Figure 1.
Effects of oleate on energization and respiration of permeabilized tubules: comparison of complex I and complex II-dependent substrates.
Direct effects of oleate on energization of permeabilized rabbit tubules measured with safranin O uptake (panels A–C) and respiration (D) supported by either succinate or the combination of complex I dependent substrates, α-ketoglutarate, malate, and glutamate (AMG). Sets of typical safranin O uptake tracings (inverted fluorescence) are shown in panels A and B and group averages for those studies are in panel C. Numbers adjacent to each tracing in panels A are the concentrations of oleate added in µM. In panel C, “Peak” indicates the maximal uptake compared to the uptake seen without added oleate using succinate as substrate measured in the presence of delipidated albumin to eliminate the effect of endogenous fatty acids. “End” indicates the final level reached at the end of the 600 second measurement period, which can be less than the peak if there has been decay of ΔΨm. The panel D respiratory rates (RR) are given as percentages relative to “Control” rates without added oleate using succinate as substrate. Shown are both the initial rate produced by oleate and then the rate at the end of 600 seconds of measurement. Values in panels C and D are means±SEM for N = 3–5 except 4 µM oleate with AMG where the N was 2. *P<0.05, #P<0.01, +P<0.001 vs. corresponding succinate group.
Figure 2.
Effects of additional substrates on oleate-induced deenergization in the presence of succinate.
A. Concentration dependence of deenergization produced by late addition of oleate to rabbit tubules. B. Representative experiment showing how modification of late deenergization by oleate was tested. 1 µM oleate was added at 710 seconds followed by a test agent at 910 seconds. Inset expands the oleate testing period to show effects of glutamate (G) and malate (M) individually. Other abbreviations are: NO – no oleate, dBSA – delipidated albumin. C. Group averages for studies such as those in panel B comparing restoration of ΔΨm. Delipidated albumin (dBSA) fully restored ΔΨm in every case. Effects of other agents were calculated relative to the level reached with dBSA. Values are means±SEM for N = 3–5. *P≤0.05, significantly different change of ΔΨm relative to the no test addition starting point. Other abbreviations are: G – glutamate A- α-ketoglutarate, M – malate, AM - α-ketoglutarate+malate, GM – glutamate, ASP – aspartate. D. Effect of malate (M) addition on succinate-supported energization in the absence of exogenous NEFA. Left panel shows tracings illustrating concentration dependence of the malate effects. Numbers adjacent to each tracing are the concentrations of malate added in mM. Right panel shows effect of dBSA.
Figure 3.
Comparison of substrate support for energization in the absence of exogenous NEFA.
Safranin O uptake by permeabilized rabbit tubules was followed in the presence of the indicated substrates. Panels A. B, D, and E are typical tracings. Panels C and F summarize the group results. In the panel A–C studies, the indicated substrates were present from the start and delipidated albumin (dBSA) was added at 600 seconds. In the panel D–F studies endogenous substrates were first depleted by allowing energization in the absence of exogenous substrate, then substrates were added as indicated either without (panel D) or with (panel E) prior dBSA. Abbreviations are: S-succinate, M-malate, A- α-ketoglutarate, G-glutamate, AM-α-ketoglutarate+malate, GM-glutamate+malate. Values in panels C and F are means±SEM for N = 3–5. @ P<0.05 vs. the corresponding group in the preceding set.
Figure 4.
Mediation of malate-induced deenergization by oxaloacetate.
A. Pathways by which glutamate and rotenone decrease mitochondrial matrix oxaloacetate accumulation. IMM – inner mitochondrial membrane, ET – electron transport, FP – flavoprotein, αKG – α-ketoglutarate. B. Effects of malate on succinate-supported respiration of rabbit tubules. Basal, ADP-stimulated, and oligomycin-suppressed respiratory rates were measured under the indicated conditions. Abbreviations are NFA – no further additions, dBSA – delipidated bovine serum albumin, M malate (4 mM), M dBSA – malate (4 mM) plus dBSA. Values are means±SEM for N = 3–5. *P<0.05, significantly different from the corresponding addition without malate. C. Representative safranin O uptake tracings showing effects of adding malate to rabbit tubules energized with succinate. Labels indicating the conditions for each experiment are in the order of the tracings from top to bottom. Malate was added at 600 seconds. Abbreviations are dBSA – delipidated bovine serum albumin, M – malate (4 mM), G – glutamate (4 mM), R – rotenone (1 µM), NFA – no further additions. D. Group results for the studies illustrated in panel C. Tubules received either malate or a sham addition at 600 seconds with measurements of the signal at 800 seconds. Data for each condition are presented relative to behavior of the corresponding dBSA sample with sham addition, where no loss of signal occurred. Values are means±SEM on studies from 5 separate preparations. *P<0.05, #P<0.01, or +P<0.001 relative to corresponding NFA group.
Figure 5.
Effects of oleate and malate on energization of mouse tubules.
A. Concentration dependence of effects of oleate on energization of permeabilized mouse tubules measured with safranin O uptake supported by either succinate or the combination of complex I dependent substrates, α-ketoglutarate, malate, and glutamate. “Peak” indicates the maximal uptake compared to the uptake seen without added oleate using succinate as substrate in the presence of delipidated albumin to eliminate the effect of endogenous fatty acids. “End” indicates the final level reached at the end of the 600 second measurement period, which can be less than the peak if there has been decay of ΔΨm. Values are means±SEM for N = 3. *P<0.05, #P<0.01, +P<0.001 vs. corresponding succinate group. B and C. Effects of malate on succinate-supported energization measured using safranin O uptake (Panel B) and respiration (Panel C). Permeabilized tubules were incubated with succinate and the indicated test agents for 350 seconds (Pre-Malate period) followed by addition of either sham malate or malate for 200 seconds with measurement of safranin uptake and respiratory rate (RR) at the end of that period. Values are compared to those measured for the no further addition (NFA) group at the end of the ‘Pre-Malate’ period. Other abbreviations are: G – glutamate, R - rotenone, G+R = glutamate+rotenone, dBSA – delipidated bovine serum albumin. For the safranin O uptakes, values are means±SEM for N = 2–3 for sham malate and 3–5 for malate. +P<0.001 for conditions with N≥3 vs. the corresponding NFA malate condition. For the respiratory rates, values are means±SEM for N = 4, *P<0.05, #P<0.01, +P<0.001 vs. corresponding NFA group.
Figure 6.
Effect of glutamate and rotenone on energization and respiration supported by succinate in rabbit and mouse tubules.
Safranin O uptakes and respiratory rates (RR) of permeabilized tubules were measured either under control conditions or in the presence of the indicated concentrations of oleate with either no further additions (NFA) or glutamate (G), rotenone (R) or glutamate+rotenone (G+R). “Peak” indicates the maximal uptake or RR during the measurement period. “End” indicates the uptake level or RR reached at the end of the second measurement period, which is less than the peak for conditions where there has been decay of ΔΨm or RR. Concentrations of oleate were chosen to give moderate deenergization (3 µM for rabbit, 4 µM for mouse) or severe deenergization (8 µM for rabbit, 10 µM for mouse). The high oleate concentration used for the rabbit RR studies was increased to 10 µM to provide more consistent decreases of the end RR to allow assessment of agents that ameliorate it. Values are means±SEM for N = 3 for both types of rabbit studies, 5–7 for the mouse safranin O uptakes and 4–5 for the mouse respiratory rates. *P<0.05, #P<0.01, +P<0.001 vs. corresponding NFA group.
Figure 7.
Effects of transaminase inhibition by aminooxyacetate on energization of mouse tubules with and without oleate addition.
Panels A–C. Studies of extended incubation without exogenous fatty acids (900 seconds). Panels A and B show representative tracings. Panel C summarizes group averages compared to the maximal uptake during the period for succinate alone. ‘Peak’ indicates maximal uptake for each condition. ‘End’ indicates uptake at 900 seconds. Agents tested and abbreviations for them are: S - succinate, M - malate, A - α-ketoglutarate, G - glutamate, AM - α-ketoglutarate+malate, GM - glutamate+malate, AMG - α-ketoglutarate+malate+glutamate, A or AOA – aminooxyacetate (4 mM), O – oleate (4 µM), R – rotenone, dB – delipidated bovine serum albumin. For the group data in panel C, values are means±SEM for N = 3–7, #P<0.01, +P<0.001 vs. corresponding group without aminooxyacetate. Panels D–J. Studies testing oleate. Panels D–I are representative tracings. Panel J summarizes group averages for behavior during the first 400 seconds in a format identical to that used for the corresponding studies without oleate in Panel C. All studies were 700 seconds in duration with a second experimental condition indicated in the panel introduced at 400 seconds. Panel D compares energization during the first 400 seconds with and without oleate for each test substrate. Panels E–G show effects of adding aminooxyacetate on the energization supported by complex I substrates. Panels H and I show effects of aminooxyacetate on succinate-supported energization with and without glutamate and rotenone. Agents tested and abbreviations used are as described for Panels A–C with additional use of O – oleate (4 µM). For the group data in panel J values are means±SEM for N = 3, *P<0.05, #P<0.01, +P<0.001 vs. corresponding group without AOA; @p<0.05 vs. corresponding ‘AM’ group.
Figure 8.
Support of energization by succinate compared to complex I substrates after H/R in rabbit and mouse tubules.
Tubules were subjected to either normoxic incubation or to 67.5(Rabbit, Panels A, C, and D) or 30 min. hypoxia (Mouse, panels, B, E, and F) followed by 60 min reoxygenation then measurement of energization using safranin O uptake with either succinate (S) glutamate+malate (GM), α-ketoglutarate+malate (AM) or α-ketoglutarate+malate+glutamate. Succinate was also studied with addition of either glutamate (SG), rotenone (1 µM, SR) or both glutamate and rotenone (SGR). Measurements were made initially without delipidated albumin (dBSA) followed by its addition (0.5 mg/ml) at the vertical marks in the tracings. Group averages±SEM for N = 5 for both rabbit and mouse are summarized in panels A and B. Typical tracings are shown in panels C–F. Statistics shown in panels A and B indicate values significantly different from the corresponding S group for the succinate studies or AM group for the complex I substrate studies at either *P<0.05, #P<0.01, or +P<0.001. Other statistical analysis indicated that all H/R conditions were significantly different (P<0.01) from the corresponding normoxic conditions except for the rabbit SG, SR and SGR groups with dBSA, which completely recovered. dBSA significantly increased energization (P<0.01) in all rabbit studies except for the normoxic succinate groups. In the mouse tubules, dBSA significantly improved energization of normoxic tubules with AM (P<0.05) and in all hypoxic groups (P<0.01) except for SR and SGR. In both normoxic and hypoxic rabbit tubules, energization with complex I substrates was poorer than with succinate (P<0.05) except for normoxic tubules in the presence of dBSA. In normoxic mouse tubules, complex I supported energization did not differ from succinate, but after H/R complex I rates energization was better than with succinate alone irrespective of the presence of dBSA (P<0.05). Relative to the other succinate conditions, complex I energization was either weaker (P<0.01 relative to SR and SGR without dBSA) or unchanged.
Figure 9.
Effects of glutamate and rotenone on succinate-supported respiration of normoxic and H/R rabbit tubules.
A. Measurements of energization using safranin O uptake done in parallel with the respiration studies on the same preparations. Abbreviations for the experimental groups testing different conditions during safranin O uptake are as for Fig. 8. Tubules were subjected to 67.5±SEM for N = 4. Figure symbols indicate #P<0.01 or +P<0.001 vs. corresponding normoxic values. Values for glutamate and rotenone-treated H/R groups without dBSA were significantly different from the corresponding group with succinate alone, P<0.05. Delipidated albumin (dBSA) significantly improved energization under all the H/R conditions (P<0.001). B. Measurements of respiration. Oxygen consumption was assessed sequentially under basal conditions, then after addition of ADP to stimulate oxidative phosphorylation, then during suppression of the ADP-induced oxidative phosphorylation by oligomycin (OLIGO), then during maximally-stimulated uncoupled respiration produced by carbonyl cyanide-m-chlorophenylhydrazone (CCCP). Studies were done with and without addition of dBSA on tubules previously subjected to either normoxic incubation or H/R. Values are means±SEM for N = 4. There were no significant effects of glutamate and/or rotenone under any condition except for a small decrease of the ADP rate in the normoxic glutamate+rotenone+dBSA group (P<0.05). Basal and oligomycin rates did not significantly differ between normoxic and H/R tubules. dBSA significantly lowered the basal and oligomycin rates in all groups (P<0.001 normoxic, P<0.01 hypoxic). ADP and CCCP rates of H/R tubules were significantly lower than the corresponding normoxic at *P<0.05 or #P<0.01 under all conditions. dBSA did not significantly affect the ADP rates, but it significantly increased the CCCP rates in the normoxic S and SR groups and the H/R S, SG, and SR groups, Ps<0.05 or better.
Figure 10.
Energization and respiration after H/R of unprotected and substrate-protected rabbit tubules supported by succinate or complex I-dependent substrates.
These experiments were done similarly to those in Fig. 9, except they compared the behavior of complex II-dependent respiration with complex I and also tested tubules that were protected by dBSA+αKG/MAL in the incubation flasks during the 60 min. reoxygenation period. A. Measurements of energization using safranin O uptake. Abbreviations for the experimental groups testing different conditions during safranin O uptake are as for Fig. 8. Values are means±SEM for N = 4. All H/R groups except protected tubules with succinate+BSA were significantly different from the corresponding normoxic groups at P<0.001 for unprotected flasks without dBSA and P<0.05 for all other groups. Statistical symbols shown in the figure indicate: *P<0.05, #P<0.01, +P<0.001 vs. corresponding protected flask. dBSA significantly increased energization in all AMG groups and in succinate groups from unprotected tubules, P<0.01. SG, SR, and SGR without dBSA had significantly better uptake than S alone in the unprotected tubules (P<0.05), and AMG without dBSA supported energization after H/R less strongly than S alone in both protected and unprotected tubules, P<0.01. B. Measurements of respiration. Oxygen consumption was measured with either succinate+glutamate+rotenone (SGR) or α-ketoglutarate+malate+glutamate (AMG) following the same experimental sequence as described for Fig. 9. Values are means±SEM for N = 4. SGR rates were significantly greater than the corresponding AMG rates under all conditions, P<0.01. Statistical symbols shown in the figure indicate: *significantly different from normoxic at P<0.01 (SGR studies) or P<0.05 (AMG), #significantly different from corresponding unprotected group at P<0.01 (SGR studies) or P<0.05 (AMG studies). All basal and oligomycin rates with dBSA were significantly lower than the corresponding rates without dBSA (P<0.02) except for the oligomycin rate of the AMG H/R group. dBSA significantly increased the succinate-supported CCCP rates for all groups and the AMG-supported CCCP rate of the normoxic tubules.
Figure 11.
Studies of energization and respiration after H/R of mouse tubules supported by succinate or complex I-dependent substrates.
These experiments were done similarly to those in Figs. 9 and 10, except tubules were subjected to 30-supported energization are as for Fig. 8. Values are means±SEM for N = 4. All H/R groups were significantly different from the corresponding normoxic groups at P<0.001. dBSA significantly increased energization only in the succinate alone (S) and AMG groups. Statistical symbols shown in the figure indicate: *P<0.05 or #P<0.01 vs. corresponding ‘S’ group. B. Measurements of respiration. Oxygen consumption was measured with either succinate alone or succinate+rotenone (SR) or α-ketoglutarate+malate+glutamate (AMG) following the same experimental sequence as described for Fig. 10. All H/R rates were significantly different from the corresponding normoxic rates, P<0.001. All succinate alone and SR rates were significantly greater than the corresponding AMG rates at P<0.001. Figure symbols indicate: #P<0.01, +P<0.001, SR group values that were significantly different from corresponding succinate alone values; @, significant effects of dBSA vs. the corresponding condition without it. The dBSA effects for basal rates were P<0.05 for normoxic succinate alone, P<0.001 for normoxic AMG and P<0.01 for all H/R groups. For oligomycin rates, they were P<0.01 for all normoxic groups and the H/R SR study. The CCCP rate with dBSA was different at P<0.05 for the succinate alone group.