High lactate levels in brain and peripheral tissues. (A) Position of the volume of interest (VOI) in cerebral cortex in (i) axial, (ii) coronal, and (iii) sagittal planes. (B) Typical ¹H-MR spectra obtained from a VOI in cerebral cortex indicating the marked increase in the lactate doublet (centered on 1.33 ppm) in mtDNA mutator mice. (C) Two-way between-subjects ANOVA on age-matched groups was performed on brain lactate concentrations (mean values ± SEM) in cerebral cortex (Upper) and striatum (Lower) from female mtDNA mutator (n = 19; red) compared with littermate wild-type (n = 18; black) mice. All mutator mice showed a significant increase in lactate levels, and ANOVA showed a significant effect for the mtDNA mutator allele both in cerebral cortex [F(1,25) = 84.1; P < 0.0001] and striatum [F(1,24) = 134; P < 0.0001] compared with controls. Posthoc analyses are denoted with ***P < 0.001. (D–G) Analyses of tissue lactate (mean values ± SEM) measured by HPLC from female 25- to 29-wk-old mtDNA mutator (n = 6; red), 25- to 29-wk-old (n = 6; black), and 86-wk-old littermate wild-type (n = 3; black) mice. (D) The 25- to 29-wk-old mtDNA mutator mice showed a threefold increase in brain lactate with [t(4) = 9.60; P = 0.0007] and without [t(4) = 4.20; P = 0.007] the presence of blood compared with controls. A twofold increase in brain lactate in 86-wk-old wild-type controls compared with 25- to 29-wk-old controls [t(4) = 16.9; P = 0.007] was revealed. (E) Plasma lactate increased sixfold in mtDNA mutator mice compared with 25- to 29-wk-old controls [t(9) = 2.71; P = 0.024]. In wild-type mice, plasma lactate levels tripled at 86 wk of age compared with 25- to 29-wk-old animals [t(7) = 3.41; P = 0.011]. (F and G) Lactate levels in cadiac muscle were also tripled in mtDNA mutator mice [t(6) = 3.15; P = 0.009], whereas levels in hepatic tissue were halved [t(6) = 3.43; P = 0.007], both compared with 25- to 29-wk-old controls. Cardiac muscle lactate levels were also increased twofold in 86-wk-old wild-type mice [t(5) = 4.90; P = 0.002] compared with 25- to 29-wk-old controls. HPLC significances were determined by two-tailed unpaired t test analysis and denoted with *P < 0.05, **P < 0.01, and ***P < 0.001.

Lactate dehydrogenase gene expression in brain. (A) In situ hybridization autoradiographic films of LDH-A and LDH-B mRNA in sections from male and female mtDNA mutator (n = 22; red) and littermate wild-type (n = 26; black) mice shown at ages 12, 46, and 130 (wild-type only) wk. (B and C) Quantification from autoradiographic films of LDH-A and LDH-B mRNA (mean values ± SEM), expressed as nCi g⁻¹, in cerebral cortex and CA1 of hippocampus from 9- to 12-wk-old and 42- to 46-wk-old mtDNA mutator compared with age-matched wild-type mice and from 130-wk-old (2.5-y-old) wild-type mice. All mtDNA mutator mice showed an up-regulation of LDH-A mRNA expression, and ANOVA showed a significant effect for the mtDNA mutator allele both in cerebral cortex [F(1,20) = 80.0; P < 0.0001] and CA1 of hippocampus [F(1,20) = 74.2; P < 0.0001] compared with controls. ANOVA also determined an overall decrease in LDH-B gene expression, caused by the mtDNA mutator allele, both in cerebral cortex [F(1,20) = 4.8; P = 0.040] and CA1 of hippocampus [F(1,20) = 31.4; P < 0.0001] compared with controls. LDH-B mRNA levels in 130-wk-old wild-type mice were significantly decreased both in cerebral cortex [t(10) = 3.06; P = 0.012] and CA1 of hippocampus [t(10) = 7.06; P < 0.0001] compared with 42- to 46-wk-old wild-type mice, whereas LDH-A mRNA expression remained constant in 130-wk-old mice. Analyses were performed by two-way between subjects ANOVA with posthoc analysis on age-matched groups (9–12 wk and 42–46 wk) and by two-tailed unpaired t test comparing 130-wk-old wild-type with 42- to 46-wk-old wild-type mice. *P < 0.05, **P < 0.01, and ***P < 0.001.

A functional metabolic shift to more lactate production. Quantification of LDH isoenzymes and their collective enzymatic activity in cerebral cortex from 23- and 45-wk-old male mtDNA mutator (n = 6; red) and age-matched littermate wild-type (n = 6; black) mice. (A) LDH isoenzyme tetrameric composition (Left) indicates the number of M and H subunits that constitute each isoenzyme. Native gel electrophoresis of LDH isoenzymes (Right) showed the expression of all five isoenzymes in mtDNA mutator and wild-type cerebral cortex. The mobility of the isoenzymes was regulated by size and charge. (B) Quantification of LDH isoenzymes. In 23-wk-old mutator mice, there was increased expression (mean ± SD) of isoenzymes comprised chiefly of M subunits [LDH-5: t(28) = 7.69; P < 0.0001 and LDH-4: t(28) = 6.85; P < 0.0001], which continued to increase with age [t(28) = 6.31; P < 0.0001 and t(28) = 16.5; P < 0.0001, respectively], and decreased expression in isoenzymes heavily composed of H subunits [LDH-1: t(28) = 7.38; P < 0.0001 and LDH-2: t(28) = 6.33; P < 0.0001], which continued to decrease with age [t(28) = 8.14; P < 0.0001 and t(28) = 16.1; P < 0.0001, respectively]. (C) Quantification of LDH activity determined from both the lactate and pyruvate sides of the reaction. Data, shown as percent change (mean ± SEM) calculated from IU mg⁻¹ protein, indicated a metabolic shift to more lactate production in mtDNA mutator mice, which increased with age from 11% to 33%. (D) The P:L ratio (mean ± SEM) is a simplified reflection of the LDH activity (IU mg⁻¹ protein) of all isoenzymes during interconversion of P ↔ L. The P:L ratio increased in 23-wk-old [t(8) = 3.69; P = 0.006] and 45-wk-old [t(8) = 6.84; P = 0.0001] mutator mice, showing higher LDH activity during P → L conversion. Analyses were performed by two-tailed unpaired t test to compare age-matched groups (23 and 45 wk). *P < 0.05, **P < 0.01, and ***P < 0.001.

Mitochondrial dysfunction in aging. (A) COX/succinate dehydrogenase (SDH) double labeling to visualize respiratory chain deficiencies, indicated by blue staining, in brains of 9- to 46-wk-old male and female mtDNA mutator (n = 24; red) and 9- to 130-wk-old littermate wild-type (n = 30; black) mice. (Scale bar: 1.00 mm.) (B) Semiquantification (mean values ± SEM) of COX deficiency on a scale of 0–4 (0, no blue staining; 4, only blue staining; recorded blindly) shows that mitochondrial respiratory chain dysfunction begins at 9–12 wk in mtDNA mutator mice and becomes widespread in cerebral cortex [H(4) = 11.7; P = 0.008], hippocampus [H(4) = 11.8; P = 0.008], nucleus accumbens [H(4) = 9.59; P = 0.022], striatum [H(4) = 10.4; P = 0.016], and thalamus [H(4) = 9.87; P = 0.019] as these mice age. In wild-type mice, there was increased COX deficiency in similar brain regions, indicating respiratory chain dysfunction in normal aging: cerebral cortex [H(5) = 21.7; P = 0.0002], hippocampus [H(5) = 18.4; P = 0.001], nucleus accumbens [H(5) = 21.4; P = 0.0003], striatum [H(5) = 18.3; P = 0.001], and thalamus [H(5) = 17.1; P = 0.019]. In wild-type mice, there was also increased COX deficiency in hippocampus [H(4) = 8.36; P = 0.039] and striatum [H(4) = 9.10; P = 0.028] over time from the 9- to 12-wk group to the 42- to 46-wk group. Observations were made at all time points, and short lines indicate 0 ratings (no blue staining). Significances from nonparametric data were determined by one-way Kruskal–Wallis ANOVA denoted with *P < 0.05, **P < 0.01, and ***P < 0.001.

Lactate dehydrogenase gene expression in peripheral tissues. (A) In situ hybridization of LDH-A and LDH-B mRNA in cardiac muscle and hepatic tissue sections from male 23-wk-old and 45-wk-old mtDNA mutator (n = 6; red) and age-matched littermate wild-type (n = 6; black) mice. (B) Quantification from autoradiographic films of LDH-A and LDH-B mRNA (mean values ± SEM) expressed as nCi g⁻¹. In cardiac muscle, mtDNA mutator mice showed an up-regulation of LDH-A [F(1,8) = 143; P < 0.0001] and a down-regulation of LDH-B genes [F(1,8) = 53.7; P < 0.0001]. In both mtDNA mutator and wild-type mice, there was an increase in LDH-A mRNA levels as they aged [t(4) = 15.1; P = 0.0001 and t(4) = 6.83; P = 0.002, respectively], while LDH-B levels decreased with age only in mutator mice [t(4) = 3.34; P = 0.029]. In hepatic tissue, both LDH-A and LDH-B gene expression levels were up-regulated in mtDNA mutator mice [F(1,8) = 55.1; P < 0.0001 and F(1,8)=186; P < 0.0001, respectively]. Two-way between subjects ANOVA with posthoc analysis on age-matched groups (23 and 45 wk), and two-tailed unpaired t test to compare these groups. *P < 0.05, **P < 0.01, and ***P < 0.001.