Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity

<div><p>Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.</p></div

UCP2 promotes fatty acid oxidation in mtDNA mutator mitochondria.

<p>(A) Steady-state levels of mitochondrial glucose and fatty acid transporters: insulin-regulated glucose transporter (GLUT4), basal glucose transporter 1 (GLUT1) and mitochondrial carnitine palmitoyltransferase I (CPT1B) in wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice. Cytoplasmic calnexin was used as loading control. (B) Quantification of Western blots from (A). (C) Oxygen consumption rates in intact mitochondria in presence of pyruvate-glutamate-malate as substrates. State III (substrates+ADP); State IV (+oligomycin); MAX (+CCCP). (D) Oxygen consumption rates in intact mitochondria in the presence of medium (OC - octanoyl-carnitine) or long chain (PALM - palmitoyl-carnitine) fatty acids as substrates. (E) Maximal oxygen consumption rates upon addition of octanoyl-carnitine+glutamate (OC+Glut) or palmitoyl-carnitine+glutamate (PALM+Glut) to intact mitochondria. (n = 4–6). Bars indicate mean levels ± standard error of the mean (S.E.M.). Statistically significant differences between mut and dm are presented with thick lines. Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).</p

UCP2 deficiency does not affect proton leak kinetics or ROS production.

<p>(A) Flow-force relationship in heart mitochondria of 25-week-old wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice examined in the presence of succinate and increasing amounts of malonate (n = 6). Data points indicate mean levels ± standard error of the mean (S.E.M.). (B) Hydrogen peroxide production rate in heart mitochondria in the presence of succinate (S); Pyruvate-Glutamate-Malate (PGM) or Pyruvate-Glutamate-Malate-Succinate (PGMS) as substrates. Measurement in the presence of succinate alone detects reverse flow ROS production from both Complex I and Complex III. When mitochondria are incubated in the presence of pyruvate-glutamate-malate (PGM), the ROS production mainly originates from complex III and therefore it is usually lower. Treatment with Antimycin A (+AA), an inhibitor of CO III, was used as a positive control, to further induce ROS production (n = 4–5). Bars indicate mean level ± standard error of the mean (S.E.M.). (C) Western blot analyses of Manganese Superoxide Dismutase (MnSOD) in heart lysates of 25- and 40-week-old mice (n = 4). (D) Analysis of 4-Hydroxynonenal as measure for oxidative stress induced lipid peroxidation in isolated mitochondria (upper panel) and tissue lysates (lower panel) of 25-week-old mouse hearts. The mitochondrial Complex II 70 kDa protein (C II) was used as loading control in isolated mitochondria and the Heat shock 70 kDa protein 8 (HSC 70) in heart tissue lysates (n = 4). (E) Fold change of <i>Ucp2</i> transcript levels in primary MEFs (P1–P3). (F) ROS production in intact cells was assessed by flow cytometric analyses of primary (passage 1–3) mouse embryonic fibroblasts (MEFs) stained with CM-H₂DCFDA that upon oxidation by ROS, particularly hydrogen peroxide (H₂O₂) and the hydroxyl radical (·OH), yields the fluorescent DCF product. Data are expressed as median values of fluorescence intensity ± standard error of the mean (S.E.M.). Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).</p

Characterization of mitochondrial cardiomyopathy in wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice.

<p>(A) Histological examination of cardiomyocytes from mut and dm mice. TEM – transmission electron micrographs. Arrows indicate lipid droplets, arrowheads abnormal mitochondria. COX/SDH - Enzyme histochemical staining for cytochrome <i>c</i> oxidase (COX) and succinate dehydrogenase (SDH) activities in heart. (B) Quantification of mitochondrial mass in transmission electron micrographs. (C) Relative expression levels of <i>Nppa</i> and <i>Nppb</i>, markers of heart failure. (D) Relative <i>Fgf21</i> mRNA expression levels in heart. (E) Relative <i>Fgf21</i> mRNA expression levels in liver and skeletal muscle. All analyses were performed on 25-week-old mice. Bars indicate mean level ± standard error of the mean (S.E.M.). Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).</p

MtDNA mutator mice have lower body mass and upregulated UCP2 levels.

<p>Body weight (A) and daily food consumption (B) in mtDNA mutator (mut) mice in comparison to littermate controls (wt) (n = 11). (C) Northern blot analyses of <i>Ucp2</i> levels in spleen, heart, brain and liver. (D) Fold change of <i>Ucp2</i> transcript levels in spleen, heart, brain, liver and hypothalamus for wt and mut (n = 4). (E) Western blot analyses of UCP2 in spleen and heart mitochondria of wt, mut and UCP2-deficient (ko) mice. (F) Proton motive force (bars) and mitochondrial matrix volume (lines) in liver and spleen mitochondria (n = 3). (G) Mean lifespan of wt, mut, ko and dm mice. All analyses were performed on 25-week-old mice. Bars indicate mean level ± standard error of the mean (S.E.M.). Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).</p

Characterization of blood metabolites in wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice.

<p>(A) Blood glucose and lactate concentrations at 25 weeks of age. (n = 6). (B) Circulating free fatty acid levels in 25- and 40-week-old animals. (n = 5–6). (C) Serum insulin levels of 30-week-old mice. (D) Glucose tolerance test in 30-week-old mice. After 16 hours of starvation period, mice were injected with 2g/kg body weight glucose and clearance was measured after 0, 15, 30, 60 and 120 minutes (n = 7). (E) Insulin tolerance test of 30-week-old random fed mice. Mice were injected with 0.75 U/kg body weight of insulin and glucose levels were measured after 0, 15, 30 60 and 120 minutes (n = 7). (F–L) Analyses of metabolic markers in the serum: (F) Leptin; (G) Ghrelin; (H) Glucagon; (I) Glucagon-like peptide 1 (GLP-1); (J) Glucose-dependent insulinotropic polypeptide (GIP); (K) Plasminogen activator inhibitor-1 (PAI-1); (L) Resistin. All measurements were performed in 30-week-old mice. (n = 4). Bars indicate mean levels ± standard error of the mean (S.E.M.). Statistically significant differences between mut and dm are presented with thick lines. Asterisks indicate level of statistical significance (*p<0.05; **p<0.005; ***p<0.001, Student's <i>t</i>-test).</p