Stable isotope labeling by amino acids in cell culture (SILAC) is widely used to quantify protein abundance in tissue culture cells. Until now, the only multicellular organism completely labeled at the amino acid level was the laboratory mouse. The fruit fly Drosophila melanogaster is one of the most widely used small animal models in biology. Here, we show that feeding flies with SILAC-labeled yeast leads to almost complete labeling in the first filial generation. We used these "SILAC flies" to investigate sexual dimorphism of protein abundance in D. melanogaster. Quantitative proteome comparison of adult male and female flies revealed distinct biological processes specific for each sex. Using a tudor mutant that is defective for germ cell generation allowed us to differentiate between sex-specific protein expression in the germ line and somatic tissue. We identified many proteins with known sexspecific expression bias. In addition, several new proteins with a potential role in sexual dimorphism were identified. Collectively, our data show that the SILAC fly can be used to accurately quantify protein abundance in vivo. The approach is simple, fast, and cost-effective, making SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics. Molecular & Cellular Proteomics 9:2173-2183, 2010.Mass spectrometry-based quantitative proteomics has emerged as a highly successful approach to study biological processes in health and disease (1-3). Most studies have so far been limited to in vitro systems such as cell culture models. Although tremendously useful, these models cannot appropriately reflect relevant regulatory mechanisms of multicellular eukaryotes in vivo. This is particularly relevant for complex processes involving interactions between different cell types such as differentiation and development (4).Relative changes in protein abundance are most accurately measured by comparing the natural form of a peptide with its stable isotope-labeled analog. Several different approaches enable stable isotope labeling of peptides either by chemical reactions or metabolic incorporation of the label (5, 6). Metabolic labeling has several advantages such as high labeling efficiency and intrinsically higher precision. For example, metabolically labeled samples can be combined before further processing steps so that protein quantification is not affected by differences in sample preparation. Labeling of organisms with stable isotope tracers was pioneered by Rudolf Schoenheimer 75 years ago (7,8). Since then, several model organisms ranging from prokaryotes to mammals have been labeled metabolically (for an excellent review, see Ref. 9). For example, Caenorhabditis elegans and Drosophila melanogaster have successfully been labeled with 15 N (10), and 15 N-labeled flies were recently used to study maternal-tozygotic transition (11) and seminal fluid proteins (sfps) 1 transferred at mating (12).15 N has also been used to label entire rats, particularly for quantitative brain proteomics (13,14). ...

Argonaute2 (Ago2) is an established component of the microRNA-induced silencing complex. Similar to miR-375 loss-of-function studies, inhibition of Ago2 in the pancreatic ␤-cell resulted in enhanced insulin release underlining the relationship between these two genes. Moreover, as the most abundant microRNA in pancreatic endocrine cells, miR-375 was also observed to be enriched in Ago2-associated complexes. Both Ago2 and miR-375 regulate the pancreatic ␤-cell secretome, and by using quantitative mass spectrometry, we identified the enhanced release of a set of proteins or secretion "signatures " in response to a glucose stimulus using the murine ␤-cell line MIN6. In addition, the loss of Ago2 resulted in the increased expression of miR-375 target genes, including gephyrin and ywhaz. The four mammalian Argonaute proteins mediate the microRNA pathway in mammalian cells by recruiting the noncoding RNAs to interact with their target mRNAs (1, 2). Whereas 60% of all mRNAs are predicted to be targets of microRNAs, the function of individual Argonaute proteins in this process is largely uncharacterized (3). Interestingly, only total loss of Argonaute2 (Ago2) expression results in embryonic lethality, although genetic deletion of the other family members does not appear to influence embryonic development in the mouse (4). In addition, Ago2 has been shown to constitute a rate-limiting determinant in RNAi efficacy (5). Although the role of the Argonaute family has not been elucidated in the pancreatic ␤-cell, miR-375, the most abundant microRNA in the pancreatic islet, has been shown to regulate both the growth and function of the ␤-cell (6, 7).Within the endocrine pancreas, several cell types (␣, ␤, ␦, and pancreatic polypeptide) release hormones into the circulation to regulate a broad spectrum of physiologic pathways (8). The pancreatic ␤-cell is the only source of insulin in the body and continuously responds to both glucose and electrical stimuli by releasing proteins into the blood (9). Processing of the proinsulin molecule occurs during assembly of the secretory granule when insulin is crystallized leading to the formation of mature dense-core granules (8). Several reports suggest the number of proteins in the insulin secretory granules to range between 50 and 150 as identified using mass spectrometry (10 -12). The dense granules are transported and stored to comprise a readily releasable pool and, upon stimulation, fuse to the plasma membrane and constitute first-phase insulin release (13).In this study, we address the regulatory role of Ago2 in the secretion pathway of the pancreatic ␤-cell. Proteins exocytosed from the murine insulinoma cell line MIN6 in response to glucose into the extracellular environment were quantified using a stable isotope labeling with amino acids in cell culture (SILAC) 1 -based approach (14,15). Comparison of the proteins detected in the supernatant after induction by high glucose with those inhibited after loss of glucokinase expression includes a set of secreted ␤-cell proteins or se...

Background: Upon entering the pancreatic ␤-cell, glucose is metabolized to ultimately induce both proliferation and the release of insulin. Results: miR-184 targets Argonaute2 to impact the microRNA pathway according to glucose metabolism. Conclusion: miR-184 is a highly regulated microRNA impacting the growth and function of the ␤-cell. Significance: These results highlight the adaptive role of the microRNA pathway based on metabolic state.

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Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-triphosphate (PIP 3 ) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP 3 and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.