BackgroundCaloric 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 FindingsThe 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/m2) 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.ConclusionsThe 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.

Insulin resistance is associated with impaired skeletal muscle oxidation capacity and reduced mitochondrial number and function. Here, we report that adiponectin signaling regulates mitochondrial bioenergetics in skeletal muscle. Individuals with a family history of type 2 diabetes display skeletal muscle insulin resistance and mitochondrial dysfunction; adiponectin levels strongly correlate with mtDNA content. Knockout of the adiponectin gene in mice is associated with insulin resistance and low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle. Adiponectin treatment of human myotubes in primary culture induces mitochondrial biogenesis, palmitate oxidation, and citrate synthase activity, and reduces the production of reactive oxygen species. The inhibition of adiponectin receptor expression by siRNA, or of AMPK by a pharmacological agent, blunts adiponectin induction of mitochondrial function. Our findings define a skeletal muscle pathway by which adiponectin increases mitochondrial number and function and exerts antidiabetic effects.

Aims/hypothesis. The recent discovery of two adiponectin receptors (AdipoR1 and AdipoR2) will improve our understanding of the molecular mechanisms underlying the insulin-sensitising effect of adiponectin. The aim of this study was to determine for the first time whether skeletal muscle AdipoR1 and/or AdipoR2 gene expression levels are associated with insulin resistance. Methods. Using RT-PCR and northern analysis we measured AdipoR1 and AdipoR2 gene expression in skeletal muscle from healthy Mexican Americans with normal glucose tolerance who had (n=8) or did not have (n=10) a family history of Type 2 diabetes. Results. Gene expression profiling indicated that the AdipoR1 and AdipoR2 isoforms are highly expressed in human skeletal muscle, unlike in mice where AdipoR2 expression was highest in the liver, and AdipoR1 was highest in skeletal muscle. In the study subjects, the expression levels of AdipoR1 (p=0.004) and AdipoR2 (p=0.04), as well as plasma adiponectin concentration (p=0.03) were lower in people with a family history of Type 2 diabetes than in those with no family history of the disease. Importantly, the expression levels of both receptors correlated positively with insulin sensitivity (r=0. 64, p=0.004 and r=0.47, p=0.048 respectively). Conclusions/interpretation. Collectively, these data indicate that both isoforms of the adiponectin receptor play a role in the insulin-sensitising effect of adiponectin.

Objective: Alternate day fasting may extend lifespan in rodents and is feasible for short periods in nonobese humans. The aim of this study was to examine the effects of 3 weeks of alternate day fasting on glucose tolerance and skeletal muscle expression of genes involved in fatty acid transport/oxidation, mitochondrial biogenesis, and stress response. Research Methods and Procedures:Glucose and insulin responses to a standard meal were tested in nonobese subjects (eight men and eight women; BMI, 20 to 30 kg/m 2 ) at baseline and after 22 days of alternate day fasting (36 hour fast). Muscle biopsies were obtained from a subset of subjects (n ϭ 11) at baseline and on day 21 (12-hour fast). Results: Glucose response to a meal was slightly impaired in women after 3 weeks of treatment (p Ͻ 0.01), but insulin response was unchanged. However, men had no change in glucose response and a significant reduction in insulin response (p Ͻ 0.03). There were no significant changes in the expression of genes involved in mitochondrial biogenesis or fatty acid transport/oxidation, although a trend toward increased CPT1 expression was observed (p Ͻ 0.08). SIRT1 mRNA expression was increased after alternate day fasting (p ϭ 0.01).Discussion: Alternate day fasting may adversely affect glucose tolerance in nonobese women but not in nonobese men. The gene expression results indicate that fatty acid oxidation and mitochondrial biogenesis are unaffected by alternate day fasting. However, the increased expression in SIRT1 suggests that alternate day fasting may improve stress resistance, a commonly observed feature of calorierestricted rodents.

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 Ϯ 0.6 yr; body mass index: 23.8 Ϯ 1.0 kg/m 2 ; maximal O2 consumption: 3.85 Ϯ 0.21 l/min). Plasma FFA concentration increased during exercise (P Ͻ 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 Ͻ 0.05). Except for a significant decrease in the expression of pyruvate dehydrogenase kinase-4 (P Ͻ 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 5Ј-AMP-activated protein kinase-␣ 2; P Ͻ 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. skeletal muscle gene expression; exercise-diet interaction SKELETAL MUSCLE HAS THE REMARKABLE CAPACITY to respond and adapt to the physical and metabolic loads imposed by exercise. At the molecular level, these adaptations include the increased capacity for oxidative metabolism of both carbohydrate (CHO) and fatty acids (FA). An enhanced capacity for CHO oxidation after exercise training is associated with enhanced insulin receptor substrate-1 (IRS-1; see Refs. 17 and 30) and phosphatidylinositol 3-kinase (PI 3-kinase) signaling (16) and increased glucose transporter protein 4 (GLUT4; see Ref. 14).