at some time points in incubations with inhibitor 
the NAD+ and NADH levels became simply too low to accurately calculate a ratio. Whether the fluctuations observed are an experimental R 115777 artifact or a feature that is associated with oscillations in cellular redox state, as has been described for yeast cells, remains to be determined. FK866 Inhibition of NAMPT Reduces Glycolytic Flux but not Intracellular ATP Since the NAD profiles of the two macrophage cell lines were highly similar and FK866 affected the NAD levels to the same extent, we chose to continue our study with main focus on the RAW 264.7 cell line and made comparisons with the MafDKO cell line where appropriate. In order to determine the metabolic effect of NAD+-depletion we assessed glycolytic activity and ATP production. Other studies already demonstrated that macrophages depend strongly on glycolysis for cellular ATP production. Instead of complete oxidation via the mitochondrial route through the TCA cycle and oxidative phosphorylation, 80% of the glucose molecules that are imported, are ultimately converted into lactate. For the RAW 264.7 lineage we confirmed this picture and found that,75% of all glucose used is converted into lactate, yielding approximately 1.5 moles of lactate per mole glucose. In glycolysis, NAD+ is converted into NADH by glyceraldehyde-3-phosphate-dehydrogenase, producing 1,3-biphosphoglycerate from glyceraldehyde-3-phosphate. In concert therewith, the end reaction of glycolysis, helps to restore the cytoplasmic NAD+/NADH balance. Therefore, in view of the crucial role of NAD+ availability in maintenance of glycolytic flux, and on the basis of our data shown NAD+ Controls Macrophage Morphodynamics 6 NAD+ Controls Macrophage Morphodynamics nucleotide levels in Maf-DKO cells incubated for the indicated time periods with or without 5 nM FK866. D,G,J,M, NAD+/NADH and NADPH/NADP+ ratios were calculated from the data in A&B, E&F, H&I, and K&L. Data in B-M represent means of three experiments performed in triplicate. ND, not detected.. doi:10.1371/journal.pone.0097378.g001 in Inhibition of NAMPT-mediated NAD+ Synthesis does not Affect Mitochondrial Respiration A large fraction of the cell’s content of reduced pyridine nucleotides is compartmentalized in the mitochondria. Cellular NADH autofluorescence is therefore considered a reliable measure for assessing the mitochondrial NADH status. We utilized this parameter to determine whether FK866 mediated inhibition of NAMPT affected mitochondrial NAD pools of RAW 264.7 cells. Although the autofluorescence intensity of 24 hour FK866-treated cells was reduced by 38%, it did not mirror the dramatic reduction observed in total cellular NADH after 24 hours. One possible explanation for this apparent discrepancy may be that FK866 hardly affects mitochondrial NAD levels. However, controversy exists around the source of mitochondrial NAD+, as the level of this pool appears still to be linked – directly or indirectly – to the level of cytosolic NAD+. Therefore, by reducing cytosolic NAD+, NAMPT 7 NAD+ Controls Macrophage Morphodynamics inhibition may ultimately affect the mitochondrial pool, but with differential kinetics. Another explanation PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19656855 for the roughly one-third shift in autofluorescence signal would be that adaptation in mitochondrial OXPHOS occurs, to compensate for the loss in glycolytic ATPproduction. Thereby a situation would be created wherein an increased fraction of total mitochondrial NAD would appear in th