Mitochondrial uncouplers with an extraordinary dynamic range

Abstract

We have discovered that some weak uncouplers (typified by butylated hydroxytoluene) have a dynamic range of more than 10(6) in vitro: the concentration giving measurable uncoupling is less than one millionth of the concentration causing full uncoupling. They achieve this through a high-affinity interaction with the mitochondrial adenine nucleotide translocase that causes significant but limited uncoupling at extremely low uncoupler concentrations, together with more conventional uncoupling at much higher concentrations. Uncoupling at the translocase is not by a conventional weak acid/anion cycling mechanism since it is also caused by substituted triphenylphosphonium molecules, which are not anionic and cannot protonate. Covalent attachment of the uncoupler to a mitochondrially targeted hydrophobic cation sensitizes it to membrane potential, giving a small additional effect. The wide dynamic range of these uncouplers in isolated mitochondria and intact cells reveals a novel allosteric activation of proton transport through the adenine nucleotide translocase and provides a promising starting point for designing safer uncouplers for obesity therapy.

Figure 1. Structures of compounds used

‘n’ denotes the number of alkyl chain carbon atoms. tBu, tertiary butyl.

Figure 2. Effect of different uncouplers on the respiration of rat liver mitochondria

(A and B) Representative traces of oxygen consumption in the presence of (A) CCCP or (B) BHT. Succinate (4 mM) was added to initiate respiration (indicated by the arrowhead). DMSO, different concentrations of CCCP or BHT, or excess (0.3 μM) FCCP were present where indicated. (C–F) Double logarithmic plots of uncoupling. Open or grey circles, titration of respiration rate with CCCP, DNP, BHT or benzoic acid. Closed circles, the respiration rate at each uncoupler concentration after subsequent addition of 0.3 μM FCCP, giving the maximum respiratory capacity of the mitochondria. Values are means±S.E.M. for four independent experiments. Lines were fitted by regression [to the grey points in (F)].

Figure 3. Effect of different uncouplers on the respiration of rat skeletal muscle mitochondria

Double logarithmic plots of uncoupling in skeletal muscle mitochondria by (A) CCCP, (B) BHT, (C) benzoic acid and (D) mitoBHT. Open circles, titration of respiration rate with CCCP, BHT, benzoic acid or mitoBHT. Closed circles, respiration rate at each uncoupler concentration after subsequent addition of 0.3 μM FCCP, giving the maximum respiratory capacity of the mitochondria. Values are means±S.E.M. for four independent experiments. Lines were fitted by regression.

Figure 4. Effect of membrane potential on uncoupling of rat liver mitochondria by BHT

(A) Kinetic response of proton leak rate to membrane potential in the presence of DMSO or different concentrations of BHT as indicated. Proton leak rate was measured as the respiration rate used to drive it; membrane potential was varied by adding different concentrations of malonate. Values are means±S.E.M. for four independent experiments. (B) Uncoupling by BHT. Data taken from (A) at the highest common membrane potential of 164.3 mV (solid line; slope=0.20±0.02; error bars for interpolated points are the weighted means of the error bars on flanking experimental points), or at the highest potential for each uncoupler concentration [broken line; slope=0.13±0.04; points and error bars as in (A) but omitted here for clarity]. The slopes are significantly different; P<0.05.

Figure 5. Effect of different uncouplers on the respiration of rat liver mitochondria in the presence of CAT

(A–D) Double logarithmic plots of uncoupling in the presence of 2 nmol of CAT/mg of protein (added before succinate). Open circles, titration of respiration rate with CCCP, DNP, BHT or benzoic acid. Closed circles, respiration rate at each uncoupler concentration after subsequent addition of 0.3 μM FCCP. Values are means±S.E.M. for four independent experiments. Lines were fitted by regression. (E–H) CAT-sensitive stimulation of mitochondrial respiration rate in linear co-ordinates. CAT-sensitive stimulation of respiration rate (open circles; shaded profile; line fitted as a rectangular hyperbola) was estimated as total uncoupling (filled circles; data from Figures 2C–2F where CAT was absent; line fitted as the sum of the other two lines) minus CAT-insensitive respiration rates (open triangles; data from Figures 5A–5D where CAT was present; linear fit).

Figure 6. Effect of different uncouplers on mitochondrial ATP synthesis and the respiration of rat thymocytes

(A) State 3 respiration in rat liver mitochondria with DMSO (vehicle) and at different BHT concentrations in the presence of 50 μM p¹,p⁵-di(adenosine-5′)pentaphosphate (an adenylate kinase inhibitor). State 3 respiration rates were measured after the addition of succinate and 0.1 mM ADP. State 3 respiration rates without DMSO are included for comparison. Values are means±S.D. for two independent experiments. (B–E) Double logarithmic plots of uncoupling of thymocyte respiration by CCCP, DNP, BHT or benzoic acid. Values are means±S.E.M. for three or four independent experiments. Lines were fitted by regression. The slopes are (B) 1.07±0.15, (C) 0.78±0.20, (D) 0.11±0.07 and (E) 0.10±0.04. For BHT and benzoic acid, lines were fitted only between 10⁻¹² and 10⁻⁹ M (shaded diamonds). (F) Gramicidin titration of thymocyte respiration. Gramicidin was sequentially added to the cells after a steady baseline was achieved in the presence (filled squares) or absence (open squares) of 1 mM ouabain. Values are means±S.E.M. for four experiments. (G) Ouabain-sensitive respiration driving the Na⁺/K⁺-ATPase at different BHT concentrations. Gramicidin at 0.57 μg/ml and ouabain at 1 mM were added before BHT, and the cellular respiration rate was measured for 10 min. Basal cell respiration rates with and without ouabain are included for comparison. Values are means±S.E.M. for three to five independent experiments. Rates and differences were not significantly affected by BHT (as analysed by ANOVA).

Figure 7. Uncoupling effects of mitoBHT in rat liver mitochondria and rat thymocytes

(A) Uptake and release of mitoBHT. The electrode was calibrated by five additions of 0.5 μM mitoBHT in the presence of mitochondria. Addition of 4 mM succinate induced membrane potential and caused a decrease in the external mitoBHT concentration. The external mitoBHT concentration was largely restored when the membrane potential was dissipated with 0.3 μM FCCP. (B) Representative traces of mitochondrial oxygen consumption in the presence of mitoBHT. DMSO, different concentrations of mitoBHT or 0.3 μM FCCP were present where indicated. (C) Double logarithmic plots of uncoupling in rat liver mitochondria. The titration of respiration rates was with mitoBHT (open circles), mitoDNP (open triangles) or TPMP (squares). Closed circles, respiration rate at each mitoBHT concentration after subsequent addition of 0.3 μM FCCP. Values are means±S.E.M. for four independent experiments. Lines were fitted by regression. (D) Effect of mitoBHT on the respiration of rat liver mitochondria in the presence of CAT. Double logarithmic plots of uncoupling in the presence of 2 nmol of CAT/mg of protein (added before succinate). Open circles, the titration of respiration rate with mitoBHT. Closed circles, respiration rate at each mitoBHT concentration after subsequent addition of 0.3 μM FCCP. Values are means±S.E.M. for four independent experiments. The line was fitted by regression. (E) CAT-sensitive stimulation of mitochondrial respiration rate by mitoBHT in linear co-ordinates. CAT-sensitive stimulation of respiration rate (open circles, shaded profile) was estimated as total uncoupling [filled circles; data from (C) where CAT was absent] minus CAT-insensitive respiration [open triangles; data from (D) where CAT was present]. Lines were fitted as in Figures 5(E)–5(H). (F) Kinetic response of proton leak to membrane potential of rat liver mitochondria incubated with 2 nmol of CAT/mg of protein, in the presence of DMSO and different concentrations of mitoBHT. Values are means±S.E.M. for four or five independent experiments. (G) Potential-independent uncoupling of rat liver mitochondria by mitoBHT. Data taken from (F) at the highest common membrane potential of 152 mV (solid line; slope=0.69±0.10; error bars for interpolated points are the weighted means of the error bars on flanking experimental points), or at the highest potential for each uncoupler concentration [broken line; slope=0.39±0.04; points and error bars as in (F) but omitted here for clarity]. The slopes are significantly different. (H) Effect of mitoBHT on the respiration of rat thymocytes, presented as a double logarithmic plot. Values are means±S.E.M. for three independent experiments. The slope of the regression line is 0.13±0.05.

Figure 8. Effect of mitoQ and TPP compounds on the respiration of rat liver mitochondria

(A and B) Double logarithmic plots of uncoupling in the presence (squares) and absence (circles) of 2 nmol of CAT/mg of protein (added before succinate). Open symbols, titration of respiration rate with (A) mitoQ₁₀ or (B) decylTPP. In the presence of CAT, concentrations of mitoQ₁₀ or decylTPP below 10⁻⁷ M gave a less than 1% increase in respiration and are off-scale. Closed symbols, rate at each mitoQ₁₀ or decylTPP concentration after subsequent addition of 0.3 μM FCCP. Values are means±S.E.M. for three to four independent experiments. Lines (no CAT, dashed line; with CAT, dotted line) were fitted to the points shown by regression. (C and D) Comparison of uncoupling by a series of (C) mitoQ compounds and (D) TPP cations with different numbers of carbon atoms (from three to ten) in the alkyl chain (grey bars). The concentration used for this comparison was 10⁻⁹ M. At this concentration, CAT inhibited any uncoupling effect by these compounds (black bars). Data with mitoBHT (Figures 7C and 7D) and TPMP (MethylTPP; Figure 7C) are included for comparison.

Figure 9. Hypothesis for the observed wide dynamic range of uncoupling by BHT and other compounds, illustrated as double logarithmic and linear plots

Two main effects are responsible: (i) saturable, medium-capacity, high-affinity, CAT-sensitive uncoupling through the ANT at low BHT concentrations (modelled as a rectangular hyperbola with arbitrary K_m and V_max), and (ii) non-saturable, high-capacity, low-affinity, CAT-insensitive uncoupling through other pathways (such as conventional uncoupler cycling) at high BHT concentrations (modelled as a linear relationship). The wide dynamic range (i + ii) is the result of an overlap of these two effects. The right-hand panel shows these effects in linear co-ordinates; the left-hand panel shows the same values plotted in log–log co-ordinates, illustrating how they can combine to give the shallow pseudo-linear overall relationship observed in the other Figures.