Calorie Restriction Increases Muscle Mitochondrial Biogenesis in Healthy Humans

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

Regulation of skeletal muscle oxidative capacity and insulin signaling by the Mitochondrial Rhomboid Protease PARL

Type 2 diabetes mellitus (T2DM) and aging are characterized by insulin resistance and impaired mitochondrial energetics. In lower organisms, remodeling by the protease pcp1 (PARL ortholog) maintains the function and lifecycle of mitochondria. We examined whether variation in PARL protein content is associated with mitochondrial abnormalities and insulin resistance. PARL mRNA and mitochondrial mass were both reduced in elderly subjects and in subjects with T2DM. Muscle knockdown of PARL in mice resulted in malformed mitochondrial cristae, lower mitochondrial content, decreased PGC1&alpha; protein levels, and impaired insulin signaling. Suppression of PARL protein in healthy myotubes lowered mitochondrial mass and insulin-stimulated glycogen synthesis and increased reactive oxygen species production. We propose that lower PARL expression may contribute to the mitochondrial abnormalities seen in aging and T2DM.<br /

Remodeling Lipid Metabolism and Improving Insulin Responsiveness in Human Primary Myotubes

OBJECTIVE: Disturbances in lipid metabolism are strongly associated with insulin resistance and type 2 diabetes (T2D). We hypothesized that activation of cAMP/PKA and calcium signaling pathways in cultured human myotubes would provide further insight into regulation of lipid storage, lipolysis, lipid oxidation and insulin responsiveness. METHODS: Human myoblasts were isolated from vastus lateralis, purified, cultured and differentiated into myotubes. All cells were incubated with palmitate during differentiation. Treatment cells were pulsed 1 hour each day with forskolin and ionomycin (PFI) during the final 3 days of differentiation to activate the cAMP/PKA and calcium signaling pathways. Control cells were not pulsed (control). Mitochondrial content, (14)C lipid oxidation and storage were measured, as well as lipolysis and insulin-stimulated glycogen storage. Myotubes were stained for lipids and gene expression measured. RESULTS: PFI increased oxidation of oleate and palmitate to CO(2) (p<0.001), isoproterenol-stimulated lipolysis (p = 0.01), triacylglycerol (TAG) storage (p<0.05) and mitochondrial DNA copy number (p = 0.01) and related enzyme activities. Candidate gene and microarray analysis revealed increased expression of genes involved in lipolysis, TAG synthesis and mitochondrial biogenesis. PFI increased the organization of lipid droplets along the myofibrillar apparatus. These changes in lipid metabolism were associated with an increase in insulin-mediated glycogen storage (p<0.001). CONCLUSIONS: Activation of cAMP/PKA and calcium signaling pathways in myotubes induces a remodeling of lipid droplets and functional changes in lipid metabolism. These results provide a novel pharmacological approach to promote lipid metabolism and improve insulin responsiveness in myotubes, which may be of therapeutic importance for obesity and type 2 diabetes

Glucose ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat oxidative genes.

Ingestion of carbohydrate during exercise may blunt the stimulation of fat oxidative pathways by raising plasma insulin and glucose concentrations and lowering plasma free fatty acid (FFA) levels, thereby causing a marked shift in substrate oxidation. We investigated the effects of a single 2-h bout of moderate-intensity exercise on the expression of key genes involved in fat and carbohydrate metabolism with or without glucose ingestion in seven healthy untrained men (22.7 &plusmn; 0.6 yr; body mass index: 23.8 &plusmn; 1.0 kg/m2; maximal O2 consumption: 3.85 &plusmn; 0.21 l/min). Plasma FFA concentration increased during exercise (P &lt; 0.01) in the fasted state but remained unchanged after glucose ingestion, whereas fat oxidation (indirect calorimetry) was higher in the fasted state vs. glucose feeding (P &lt; 0.05). Except for a significant decrease in the expression of pyruvate dehydrogenase kinase-4 (P &lt; 0.05), glucose ingestion during exercise produced minimal effects on the expression of genes involved in carbohydrate utilization. However, glucose ingestion resulted in a decrease in the expression of genes involved in fatty acid transport and oxidation (CD36, carnitine palmitoyltransferase-1, uncoupling protein 3, and 5\u27-AMP-activated protein kinase-&alpha;2; P &lt; 0.05). In conclusion, glucose ingestion during exercise decreases the expression of genes involved in lipid metabolism rather than increasing genes involved in carbohydrate metabolism.<br /

The Effects of Caloric Restriction on Mitochondrial Bionenergetics

<p>(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%, ^*<i>p</i> = 0.005) and the CREX group (21% ± 4%, ^#<i>p</i> < 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 <i>SIRT1</i> and <i>PPARGC1A</i> mRNAs from baseline in control (○), <i>r</i> = 0.83, <i>p</i> < 0.05; CR (□), <i>r</i> = 0.95, <i>p</i> < 0.01; and CREX participants (▵), <i>r</i> = 0.76, <i>p</i> < 0.05). The linear correlation between the change in <i>SIRT1</i> mRNA and <i>PPARGC1A</i> mRNA from baseline in the CR group (□) remained significant after exclusion of the outlier (<i>r</i> = 0.81, <i>p</i> < 0.01). Changes from baseline to month 6 were analyzed by analysis of variance with baseline values included as covariates.</p

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

<div><p>(A–C) Effects of 96 h of 50 μM DETA-NO treatment on mitochondrial content (using MitoTracker Green, <i>p</i> = 0.002) (A), electron transport chain activity (COX, <i>p</i> = 0.018) (B), and mitochondrial membrane potential (TMRE, <i>n</i> = 6, <i>p</i> = 0.042) (C). Treatment effect was determined using independent sample t-test. OD, optical density.</p> <p>(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.</p></div