Respiration in adipocytes is inhibited by reactive oxygen species

Abstract

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat-fed mice. Maximum O(2) consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Delta psi(M)) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N-acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Delta psi(M) and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O(2) consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O(2) consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O(2) consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O(2) consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.

Figure 1

O₂ consumption of isolated rat white adipocytes in the presence of different compounds. (a) Concentration dependence of pyruvate stimulated uncoupled O₂ consumption. O₂ consumption in intact adipocytes was measured at 37 °C in 1 ml of adipocyte suspension (adipocytes: KRP buffer 1:3, vol/vol), with 5 mmol/l glucose and 30 µmol/l carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) plus the indicated concentrations of pyruvate. Data are means ± s.e. of experiments from three separate preparations. (b) Concentration dependence of FCCP stimulated O₂ consumption. O₂ consumption in intact adipocytes was measured as described above with 5 mmol/l pyruvate plus the indicated concentrations of FCCP. Data are means ± s.e. of experiments from three separate preparations. (c) The influence of different substrates and inhibitors on adipocyte O₂ consumption. O₂ consumption was measured under basal conditions or following addition of FCCP (30 µmol/l), Cyncin (5 mmol/l), pyruvate (5 mmol/l), MePyr (5 mmol/l) separately, or in combinations indicated. Data are means ± s.e., n = 4. (d) N-acetylcysteine (NAC) stimulated O₂ consumption. O₂ consumption was measured under basal conditions or following addition of 10 mmol/l NAC. Data are means ± s.e., n = 4. *P < 0.05.

Figure 2

Adipocyte mitochondrial membrane potential, adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, and reactive oxygen species (ROS). (a) Left, three dimensional reconstruction of adipocyte mitochondria identified with MitoTracker Green (MTG). Single adipocytes exhibited a high concentration of mitochondria in the vicinity of the nucleus and sparse distribution of mitochondria around the equator. Middle and right, illustration of decreased mitochondrial membrane potential in adipocytes following carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) addition. A single plane through an immobilized adipocyte before (middle) and after (right) FCCP addition. Adipocytes were loaded with MTG to identify mitochondria (green image in upper left panels) and tetramethylrhodamine ethyl ester perchlorate (TMRE) to measure mitochondrial ΔΨ (red image upper right panels). The merged images are shown in the lower right panels. Illustration is from one of three separate preparations. (b) Mitochondrial membrane potential using a microplate assay. Please refer to the Methods section for data normalization. Data are means ± s.e., n = 3. *P < 0.05. (c) The influence of FCCP, pyruvate, N-acetylcysteine (NAC), and combinations on the ATP/ADP ratio. The ATP/ADP ratio was determined in adipocytes following incubation with KRP buffer (adipocytes:KRP buffer 1:3, vol/vol) for 10 min in the presence of FCCP (30 µmol/l), pyruvate (5 mmol/l), pyruvate plus FCCP, NAC (10 mmol/l), or NAC plus FCCP. Data are means ± s.e., n = 3. *P < 0.05. (d)The effect of pyruvate, NAC, FCCP, or H₂O₂ on ROS levels measured using CM-H₂DCF. ROS levels were measured using CM-DCF at excitation, 490 nm, emission, 529 nm. Cells were first loaded with CM-H₂DCF and then incubated with pyruvate (5 mmol/l), NAC (10 or 20 mmol/l) or H₂O₂ (100 mmol/l) in KRP buffer (5 mmol/l glucose, 1% BSA) as described in methods. Graphs represent experiments that were repeated 5–11 times. Data are means ± s.e. *P < 0.05.

Figure 3

Effect of inhibiting pyruvate oxidation on respiration with methyl-succinate (MeS) as an alternative substrate. Reagents shown on the x axis were added sequentially and their concentrations are indicated. (a) Rotenone dose-dependently inhibited uncoupled O₂ consumption driven by pyruvate. Data shown are fold-change of O₂ consumption rate over basal condition. (b) MeS stimulated O₂ consumption in the presence of rotenone and carbonylcyanide p-trifluoromethoxyphenylhydrazone, with (black bar) or without (gray bar) pyruvate. Data shown are relative change of O₂ consumption rate from basal respiration. Data are means ± s.e., n = 3. *P < 0.05.

Figure 4

Effect of N-acetylcysteine (NAC) in vivo. (a) Body fat and body weight. Postweaning male mice were fed a high-fat diet (35% fat-derived energy). NAC (3 mg/ml) was added to their drinking water and replaced daily (black bars). On days 1 and 10 mice were weighed and analyzed by NMR for fat mass. (b) O₂ consumption and CO₂ production. Between day 12 and day 15, mice were analyzed by indirect calorimetry for respiratory rate and CO₂ production. Data are means ± s.e. for four mice. *P < 0.05.