Figure 1.
Effect of mitochondrial sirtuin expression on the cellular oxygen consumption rate.
(A) HEK293 cells stably transfected with either HA-tagged SIRT3, SIRT4, SIRT5, or the empty plasmid vector (V) were fractionated into mitochondria and cytosol and subjected to western blotting with anti-HA to visualize the tagged sirtuins. Anti-cytochrome-C (Cyt-C) and anti-very long-chain acyl-CoA dehydrogenase (VLCAD) antibodies were used as markers to demonstrate that the mitochondria were intact. Ponceau-S staining served as a loading control. (B) Anti-sirtuin western blotting of stably transfected (Tr) HEK293 cell lines versus vector (V) transfected control cells picks up the same isoforms as anti-HA blotting in panel A, and also visualizes weak expression of endogenous SIRT3 (indicated with arrow). No endogenous SIRT4 or SIRT5 were detected under the conditions used. (C) Equal numbers of the stably transfected HEK293 cells were subjected to oxygen consumption measurements in a Seahorse XF24 extracellular flux analyzer, with sequential additions of the metabolic inhibitors/activators oligomycin (O), FCCP (F), 2-deoxyglucose (2DG), and rotenone (R). The measurements were done in quadruplicate wells of cells. The experiment was repeated with similar results. Oxygen consumption data over the first 30 minutes was averaged to yield the basal oxygen consumption parameter (D). Proton leak (E), defined as oligomycin-insensitive oxygen consumption was calculated by averaging the data over the second 30 minute period (following oligomycin injection). All graphs depict means and standard deviations, and *P<0.01.
Figure 2.
Sirtuin expression in HEK293 does not affect (A) the rate of cellular growth; (B) mitochondrial mass as judged by western blotting of electron transport chain components; (C)steady-state ATP under basal conditions or after the addition of the metabolic inhibitors etomoxir (Eto, 100 µM), oligomycin (oligo, 1 µM), 2-deoxyglucose (2DG, 100 mM), or combinations thereof; or (D) intramitochondrial NAD+.
Growth was measured in quadruplicate wells in two separate experiments which were averaged. ATP was measured in triplicate wells containing equal numbers of cells in two separate experiments which were averaged. NAD+ was measured in three separate preparations of mitochondria and the results averaged. All data are means and standard deviations.
Figure 3.
Effect of mitochondrial sirtuin expression on glycolysis.
Seahorse extracellular acidification rates (A) were measured in quadruplicate wells containing equal numbers of cells. The experiment was repeated with similar results. Data collected over the first 30 minutes were averaged to yield the basal glycolytic rate (B). Oligomycin-stimulated glycolysis (C) was calculated by subtracting the basal values from the maximum values obtained immediately after oligomycin injection. The oxygen consumption/extracellular acidification ratio (D) was calculated by dividing the values shown in Figure 1D by those in Figure 3B. All graphs depict means and standard deviations, and *P<0.05. mpH = milli pH units.
Figure 4.
Effect of mitochondrial sirtuin expression on superoxide and mitochondrial membrane potential.
(A) Superoxide measured with MitoSox. (B) Membrane potential measured with TMRM. Graphs depict means and standard deviations, and *P<0.05.
Figure 5.
Effect of mitochondrial sirtuin expression on substrate flux.
(A) Glucose oxidation to 14CO2. (B) 3H-palmitate oxidation to 3H2O. (C) 14C-acetate conversion to fatty acids. All graphs depict means and standard deviations, and *P<0.05.
Figure 6.
Seahorse XF24 extracellular flux analysis of sirtuin-expressing HEK293 cells under 5 mm glucose conditions.
(A) Oxygen consumption and (B) extracellular acidification rates were measured under the same protocol as described in Figures 1 and 3. All graphs depict means and standard deviations. mpH = milli pH units.