Calorie restriction increases muscle mitochondrial biogenesis in healthy humans

Abstract

Background: Caloric restriction without malnutrition extends life span in a range of organisms including insects and mammals and lowers free radical production by the mitochondria. However, the mechanism responsible for this adaptation are poorly understood.

Methods and findings: The current study was undertaken to examine muscle mitochondrial bioenergetics in response to caloric restriction alone or in combination with exercise in 36 young (36.8 +/- 1.0 y), overweight (body mass index, 27.8 +/- 0.7 kg/m(2)) individuals randomized into one of three groups for a 6-mo intervention: Control, 100% of energy requirements; CR, 25% caloric restriction; and CREX, caloric restriction with exercise (CREX), 12.5% CR + 12.5% increased energy expenditure (EE). In the controls, 24-h EE was unchanged, but in CR and CREX it was significantly reduced from baseline even after adjustment for the loss of metabolic mass (CR, -135 +/- 42 kcal/d, p = 0.002 and CREX, -117 +/- 52 kcal/d, p = 0.008). Participants in the CR and CREX groups had increased expression of genes encoding proteins involved in mitochondrial function such as PPARGC1A, TFAM, eNOS, SIRT1, and PARL (all, p < 0.05). In parallel, mitochondrial DNA content increased by 35% +/- 5% in the CR group (p = 0.005) and 21% +/- 4% in the CREX group (p < 0.004), with no change in the control group (2% +/- 2%). However, the activity of key mitochondrial enzymes of the TCA (tricarboxylic acid) cycle (citrate synthase), beta-oxidation (beta-hydroxyacyl-CoA dehydrogenase), and electron transport chain (cytochrome C oxidase II) was unchanged. DNA damage was reduced from baseline in the CR (-0.56 +/- 0.11 arbitrary units, p = 0.003) and CREX (-0.45 +/- 0.12 arbitrary units, p = 0.011), but not in the controls. In primary cultures of human myotubes, a nitric oxide donor (mimicking eNOS signaling) induced mitochondrial biogenesis but failed to induce SIRT1 protein expression, suggesting that additional factors may regulate SIRT1 content during CR.

Conclusions: The observed increase in muscle mitochondrial DNA in association with a decrease in whole body oxygen consumption and DNA damage suggests that caloric restriction improves mitochondrial function in young non-obese adults.

Figure 1. Changes in Skeletal Muscle Gene Expression for Key Mitochondrial Proteins

(A) TFAM: ^*CR, p = 0.001; ^#CREX, p = 0.014. (B) PPARGC1A: ^*CR, p = 0.004; ^#CREX, p = 0.002. (C) SIRT1: ^*CR, p = 0.016; ^#CREX, p = 0.023. (D) eNOS: ^*CR, p = 0.002; ^#CREX, p = 0.039. Graphs show six-month changes in enzyme expression in response to each intervention. The y-axis represents the relative gene expression change from baseline for each study group. Each box plot shows the distribution of expression levels from 25th to 75th percentile, and the lines inside the boxes denote the medians. The whiskers denote the interval between the 10th and 90th percentiles. The filled circles mark the data points outside the 10th and 90th percentiles. Molecular analysis was performed in 11 of 12 volunteers per group from whom there was sufficient pre- and postintervention sample for this determination. Changes from baseline to month 6 were analyzed by analysis of variance with baseline values included as covariates.

Figure 2. The Effects of Caloric Restriction on Mitochondrial Bionenergetics

(A and B) Each box plot shows the distribution of expression levels from 25th to 75th percentile and the lines inside the boxes denote the medians. The whiskers denote the interval between the 10th and 90th percentiles. The filled circles mark the data points outside the 10th and 90th percentiles. (A) Caloric deficit–induced mitochondrial biogenesis in the CR group (35% ± 5%, ^* p = 0.005) and the CREX group (21% ± 4%, ^# p < 0.004), with no change in the control group (2% ± 2%). The y-axis represents the relative change from baseline in mtDNA for each study group. (B) Analysis of mitochondrial enzyme activity; β-HAD (β-oxidation); CS (TCA cycle), and COX (electron transport chain). The y-axis represents the relative change from baseline in mitochondrial enzyme activity for each study group. (C) Linear correlation between the change from baseline in SIRT1 and PPARGC1A mRNAs from baseline in control (○), r = 0.83, p < 0.05; CR (□), r = 0.95, p < 0.01; and CREX participants (▵), r = 0.76, p < 0.05). The linear correlation between the change in SIRT1 mRNA and PPARGC1A mRNA from baseline in the CR group (□) remained significant after exclusion of the outlier (r = 0.81, p < 0.01). Changes from baseline to month 6 were analyzed by analysis of variance with baseline values included as covariates.

Figure 3. Statistical Dot Plots Showing the Effects of DETA-NO and Adiponectin Treatment on Mitochondrial Content and SIRT1 Protein in Primary Human Myotubes

(A–C) Effects of 96 h of 50 μM DETA-NO treatment on mitochondrial content (using MitoTracker Green, p = 0.002) (A), electron transport chain activity (COX, p = 0.018) (B), and mitochondrial membrane potential (TMRE, n = 6, p = 0.042) (C). Treatment effect was determined using independent sample t-test. OD, optical density. (D) Effects of 50 μM DETA-NO on SIRT1 and β-actin protein (top blots); effects of 0.5 μg/ml of globular adiponectin (gAD) and adiponectin receptor R1- and R2-siRNA on SIRT1 and β-actin protein expression (bottom blots). Immunoblotting was undertaken in three participants and data are shown as a representative blot. Means are denoted by the solid black bars.