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. Author manuscript; available in PMC: 2016 Nov 10.
Published in final edited form as: Cell Rep. 2016 Sep 27;17(1):69–85. doi: 10.1016/j.celrep.2016.08.073

Figure 5.

Figure 5

NAMPT inhibition causes metabolic dysfunction and photoreceptor death. (A–B) NAMPT inhibition with 20 μM FK866 caused loss of reductive capacity in 661W cone photoreceptor-like cells by 24 hours and 48 hours (n=15/group from three independent experiments; 1-way ANOVA with Tukey post-hoc test). (C–D) This metabolic dysfunction caused cell death by 48 hours (n=14/group from three independent experiments; 1-way ANOVA with Tukey post-hoc test). The effects of NAMPT inhibition were rescued with 100 μM NMN (A–B, D). (E) NAMPT inhibition with 20 μM FK866 led to a significant reduction in total NAD+ in photoreceptor cells by 6 hours, which was restored to near-normal levels with 100 μM NMN (n=3/group; 1-way ANOVA with Tukey post-hoc test). (F) 24 hours of 20 μM FK866 treatment led to undetectable levels of NAD+ (N.D. = not detected), which once again was restored with 100 μM NMN (n=5–7/group). (G) NAMPT inhibition also caused ATP depletion but in a delayed time frame relative to NAD+ depletion (n=4/group). (H–I) FK866-treated photoreceptor cells had reduced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) at baseline, impaired ECAR acceleration after oligomycin treatment, and impaired OCR acceleration after FCCP treatment (n=15–16/group from representative experiment; 2-way mixed ANOVA with Bonferroni post-hoc test). 100 μM NMN restored normal metabolic responses (H–I). (J) NAD+-dependent isocitrate dehydrogenase (NAD-IDH/IDH3) activity was reduced in rods isolated from Nampt−rod/−rod mice compared to those isolated from NamptF/F mice, even when sufficient NAD+ was added to the reaction mixture (n=6/group from three independent experiments; 2-tailed, unpaired t-test) and despite similar Idh3a expression levels (K; n=11–13/group from three independent experiments; 2-tailed, unpaired t-test). (L–M) The activities of other NAD+-dependent enzymes alpha-ketoglutarate dehydrogenase (AGDH; n=5/group from three independent experiments; Mann-Whitney U test) and malate dehydrogenase (MDH; n=7/group from three independent experiments; 2-tailed, unpaired t-test) in rods from Nampt−rod/−rod mice were restored with exogenous NAD+. Graphs depict mean + S.E.M. (A–F, J–M) or mean ± S.E.M. (G–I) (* p < .05; ** p < .01; *** p < .001; # p < .0001; red: vehicle versus FK; blue: FK versus FK+NMN).