Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates

Abstract

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.

Figure 1. Mitochondrial oxygen consumption (A, D), heat release (B, E) and ATP synthesis (C, F) in response to pyruvate/malate (A–C) or succinate (D–F) as the respiratory substrate

The assay medium consisted of 20 mM Hepes buffer (pH 7.4), 100 mM KCl, 0.2 mM ADP, 3 mM MgCl₂, 2 mM Pi, 1 mM EGTA and 20–50 μg of mitochondrial protein/ml. The assay conditions are described in the Materials and methods section. (open bar) 0.2 mM ADP, (hatched bar) 0.2 mM ADP plus 1 mg/ml of faf-BSA and (grey bar) 0.2 mM ADP, 3 mM GDP and 1 mg/ml of faf-BSA, (black bar) 0.2 mM ADP, 3 mM GDP, 1 mg/ml of faf-BSA and 60 μM oleate. Statistical analysis: *P<0.01 and **P<0.001 compared with ADP; ^&P<0.001 compared with ADP+faf-BSA+GDP.

Figure 2. Mitochondrial membrane potential in the presence of pyruvate/malate (A) or succinate (B)

The assay medium was composed of 20 mM Hepes buffer (pH 7.4), 100 mM KCl, 0.2 mM ADP, 3 mM MgCl₂, 2 mM P_i, 1 mM EGTA, 10 μM Safranine O and 300 μg of mitochondrial protein/ml. λ_excitation was 495 nm and λ_emmission was 586 nm. The assay was performed at 35°C. The arrows indicate additions of P/M (1 mM pyruvate and 1 mM malate), 2 mM succinate, 3 mM GDP, 1 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 1 mg/ml of faf-BSA.

Figure 3. Representative experiment showing the BAT oxygen consumption in the presence of pyruvate/malate (A) or succinate (B)

The assay medium and conditions are described in the Materials and methods section.

Figure 4. Effect of oleate on mitochondrial oxygen consumption (Δ), heat release (○) and ATP synthesis (□) in the presence of pyruvate/malate (A) or succinate (B) as respiratory substrates

The assay medium was composed of 20 mM Hepes buffer (pH 7.4), 100 mM KCl, 0.2 mM ADP, 3 mM MgCl₂, 2 mM P_i, 1 mM EGTA, 0.2 mM ADP, 3 mM GDP, 1 mg/ml of faf-BSA and 20–50 μg of mitochondrial protein/ml. The conditions are described in the Experimental section.

Figure 5. Representative experiment demonstrating H₂O₂ generation by BAT mitochondria using succinate as a substrate

The white and black symbols indicate the absence or presence of rotenone respectively. Mitochondria were uncoupled (A) or coupled with faf-BSA and GDP (B).

Figure 6. Representative experiment illustrating the heat released by BAT mitochondria using pyruvate/malate (○, ●;) or succinate (Δ, ▲;) as a substrate

The white and black symbols indicate the absence or presence of rotenone, respectively. Mitochondria were uncoupled (A) or coupled with BSA-faf and GDP (B).