolism of labeled glucose will result in the incorporation of 13C into other metabolites, which can be measured using either MS or NMR. For example, lactate and pyruvate molecules derived from labeled carbon will be 3 amu heavier, or m+3, than unlabeled molecules. Pyruvate derived from labeled glucose can enter the TCA cycle via formation of acetyl‐CoA, which will result in m+2 citrate. As labeled carbons move throughout the cycle, the mass of intermediates will become more complex, resulting in m+3 and m+4 labeled compounds. The end result is represented by an isotopologue distribution.
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A diagram illustrating common problems associated with using the glucose oxidase (GO) method for measuring larval trehalose. The GO assay oxidizes free glucose to generate a red product. The intensity of the colored product is measured by absorbance at 540 nm. (a) All trehalose assays must include a standard curve for both glucose and trehalose. An acceptable assay will display a linear increase in absorbance that reflects the increasing concentrations of both sugars. Failure to observe a concentration dependent increase in trehalose measurements is the result of inactive trehalase, which can stem from problems with either the buffer or enzyme stock. Expired GO reagent will result in abnormally low readings for both the glucose and trehalose standard cure. (b) Problems associated with measuring trehalose in both a control strain and a mutant strain with a hypertrehalosic phenotype. Improperly prepared samples will result in glycogen degradation, which leads to abnormally high free glucose levels in the no trehalase control (−) of both strains (free glucose levels are normally very low in larval measurements). Trehalose concentrations that fall beyond the linear range of the GO assay generate a purple color, which alters the absorption spectra of the sample and generates inaccurate measurements. Properly prepared samples will exhibit a red hue.
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