Al., 2013). However, muscle- or liver-specific deletion of SIRT3 didn’t result
Al., 2013). Having said that, muscle- or liver-specific deletion of SIRT3 did not result in changes in ATP levels, suggesting that SIRT3 deletion in a tissue-specific manner doesn’t influence cellular energy levels (Fernandez-Marcos et al., 2012). In this study, we have applied Drosophila as a model and performed mass spectrometric analyses on wild-type and dsirt2 mutant flies to determine the Drosophila mitochondrial and dSirt2-regulated acetylome. Our proteomic experiments show Drosophila Sirt2 is an essential regulator of mitochondrial function and is definitely the functional homologue of mammalian SIRT3. These experiments also offer a complete view on the impact of acetylation on OXPHOS and its regulation by dSirt2. We demonstrate that ATP synthase , the catalytic subunit of complicated V, is an acetylated protein, and it is a substrate of Drosophila Sirt2 and human SIRT3.290 JCB VOLUME 206 Number 2 Within this study, we also reveal a novel connection amongst NAD metabolism, sirtuins, plus the sphingolipid ceramide. MAO-A drug sphingolipids are an crucial class of lipids which might be creating blocks for membranes and serve as transducers in signaling cascades that regulate cell growth and death (Hannun and Obeid, 2008). Ceramide, a central intermediate in sphingolipid metabolism, mediates many stress responses, and current literature highlights that perturbations in ceramide levels can impact glucose and fat metabolism (Bikman and Summers, 2011). How ceramide and also other sphingolipids have an effect on cellular metabolism, what metabolic pathways they impinge on, and identification from the ensuing functional consequences are only starting to become explored. We show that Drosophila mutants of sphingolipid metabolism, especially, ceramide kinase mutants (dcerk1), have enhanced levels of ceramide and decreased levels of NAD. This results in BRD2 Source reduced dSirt2 activity in dcerk1 mutants, leading to acetylation of quite a few subunits of complex V, which includes ATP synthase and decreased complex V activity. These experiments reveal a novel axis involving ceramide, NAD, and sirtuins.ResultsCeramide improve impacts NAD level and sirtuin activityWe performed metabolomic profiling on sphingolipid mutants that accumulate ceramide to gain insight into metabolic pathways that could possibly be altered in these mutants. Our earlier study combined metabolomic profiling with genetic and biochemical approaches and demonstrated that dcerk1 mutants show an increased reliance on glycolysis, which results in a rise in lactate to compensate for the decreased production of ATP via OXPHOS (Nirala et al., 2013). The raise in glycolytic flux is also observed in a mammalian model of ceramide improve, mice heterozygous for the ceramide transfer protein (Wang et al., 2009; Nirala et al., 2013). In addition to modifications in glycolytic intermediates, metabolomic profiling revealed that dcerk1 mutants have a considerably decreased degree of NAD compared with that in w1118 (manage) flies (Fig. 1 A). The NAD level is controlled by balancing synthesis, salvage, and consumption pathways (Fig. 1 B). Like in mammals, NAD might be synthesized in Drosophila in the salvage pathway from nicotinic acid, nicotinamide, and nicotinamide riboside (nicotinamide mononucleotide) and by the de novo pathway from tryptophan (Zhai et al., 2006; Campesan et al., 2011). We utilized mass spectrometry (MS) to measure the levels of intermediates in these pathways and associated metabolites. The levels of important intermediates, for example nicotinamide riboside in the.