Increase in ceramide levels results in depletion of NAD⁺ and decrease in sirtuin activity leading to hyperacetylation of proteins in different cellular compartments. (A) dcerk¹ fly extracts show 65% reduction in NAD⁺ level compared with w¹¹¹⁸ control. n = 3. (B) NAD synthesis and salvage pathways. TDO, tryptophan-2,3-dioxygenase; KMO, kynurenine 3-monooxygenase; QPRTase, quinolinate phosphoribosyltransferase; NaMNAT, nicotinic acid mononucleotide adenyltransferase; NADS, NAD synthetase; NMNAT, nicotinamide mononucleotide adenyltransferase; NAmPRTase, nicotinamide phosphoribosyl transferase; NDase, nicotinamidase; NaPRTase, nicotinic acid phosphoribosyltransferase. (C) Mass spectrometric measurements of metabolites in the salvage and the de novo pathways for synthesis of NAD⁺. n = 3. (D) Soluble, mitochondrial, and nuclear extracts were prepared from w¹¹¹⁸ and dcerk¹ mutant flies and separated by PAGE. Protein acetylation was monitored by Western blotting using an anti–acetyl-Lys antibody. The individual blots were probed with antibodies to actin, porin, and H2A as loading controls. dcerk¹ mutants show protein hyperacetylation in the different cellular compartments. Arrows indicate proteins that are hyperacetylated in dcerk¹ compared with w¹¹¹⁸. MM, molecular mass. (E) Mitochondrial NAD⁺ levels are decreased in dcerk¹ compared with control. (F) d14 long chain base ceramides with different fatty acids were estimated by MS in sphingolipid-enriched fractions prepared from w¹¹¹⁸ and dcerk¹ mitochondria. C denotes the carbon chain length of fatty acids in the different ceramides. The amount of ceramide is normalized to total carbon content, and the level in w¹¹¹⁸ is taken as 100%. Many ceramides show significant increase in the mutant mitochondria compared with w¹¹¹⁸. n = 3. Error bars represent SDs. *, P ≤ 0.05–0.01; **, P ≤ 0.01–0.001; ***, P ≤ 0.001–0.0001 in Student’s t test.