Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster α-Esterase-7.

Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such as xenobiotic detoxi fi cation, lipid metabolism and reproduction. Phylogenetically, many insect carbox- ylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene clusters such as the a -Esterase cluster. Much in contrast to the vital importance attributed to carbox- ylesterases in general, the in vivo function of individual a -Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fl y Drosophila melanogaster fat body. One of the fat body carboxylesterases, a -Esterase-7 , was selected for mutational analysis by gene targeting to generate a deletion mutant fl y. Phenotypic characterization of a -Esterase-7 null mutants and transgenic fl ies, which overexpress a chimeric a -Esterase-7:EGFP gene, reveals important functions of a -Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The presented fi rst deletion mutant of any a -Esterase in the model insect D. melanogaster generated by gene targeting not only provides experimental evidence for the endogenous functions of this gene family. It also offers an entry point for in vivo structure-function analyses of a -Esterase- 7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species

Assays for insulin and insulin-like activity based on adipocytes.

Data from the metabolic assays (and signaling assays; see below) are calculated as stimulation factor above basal activity (absence of insulin/compound/drug candidate) for processes stimulated (e.g., lipogenesis, glucose transport, and GLUT4 translocation) or as difference between the basal and insulin/compound/drug candidate-induced values for processes downregulated (e.g., lipolysis). In each case, these data, which reflect the responsiveness of the metabolic effector system studied toward the respective stimulus (insulin/compound/drug candidate), are normalized to the basal (set at 0 %) and maximal insulin action (set at 100 %; elicited by maximally effective concentration of insulin). For characterization of the sensitivity of the metabolic effector system toward the respective stimulus, effective concentrations for the induction of 150 % (or higher) of the basal activity (set at 100 %) can be given. These so-called EC150-values facilitate the insulin-independent comparison of the relative potency of the insulin-like activity between compounds/drug candidates, in general, and in particular for those frequently observed stimuli, which do not elicit the same maximal response in % stimulation or inhibition and/or fail to approach the maximal insulin response