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This study aimed to investigate the long-term effects of training intervention and resting on protein expression and stability of peroxisome proliferator-activated receptor &beta, /&delta, (PPAR&beta, ), peroxisome proliferator-activated receptor gamma coactivator 1-&alpha, (PGC1&alpha, ), glucose transporter type 4 (GLUT4), and mitochondrial proteins, and determine whether glucose homeostasis can be regulated through stable expression of these proteins after training. Rats swam daily for 3, 6, 9, 14, or 28 days, and then allowed to rest for 5 days post-training. Protein and mRNA levels were measured in the skeletal muscles of these rats. PPAR&beta, was overexpressed and knocked down in myotubes in the skeletal muscle to investigate the effects of swimming training on various signaling cascades of PGC-1&alpha, transcription, insulin signaling, and glucose uptake. Exercise training (Ext) upregulated PPAR&beta, PGC-1&alpha, GLUT4, and mitochondrial enzymes, including NADH-ubiquinone oxidoreductase (NUO), cytochrome c oxidase subunit I (COX1), citrate synthase (CS), and cytochrome c (Cyto C) in a time-dependent manner and promoted the protein stability of PPAR&beta, GLUT4, NUO, CS, and Cyto C, such that they were significantly upregulated 5 days after training cessation. PPAR&beta, overexpression increased the PGC-1&alpha, protein levels post-translation and improved insulin-induced signaling responsiveness and glucose uptake. The present results indicate that Ext promotes the protein stability of key mitochondria enzymes GLUT4, PGC-1&alpha, and PPAR&beta, even after Ext cessation.

Summary Background For several cardiometabolic risk factors, values considered within normal range are associated with an increased risk of cardiovascular morbidity and mortality. We aimed to investigate the short-term and long-term effects of calorie restriction with adequate nutrition on these risk factors in healthy, lean, or slightly overweight young and middle-aged individuals. Methods CALERIE was a phase 2, multicentre, randomised controlled trial in young and middle-aged (21–50 years), healthy non-obese (BMI 22·0–27·9 kg/m2) men and women done in three clinical centres in the USA. Participants were randomly assigned (2:1) to a 25% calorie restriction diet or an ad libitum control diet. Exploratory cardiometabolic risk factor responses to a prescribed 25% calorie restriction diet for 2 years were evaluated (systolic, diastolic, and mean blood pressure; plasma lipids; high-sensitivity C-reactive protein; metabolic syndrome score; and glucose homoeostasis measures of fasting insulin, glucose, insulin resistance, and 2-h glucose, area-under-the curve for glucose, and insulin from an oral glucose tolerance test) analysed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT00427193. Findings From May 8, 2007, to Feb 26, 2010, of 238 participants that were assessed, 218 were randomly assigned to and started a 25% calorie restriction diet (n=143, 66%) or an ad libitum control diet (n=75, 34%). Individuals in the calorie restriction group achieved a mean reduction in calorie intake of 11·9% (SE 0·7; from 2467 kcal to 2170 kcal) versus 0·8% (1·0) in the control group, and a sustained mean weight reduction of 7·5 kg (SE 0·4) versus an increase of 0·1 kg (0·5) in the control group, of which 71% (mean change in fat mass 5·3 kg [SE 0·3] divided by mean change in weight 7·5 kg [0·4]) was fat mass loss. Calorie restriction caused a persistent and significant reduction from baseline to 2 years of all measured conventional cardiometabolic risk factors, including change scores for LDL-cholesterol (p Interpretation 2 years of moderate calorie restriction significantly reduced multiple cardiometabolic risk factors in young, non-obese adults. These findings suggest the potential for a substantial advantage for cardiovascular health of practicing moderate calorie restriction in young and middle-aged healthy individuals, and they offer promise for pronounced long-term population health benefits. Funding National Institute on Aging and National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.

Summary Young‐onset calorie restriction (CR) in rodents decreases serum IGF‐1 concentration and increases serum corticosterone levels, which have been hypothesized to play major roles in mediating its anticancer and anti‐aging effects. However, little is known on the effects of CR on the IGF‐1 system and cortisol in humans. To test the sustained effects of CR on these key hormonal adaptations, we performed a multicenter randomized trial of a 2‐year 25% CR intervention in 218 nonobese (body mass index between 22 and 27.8 kg m−2) young and middle‐aged (20–50 years age range) men and women. Average CR during the first 6 months was 19.5 ± 0.8% and 9.1 ± 0.7% over the next 18 months of the study. Weight loss averaged 7.6 ± 0.3 kg over the 2‐years period of which 71% was fat mass loss (P

Endurance exercise training can increase the ability to perform prolonged strenuous exercise. The major adaptation responsible for this increase in endurance is an increase in muscle mitochondria. This adaptation occurs too slowly to provide a survival advantage when there is a sudden change in environment that necessitates prolonged exercise. In the present study, we discovered another, more rapid adaptation, a downregulation of expression of the glycogenolytic and glycolytic enzymes in muscle that mediates a slowing of muscle glycogen depletion and lactic acid accumulation. This adaptation, which appears to be mediated by PGC-1α, occurs in response to a single exercise bout and is further enhanced by two additional daily exercise bouts. It is biologically significant, because glycogen depletion and lactic acid accumulation are two of the major causes of muscle fatigue and exhaustion.

After food ingestion, the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are secreted by the intestines into circulation where they act on the pancreas to promote insulin secretion. We evaluated the hypothesis that low postprandial plasma insulin levels in lean exercise-trained individuals are associated with low concentrations of incretin hormones.A cross-sectional study was performed to compare postprandial incretin hormone levels in lean endurance exercise-trained individuals (EX; n = 14, ≥40 yr) and age- and sex-matched, nonobese, sedentary control subjects (CON, n = 14). The main outcome measures were GLP-1, GIP, insulin, and glucose incremental areas under the curve (AUC) as measured in plasma samples collected during a 2-h,75-g oral glucose tolerance test (OGTT).The EX group had lower body fat percentage (14.6% ± 1.1% vs 23.3% ± 1.7%, P = 0.0002) and higher maximal oxygen uptake (53 ± 2 vs 34 ± 2, P0.0001) than CON. Glucose AUC did not differ between groups (P = 0.20). Insulin AUC was lower in EX (2.5 ± 0.5 vs 4.2 ± 1.2 μU·mL·1000 min, P = 0.02). No differences were observed between groups (EX and CON, respectively) for GLP-1 AUC (3.5 ± 0.7 vs 4.1 ± 1.1 pmol·min·100 L, P = 0.61) or GIP AUC (19.2 ± 1.4 vs 18.0 ± 1.4 pg·min·1000 mL; P = 0.56). In CON, insulin AUC was correlated with AUC for GLP-1 (r = 0.53, P = 0.05) and GIP (r = 0.71, P = 0.004), but no such correlations were observed in EX (both P ≥ 0.67).Low postprandial insulin levels in lean exercise-trained individuals are not attributable to lower incretin hormone concentrations. However, exercise may decrease the dependency of postprandial insulin levels on incretin hormones.

Dietary supplements are widely used for health purposes. However, little is known about the metabolic and cardiovascular effects of combinations of popular over-the-counter supplements, each of which has been shown to have anti-oxidant, anti-inflammatory and pro-longevity properties in cell culture or animal studies. This study was a 6-month randomized, single-blind controlled trial, in which 56 non-obese (BMI 21.0-29.9 kg/m(2)) men and women, aged 38 to 55 yr, were assigned to a dietary supplement (SUP) group or control (CON) group, with a 6-month follow-up. The SUP group took 10 dietary supplements each day (100 mg of resveratrol, a complex of 800 mg each of green, black, and white tea extract, 250 mg of pomegranate extract, 650 mg of quercetin, 500 mg of acetyl-l-carnitine, 600 mg of lipoic acid, 900 mg of curcumin, 1 g of sesamin, 1.7 g of cinnamon bark extract, and 1.0 g fish oil). Both the SUP and CON groups took a daily multivitamin/mineral supplement. The main outcome measures were arterial stiffness, endothelial function, biomarkers of inflammation and oxidative stress, and cardiometabolic risk factors. Twenty-four weeks of daily supplementation with 10 dietary supplements did not affect arterial stiffness or endothelial function in nonobese individuals. These compounds also did not alter body fat measured by DEXA, blood pressure, plasma lipids, glucose, insulin, IGF-1, and markers of inflammation and oxidative stress. In summary, supplementation with a combination of popular dietary supplements has no cardiovascular or metabolic effects in non-obese relatively healthy individuals.

There are reports that the β-adrenergic agonist clenbuterol induces a large increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in skeletal muscle. This has led to the hypothesis that the increases in PGC-1α and mitochondrial biogenesis induced in muscle by endurance exercise are mediated by catecholamines. In the present study, we evaluated this possibility and found that injecting rats with clenbuterol or norepinephrine induced large increases in PGC-1α and mitochondrial proteins in brown adipose tissue but had no effect on PGC-1α expression or mitochondrial biogenesis in skeletal muscle. In brown adipocytes, the increase in PGC-1α expression induced by β-adrenergic stimulation is mediated by activation of p38 mitogen-activated protein kinase (p38 MAPK), which phosphorylates and activates the cAMP response element binding protein (CREB) family member activating transcription factor 2 (ATF2), which binds to a cyclic AMP response element (CRE) in the PGC-1α promoter and mediates the increase in PGC-1α transcription. Phospho-CREB does not have this effect. Our results show that the reason for the lack of effect of β-adrenergic stimulation on PGC-1α expression in muscle is that catecholamines do not activate p38 or increase ATF2 phosphorylation in muscle.

The objective of this study was to evaluate the specific mechanism(s) by which PPARβ regulates mitochondrial content in skeletal muscle. We discovered that PPARβ increases PGC-1α by protecting it from degradation by binding to PGC-1α and limiting ubiquitination. PPARβ also induces an increase in nuclear respiratory factor 1 (NRF-1) expression, resulting in increases in mitochondrial respiratory chain proteins and MEF2A, for which NRF-1 is a transcription factor. There was also an increase in AMP kinase phosphorylation mediated by an NRF-1-induced increase in CAM kinase kinase-β (CaMKKβ). Knockdown of PPARβ resulted in large decreases in the levels of PGC-1α and mitochondrial proteins and a marked attenuation of the exercise-induced increase in mitochondrial biogenesis. In conclusion, PPARβ induces an increase in PGC-1α protein, and PPARβ is a transcription factor for NRF-1. Thus, PPARβ plays essential roles in the maintenance and adaptive increase in mitochondrial enzymes in skeletal muscle by exercise.

Calorie restriction (CR) inhibits inflammation and slows aging in many animal species, but in rodents housed in pathogen-free facilities, CR impairs immunity against certain pathogens. However, little is known about the effects of long-term moderate CR on immune function in humans. In this multi-center, randomized clinical trial to determine CR's effect on inflammation and cell-mediated immunity, 218 healthy non-obese adults (20-50 y), were assigned 25% CR (n=143) or an ad-libitum (AL) diet (n=75), and outcomes tested at baseline, 12, and 24 months of CR. CR induced a 10.4% weight loss over the 2-y period. Relative to AL group, CR reduced circulating inflammatory markers, including total WBC and lymphocyte counts, ICAM-1 and leptin. Serum CRP and TNF-α concentrations were about 40% and 50% lower in CR group, respectively. CR had no effect on the delayed-type hypersensitivity skin response or antibody response to vaccines, nor did it cause difference in clinically significant infections. In conclusion, long-term moderate CR without malnutrition induces a significant and persistent inhibition of inflammation without impairing key in vivo indicators of cell-mediated immunity. Given the established role of these pro-inflammatory molecules in the pathogenesis of multiple chronic diseases, these CR-induced adaptations suggest a shift toward a healthy phenotype.

Serum dehydroepiandrosterone (DHEA) concentrations decrease approximately 80% between ages 25 and 75 year. Aging also results in an increase in arterial stiffness, which is an independent predictor of cardiovascular disease (CVD) risk and mortality. Therefore, it is conceivable that DHEA replacement in older adults could reduce arterial stiffness. We sought to determine whether DHEA replacement therapy in older adults reduces carotid augmentation index (AI) and carotid-femoral pulse wave velocity (PWV) as indices of arterial stiffness. A randomized, double-blind trial was conducted to study the effects of 50 mg day(-1) DHEA replacement on AI (n = 92) and PWV (n = 51) in women and men aged 65-75 year. Inflammatory cytokines and sex hormones were measured in fasting serum. AI decreased in the DHEA group, but not in the placebo group (difference between groups, -6 ± 2 AI units, P = 0.002). Pulse wave velocity also decreased (difference between groups, -3.5 ± 1.0 m s(-1), P = 0.001); however, after adjusting for baseline values, the between-group comparison became nonsignificant (P = 0.20). The reductions in AI and PWV were accompanied by decreases in inflammatory cytokines (tumor necrosis factor α and IL-6, P < 0.05) and correlated with increases in serum DHEAS (r = -0.31 and -0.37, respectively, P < 0.05). The reductions in AI also correlated with free testosterone index (r = -0.23, P = 0.03). In conclusion, DHEA replacement in elderly men and women improves indices of arterial stiffness. Arterial stiffness increases with age and is an independent risk factor for CVD. Therefore, the improvements observed in this study suggest that DHEA replacement might partly reverse arterial aging and reduce CVD risk.

Caloric restriction (CR) retards aging in laboratory rodents. No information is available on the effects of long-term CR on physiologic markers of aging and longevity in humans. Heart rate variability (HRV) is a marker for cardiac autonomic functioning. The progressive decline in HRV with aging and the association of higher HRV with better health outcomes are well established. Heart rate variability assessment is a reliable tool by which the effects of CR on autonomic function can be assessed. Time- and frequency-domain analyses compared 24-h HRV in 22 CR individuals aged 35-82 years and 20 age-matched controls eating Western diets (WD). The CR group was significantly leaner than the WD group. Heart rate was significantly lower, and virtually, all HRV values were significantly higher in the CR group than in the WD group (P < 0.002). Heart rate variability in the CR individuals was comparable with published norms for healthy individuals 20 years younger. In addition, when differences in heart rate (HR) and HRV between CR and WD were compared with previously published changes in HRV induced in healthy adults given atenolol, percent differences in each measure were generally similar in direction and magnitude and suggested declines in sympathetic and increases in parasympathetic modulation of HR and increased circadian variability associated with CR. These findings provide evidence that CR has direct systemic effects that counter the expected age-associated changes in autonomic function so that HRV indexes in CR individuals are similar to those of individuals 20 years younger eating WDs.

Inference of the insulin secretion rate (ISR) from C-peptide measurements as a quantification of pancreatic β-cell function is clinically important in diseases related to reduced insulin sensitivity and insulin action. ISR derived from C-peptide concentration is an example of nonparametric Bayesian model selection where a proposed ISR time-course is considered to be a “model”. An inferred value of inaccessible continuous variables from discrete observable data is often problematic in biology and medicine, because it is a priori unclear how robust the inference is to the deletion of data points, and a closely related question, how much smoothness or continuity the data actually support. Predictions weighted by the posterior distribution can be cast as functional integrals as used in statistical field theory. Functional integrals are generally difficult to evaluate, especially for nonanalytic constraints such as positivity of the estimated parameters. We propose a computationally tractable method that uses the exact solution of an associated likelihood function as a prior probability distribution for a Markov-chain Monte Carlo evaluation of the posterior for the full model. As a concrete application of our method, we calculate the ISR from actual clinical C-peptide measurements in human subjects with varying degrees of insulin sensitivity. Our method demonstrates the feasibility of functional integral Bayesian model selection as a practical method for such data-driven inference, allowing the data to determine the smoothing timescale and the width of the prior probability distribution on the space of models. In particular, our model comparison method determines the discrete time-step for interpolation of the unobservable continuous variable that is supported by the data. Attempts to go to finer discrete time-steps lead to less likely models.

To identify new gene regulatory pathways controlling skeletal muscle energy metabolism, comparative studies were conducted on muscle-specific transgenic mouse lines expressing the nuclear receptors peroxisome proliferator-activated receptor α (PPARα; muscle creatine kinase [MCK]-PPARα) or PPARβ/δ (MCK-PPARβ/δ). MCK-PPARβ/δ mice are known to have enhanced exercise performance, whereas MCK-PPARα mice perform at low levels. Transcriptional profiling revealed that the lactate dehydrogenase b (Ldhb)/Ldha gene expression ratio is increased in MCK-PPARβ/δ muscle, an isoenzyme shift that diverts pyruvate into the mitochondrion for the final steps of glucose oxidation. PPARβ/δ gain- and loss-of-function studies in skeletal myotubes demonstrated that PPARβ/δ, but not PPARα, interacts with the exercise-inducible kinase AMP-activated protein kinase (AMPK) to synergistically activate Ldhb gene transcription by cooperating with myocyte enhancer factor 2A (MEF2A) in a PPARβ/δ ligand-independent manner. MCK-PPARβ/δ muscle was shown to have high glycogen stores, increased levels of GLUT4, and augmented capacity for mitochondrial pyruvate oxidation, suggesting a broad reprogramming of glucose utilization pathways. Lastly, exercise studies demonstrated that MCK-PPARβ/δ mice persistently oxidized glucose compared with nontransgenic controls, while exhibiting supranormal performance. These results identify a transcriptional regulatory mechanism that increases capacity for muscle glucose utilization in a pattern that resembles the effects of exercise training.

It has been reported that supplementation with the antioxidant vitamins C and E prevents the adaptive increases in mitochondrial biogenesis and GLUT4 expression induced by endurance exercise. We reevaluated the effects of these antioxidants on the adaptive responses of rat skeletal muscle to swimming in a short-term study consisting of 9 days of vitamins C and E with exercise during the last 3 days and a longer-term study consisting of 8 wk of antioxidant vitamins with exercise during the last 3 wk. The rats in the antioxidant groups were given 750 mg·kg body wt−1·day−1vitamin C and 150 mg·kg body wt−1·day−1vitamin E. In rats euthanized immediately after exercise, plasma TBARs were elevated in the control rats but not in the antioxidant-supplemented rats, providing evidence for an antioxidant effect. In rats euthanized 18 h after exercise there were large increases in insulin responsiveness of glucose transport in epitrochlearis muscles mediated by an approximately twofold increase in GLUT4 expression in both the short- and long-term treatment groups. The protein levels of a number of mitochondrial marker enzymes were also increased about twofold. Superoxide dismutases (SOD) 1 and 2 were increased about twofold in triceps muscle after 3 days of exercise, but only SOD2 was increased after 3 wk of exercise. There were no differences in the magnitudes of any of these adaptive responses between the control and antioxidant groups. These results show that very large doses of antioxidant vitamins do not prevent the exercise-induced adaptive responses of muscle mitochondria, GLUT4, and insulin action to exercise and have no effect on the level of these proteins in sedentary rats.

Plasma dehydroepiandrosterone (DHEA) decreases ~80% between ages 25 and 75 yr. In a preliminary study, we found that 6 mo of DHEA replacement improved insulin action in elderly individuals. The purpose of the present larger, randomized double-blind study was to determine whether a longer period of DHEA replacement improves glucose tolerance. Fifty-seven men an d 68 women aged 65 to 75 yr were randomly assigned to 50 mg DHEA or placebo once daily. Year one was a randomized, double blind trial. Year 2 was an open label continuation. DHEA replacement improved glucose tolerance in participants who had abnormal GT initially, reduced plasma triglycerides, and the infla mmatory cytokines IL6 and TNFα. This trial was registered at clinicaltrials.gov as NCT00182975.

OBJECTIVE Ginseng and its active component, ginsenoside Re, are popular herbal products that are advocated for treatment of diabetes. The purpose of this study was to determine whether ginseng or ginsenoside Re improves β-cell function and insulin sensitivity (IS) in insulin-resistant subjects. RESEARCH DESIGN AND METHODS Overweight or obese subjects (BMI = 34 ± 1 kg/m2) with impaired glucose tolerance or newly diagnosed type 2 diabetes were randomized to 30 days of treatment with ginseng root extract (8 g/day), ginsenoside Re (250–500 mg/day), or placebo. β-Cell function was assessed as the disposition index (DI) and measured by a frequently sampled oral glucose tolerance test, and IS was assessed as the relative increase in glucose disposal during a hyperinsulinemic-euglycemic clamp procedure plus stable isotope tracer infusion. RESULTS Values for DI and IS after therapy (Post) were not different from values before therapy (Pre) in the placebo (DI: Pre, 5.8 ± 0.9 × 10−3 and Post, 5.8 ± 0.8 × 10−3, P = 0.99; IS: Pre,165 ± 29% and Post, 185 ± 24%, P = 0.34), ginseng (DI: Pre, 7.7 ± 2.0 × 10−3 and Post, 6.0 ± 0.8 × 10−3, P = 0.29; IS: Pre, 171 ± 72% and Post,137 ± 59%, P = 0.88), and ginsenoside Re (DI: Pre, 7.4 ± 3.0 × 10−3 and Post, 5.9 ± 1.1 × 10−3, P = 0.50; IS: Pre, 117 ± 31% and Post, 134 ± 34%, P = 0.44) groups. Ginsenosides Re, Rb1, and Rb2 were not detectable in plasma after treatment with ginseng root extract or ginsenoside Re. CONCLUSIONS Oral ginseng or ginsenoside Re therapy does not improve β-cell function or IS in overweight/obese subjects with impaired glucose tolerance or newly diagnosed diabetes. Poor systemic bioavailability might be responsible for the absence of a therapeutic effect.

Endurance exercise training can induce large increases mitochondria and the GLUT4 isoform of the glucose transporter in skeletal muscle. For a long time after the discovery in the 1960s that exercise results in an increase in muscle mitochondria, there was no progress in elucidation of the mechanisms involved. The reason for this lack of progress was that nothing was known regarding how expression of the genes-encoding mitochondrial proteins is coordinately regulated. This situation changed rapidly after discovery of transcription factors that control transcription of genes-encoding mitochondrial proteins and, most importantly, the discovery of peroxisome proliferator-gamma coactivator-1α (PGC-1α). This transcription coactivator binds to and activates transcription factors that regulate transcription of genes-encoding mitochondrial proteins. Thus, PGC-1α activates and coordinates mitochondrial biogenesis. It is now known that exercise rapidly activates and induces increased expression of PGC-1α. The exercise-generated signals that lead to PGC-1α activation and increased expression are the increases in cytosolic Ca(2+) and decreases in ATP and creatine phosphate (∼P). Ca(2+) mediates its effect by activating CAMKII, while the decrease in ∼P mediates its effect via activation of AMPK. Expression of the GLUT4 isoform of the glucose transporter is regulated in parallel with mitochondrial biogenesis via the same signaling pathways. This review describes what is known regarding the regulation of mitochondrial biogenesis and GLUT4 expression by exercise. A major component of this review deals with the physiological and metabolic consequences of the exercise-induced increase in mitochondria and GLUT4.

Reduction of body temperature has been proposed to contribute to the increased lifespan in calorie restricted animals and mice overexpressing the uncoupling protein-2 in hypocretin neurons. However, nothing is known regarding the long-term effects of calorie restriction (CR) with adequate nutrition on body temperature in humans. In this study, 24-hour core body temperature was measured every minute by using ingested telemetric capsules in 24 men and women (mean age 53.7 ± 9.4 yrs) consuming a CR diet for an average of 6 years, 24 age- and sex-matched sedentary (WD) and 24 body fat-matched exercise-trained (EX) volunteers, who were eating Western diets. The CR and EX groups were significantly leaner than the WD group. Energy intake was lower in the CR group (1769 ± 348 kcal/d) than in the WD (2302 ± 668 kcal/d) and EX (2798 ± 760 kcal/d) groups (P0.0001). Mean 24-hour, day-time and night-time core body temperatures were all significantly lower in the CR group than in the WD and EX groups (P ≤ 0.01). Long-term CR with adequate nutrition in lean and weight-stable healthy humans is associated with a sustained reduction in core body temperature, similar to that found in CR rodents and monkeys. This adaptation is likely due to CR itself, rather than to leanness, and may be involved in slowing the rate of aging.

It has been hypothesized that insulin resistance is mediated by a deficiency of mitochondria in skeletal muscle. In keeping with this hypothesis, high-fat diets that cause insulin resistance have been reported to result in a decrease in muscle mitochondria. In contrast, we found that feeding rats high-fat diets that cause muscle insulin resistance results in a concomitant gradual increase in muscle mitochondria. This adaptation appears to be mediated by activation of peroxisome proliferator-activated receptor (PPAR)δ by fatty acids, which results in a gradual, posttranscriptionally regulated increase in PPAR γ coactivator 1α (PGC-1α) protein expression. Similarly, overexpression of PPARδ results in a large increase in PGC-1α protein in the absence of any increase in PGC-1α mRNA. We interpret our findings as evidence that raising free fatty acids results in an increase in mitochondria by activating PPARδ, which mediates a posttranscriptional increase in PGC-1α. Our findings argue against the concept that insulin resistance is mediated by a deficiency of muscle mitochondria.

Calorie restriction (CR) retards aging, acts as a hormetic intervention, and increases serum corticosterone and HSP70 expression in rodents. However, less is known regarding the effects of CR on these factors in humans. Serum cortisol and molecular chaperones and autophagic proteins were measured in the skeletal muscle of subjects on CR diets for 3-15 years and in control volunteers. Serum cortisol was higher in the CR group than in age-matched sedentary and endurance athlete groups (15.6 ± 4.6 ng/dl versus 12.3 ± 3.9 ng/dl and 11.2 ± 2.7 ng/dl, respectively; p ≤ 0.001). HSP70, Grp78, beclin-1, and LC3 mRNA and/or protein levels were higher in the skeletal muscle of the CR group compared to controls. Our data indicate that CR in humans is associated with sustained rises in serum cortisol, reduced inflammation, and increases in key molecular chaperones and autophagic mediators involved in cellular protein quality control and removal of dysfunctional proteins and organelles. © 2016 The Authors.

The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been identified as an inducible regulator of mitochondrial function. Skeletal muscle PGC-1alpha expression is induced post-exercise. Therefore, we sought to determine its role in the regulation of muscle fuel metabolism. Studies were performed using conditional, muscle-specific, PGC-1alpha gain-of-function and constitutive, generalized, loss-of-function mice. Forced expression of PGC-1alpha increased muscle glucose uptake concomitant with augmentation of glycogen stores, a metabolic response similar to post-exercise recovery. Induction of muscle PGC-1alpha expression prevented muscle glycogen depletion during exercise. Conversely, PGC-1alpha-deficient animals exhibited reduced rates of muscle glycogen repletion post-exercise. PGC-1alpha was shown to increase muscle glycogen stores via several mechanisms including stimulation of glucose import, suppression of glycolytic flux, and by down-regulation of the expression of glycogen phosphorylase and its activating kinase, phosphorylase kinase alpha. These findings identify PGC-1alpha as a critical regulator of skeletal muscle fuel stores.

Objective: To compare the inter-rater and intra-rater reliability and analysis time of two methods for quantifying visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) volumes from magnetic resonance (MR) images. Research Methods and Procedures: Ten subjects (BMI, 27.0 ± 2.1 kg/m2; 56 years of age ± 4 years) underwent MR imaging of the abdomen. Ten transverse T1-weighted images were selected from each scan and analyzed using two software packages that differ in principle. The first method, ANALYZE version 5.0, represents the manual threshold method, and the second, HIPPO version 1.3, is based on the fuzzy clustering approach. Inter-rater reliability for each method was assessed by comparing the intra-class correlation coefficients (ICCs) for VAT and SAT results from two evaluators, and intra-rater reliability for each method was assessed by comparing ICCs for VAT and SAT analyses performed 1 week apart by the same evaluator. The total time for analysis also was compared between methods. Results: The inter-rater reliability for VAT was greater with HIPPO than with ANALYZE (ICC = 0.996 vs. 0.828), whereas inter-rater reliability for SAT did not differ between methods (ICC = 0.975 and 0.987). The intra-rater reliability was equally high with HIPPO and ANALYZE for both VAT (ICC = 0.998 vs. 0.992) and SAT (ICC = 0.996 vs. 0.992). HIPPO required less than one-half as much analysis time as ANALYZE (15.9 ± 4.4 vs. 36.5 ± 8.2 minutes, p < 0.0001). Discussion: HIPPO software appears advantageous for the quantification of VAT from multislice MR images because inter-rater results are more reliable, and it is more time-efficient than less automated methods.

Increases in exercise energy expenditure without compensatory changes in food intake (EX) and restriction of calorie intake (CR) both decrease body weight and fat mass, which, in turn, improve glucoregulatory function. However, EX may provide greater benefits than can be provided through CR. Therefore, our study hypothesis was that weight loss through EX reduces visceral abdominal fat and improves glucoregulation to a greater extent than does similar weight loss through CR. Forty-eight sedentary 50- to 60-y-old men and women, most of whom were overweight, underwent 12 mo of EX, CR, or a healthy lifestyle control period. Body composition was assessed by dual-energy x-ray absorptiometry and by magnetic resonance imaging. Indices of glucoregulatory function were determined by oral glucose tolerance test and were measured ≥48 h after the last exercise bout in the EX group. Body weight, total fat mass, and visceral fat volume decreased similarly in the EX and CR groups but did not change in the HL group. Likewise, insulin sensitivity index and the oral glucose tolerance test glucose and insulin areas under the curve improved similarly in the EX and CR groups and remained unchanged in the HL group. In conclusion, weight losses induced by exercise and by CR are effective means for improving glucose tolerance and insulin action in nonobese, healthy, middle-aged men and women; however, it does not appear that exercise training-induced weight loss results in greater improvements than those that result from CR.

Exercise results in rapid increases in expression of the transcription coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) and in mitochondrial biogenesis in skeletal muscle. PGC-1alpha regulates and coordinates mitochondrial biogenesis, and overexpression of PGC-1alpha in muscle cells results in increases in mitochondrial content. In this context, it has been proposed that the increase in PGC-1alpha protein expression mediates the exercise-induced increase in mitochondrial biogenesis. However, we found that mitochondrial proteins with a short half-life increase as rapidly as, or more rapidly than, PGC-1alpha protein. This finding led us to hypothesize that activation, rather than increased expression, of PGC-1alpha mediates the initial phase of the exercise-induced increase in mitochondria. In this study, we found that most of the PGC-1alpha in resting skeletal muscle is in the cytosol. Exercise resulted in activation of p38 MAPK and movement of PGC-1alpha into the nucleus. In support of our hypothesis, binding of the transcription factor nuclear respiratory factor 1 (NRF-1) to the cytochrome c promoter and NRF-2 to the cytochrome oxidase subunit 4 promoter increased in response to exercise prior to an increase in PGC-1alpha protein. Furthermore, exercise-induced increases in the mRNAs of cytochrome c, delta-aminolevulinate synthase, and citrate synthase also occurred before an increase in PGC-1 protein. Thus, it appears that activation of PGC-1alpha may mediate the initial phase of the exercise-induced adaptive increase in muscle mitochondria, whereas the subsequent increase in PGC-1alpha protein sustains and enhances the increase in mitochondrial biogenesis.

The plasma levels of dehydroepiandrosterone (DHEA) and its sulfated form (DHEAS) decline ∼80% between the ages of 25 and 75 yr. Muscle mass and strength also decrease with aging. Published data on the effects of DHEA replacement on muscle mass and strength are conflicting. The goals of this study were to determine whether DHEA replacement increases muscle mass and strength and/or enhances the effects of heavy resistance exercise in elderly women and men. We conducted a randomized, double-blind, placebo-controlled study of the effects of 10 mo of DHEA replacement therapy with the addition of weightlifting exercise training during the last 4 mo of the study (DHEA + exercise group, n = 29; placebo + exercise group, n = 27). DHEA alone for 6 mo did not significantly increase strength or thigh muscle volume. However, DHEA therapy potentiated the effect of 4 mo of weightlifting training on muscle strength, evaluated by means of one-repetition maximum measurement and Cybex dynamometry, and on thigh muscle volume, measured by magnetic resonance imaging. Serum insulin-like growth factor concentration increased in response to DHEA replacement. This study provides evidence that DHEA replacement has the beneficial effect of enhancing the increases in muscle mass and strength induced by heavy resistance exercise in elderly individuals.

We investigated the hemodynamic determinants of the age-associated decline in maximal oxygen uptake (V̇o2 max) and the influence of gender on the decline in V̇o2 max and its determinants in old and very old men and women. Sedentary, 60- to 92-yr-old women ( n = 71) and men ( n = 29), with no evidence of cardiovascular disease, underwent maximal treadmill exercise tests during which V̇o2 max and maximal cardiac output (Q̇max) were determined. V̇o2 max and age were inversely related in both women (−23 ± 2 ml·min−1·yr−1; P < 0.0001) and men (−57 ± 5 ml·min−1·yr−1; P < 0.0001). The absolute slope of the V̇o2 max vs. age relationship was twofold steeper in men than in women ( P < 0.0001). Q̇max was also inversely related to age in a gender-specific manner (women = −87 ± 25 ml·min−1·yr−1, P = 0.0009; men = −215 ± 50 ml·min−1·yr−1, P = 0.0002; P = 0.01 women vs. men). Age-related changes in maximal exercise arteriovenous oxygen content difference (a-vDo2) were marginally different ( P = 0.08) between women (−0.12 ± 0.03 ml·dl−1·yr−1, P = 0.0003) and men (−0.22 ± 0.04 ml·dl−1·yr−1, P < 0.0001). Age-associated decreases in Q̇max and a-vDo2 contributed equally to the declines in V̇o2 max in both men and women. In the later stages of life, V̇o2 max, Q̇max, and a-vDo2 decrease with age more rapidly in older men than they do in older women. As a result, the gender differences dissipate in the later decades of life. Declines in Q̇max and a-vDo2 contribute equally to the age-related decrease in V̇o2 max in men and women.

OBJECTIVE—Physical inactivity and increased adiposity contribute to insulin resistance; less is known, however, about the relative contributions of these factors in older adults. The aim of this study was to determine whether cardiovascular fitness, whole-body adiposity, or abdominal adiposity is the strongest predictor of insulin resistance into old age. RESEARCH DESIGN AND METHODS—Subjects included 407 men and women aged 50–95 years (means ± SD 69 ± 11 years). Insulin resistance was estimated using the insulin sensitivity index (ISI) of Matsuda and DeFronzo [ISI = 10,000/square root of (fasting glucose × fasting insulin) × (mean glucose × mean insulin during an oral glucose tolerance test); lower ISI = greater insulin resistance]. Fitness was determined with a treadmill maximal oxygen consumption (Vo2max) test. Whole-body adiposity measures included BMI and percent fat by dual-energy X-ray absorptiometry or hydrodensitometry; abdominal adiposity was estimated by waist circumference. RESULTS—Waist circumference was the strongest independent correlate of ISI (r = −0.52, P < 0.0001), explaining 28% of the variance when controlling for sex, BMI, percent fat, and Vo2max. BMI (r = −0.45), percent fat (r = −0.40), and Vo2max (r = 0.22) independently predicted ISI (all P < 0.0001); however, after controlling for waist circumference, only Vo2max remained significant (r = 0.13, P = 0.009). CONCLUSIONS—Adiposity and fitness continue to be significant predictors of insulin sensitivity into old age, with abdominal obesity being the most important single factor. These findings support the measurement of waist circumference to assess health risk among older adults.

Muscle contractions and insulin stimulate glucose transport into muscle by separate pathways. The contraction-mediated increase in glucose transport is mediated by two mechanisms, one involves the activation of 5′-AMP-activated protein kinase (AMPK) and the other involves the activation of calcium/calmodulin-dependent protein kinase II (CAMKII). The steps leading from the activation of AMPK and CAMKII to the translocation of GLUT4 to the cell surface have not been identified. Studies with the use of the tyrosine kinase inhibitor genistein suggest that one or more tyrosine kinases could be involved in contraction-stimulated glucose transport. The purpose of the present study was to determine the involvement of tyrosine kinases in contraction-stimulated glucose transport in rat soleus and epitrochlearis muscles. Contraction-stimulated glucose transport was completely prevented by pretreatment with genistein (100 μM) and the related compound butein (100 μM). However, the structurally distinct tyrosine kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4-d]pyridine and herbimycin did not reduce contraction-stimulated glucose transport. Furthermore, genistein and butein inhibited glucose transport even when muscles were exposed to these compounds after being stimulated to contract. Muscle contractions did not result in increases in tyrosine phosphorylation of proteins such as proline-rich tyrosine kinase and SRC. These results provide evidence that tyrosine kinases do not mediate contraction-stimulated glucose transport and that the inhibitory effects of genistein on glucose transport result from direct inhibition of the glucose transporters at the cell surface.

Objectives We determined whether caloric restriction (CR) has cardiac-specific effects that attenuate the established aging-associated impairments in diastolic function (DF). Background Caloric restriction retards the aging process in small mammals; however, no information is available on the effects of long-term CR on human aging. In healthy individuals, Doppler echocardiography has established the pattern of aging-associated DF impairment, whereas little change is observed in systolic function (SF). Methods Diastolic function was assessed in 25 subjects (age 53 ± 12 years) practicing CR for 6.5 ± 4.6 years and 25 age- and gender-matched control subjects consuming Western diets. Diastolic function was quantified by transmitral flow, Doppler tissue imaging, and model-based image processing (MBIP) of E waves. C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta 1 (TGF-β 1 ) were also measured. Results No difference in SF was observed between groups; however, standard transmitral Doppler flow DF indexes of the CR group were similar to those of younger individuals, and MBIP-based, flow-derived DF indexes, reflecting chamber viscoelasticity and stiffness, were significantly lower than in control subjects. Blood pressure, serum CRP, TNF-α, and TGF-β 1 levels were significantly lower in the CR group (102 ± 10/61 ± 7 mm Hg, 0.3 ± 0.3 mg/l, 0.8 ± 0.5 pg/ml, 29.4 ± 6.9 ng/ml, respectively) compared with the Western diet group (131 ± 11/83 ± 6 mm Hg, 1.9 ± 2.8 mg/l, 1.5 ± 1.0 pg/ml, 35.4 ± 7.1 ng/ml, respectively). Conclusions Caloric restriction has cardiac-specific effects that ameliorate aging-associated changes in DF. These beneficial effects on cardiac function might be mediated by the effect of CR on blood pressure, systemic inflammation, and myocardial fibrosis.

Background. Progressive resistance exercise training (PRT) has been shown to increase muscle strength and fat-free mass (FFM) in elderly persons. Limited information is available regarding the effects of PRT on lean and fat mass in frail elderly persons. Methods. Ninety-one community-dwelling sedentary men and women, 78 years and older with physical frailty (defined using standardized objective criteria) were enrolled in a 9-month trial of exercise training (ET). Physical frailty was defined as having 2 of the 3 following criteria: modified Physical Performance Test score between 18 and 32, peak aerobic power between 10 and 18 ml/kg/min, or self-report of difficulty or assistance with two instrumental activities of daily living or one basic activity of daily living. Participants were randomly assigned to either a control (CTL) group that performed a low intensity home exercise program or a supervised ET group that performed 3 months of low intensity exercise and 3 months of PRT. Results. After completion of PRT, ET participants had greater improvements than did CTL participants in maximal voluntary force production for knee extension (mean þ5.3 6 13 ft/lb vs þ1.1 6 11 ft/lb, p ¼ .05), measured using isokinetic dynamometry. Total body FFM (measured using dual energy x-ray absorptiometry) increased in the ET group, but not in the CTL group (mean þ0.84 6 1.4 kg vs þ0.01 6 1.5 kg, p ¼ .005). Total, trunk, intra-abdominal, and subcutaneous fat mass (measured using dual energy x-ray absorptiometry and 1 H-magnetic resonance imaging) did not change in response to PRT. Conclusions. Three months of supervised PRT induced improvements in maximal voluntary thigh muscle strength and whole body FFM in frail, community-dwelling elderly women and men. This supervised exercise program may not be sufficient to reduce whole-body or intra-abdominal fat area in this population.

Little is known regarding the long-term effects of caloric restriction (CR) on the risk for atherosclerosis. We evaluated the effect of CR on risk factors for atherosclerosis in individuals who are restricting food intake to slow aging. We studied 18 individuals who had been on CR for an average of 6 years and 18 age-matched healthy individuals on typical American diets. We measured serum lipids and lipoproteins, fasting plasma glucose and insulin, blood pressure (BP), high-sensitivity C-reactive protein (CRP), platelet-derived growth factor AB (PDGF-AB), body composition, and carotid artery intima-media thickness (IMT). The CR group were leaner than the comparison group (body mass index, 19.6 ± 1.9 vs. 25.9 ± 3.2 kg/m 2 ; percent body fat, 8.7 ± 7% vs. 24 ± 8%). Serum total cholesterol (Tchol), low-density lipoprotein cholesterol, ratio of Tchol to high-density lipoprotein cholesterol (HDL-C), triglycerides, fasting glucose, fasting insulin, CRP, PDFG-AB, and systolic and diastolic BP were all markedly lower, whereas HDL-C was higher, in the CR than in the American diet group. Medical records indicated that the CR group had serum lipid-lipoprotein and BP levels in the usual range for individuals on typical American diets, and similar to those of the comparison group, before they began CR. Carotid artery IMT was ≈40% less in the CR group than in the comparison group. Based on a range of risk factors, it appears that long-term CR has a powerful protective effect against atherosclerosis. This interpretation is supported by the finding of a low carotid artery IMT.

To address the potential role of lipotoxicity and mitochondrial function in insulin resistance, we studied mice with high-level expression of uncoupling protein-1 in skeletal muscle (UCP-H mice). Body weight, body length, and bone mineral density were decreased in UCP-H mice compared with wild-type littermates. Forelimb grip strength and muscle mass were strikingly decreased, whereas muscle triglyceride content was increased fivefold in UCP-H mice. Electron microscopy demonstrated lipid accumulation and large mitochondria with abnormal architecture in UCP-H skeletal muscle. ATP content and key mitochondrial proteins were decreased in UCP-H muscle. Despite mitochondrial dysfunction and increased intramyocellular fat, fasting serum glucose was 22% lower and insulin-stimulated glucose transport 80% higher in UCP-H animals. These beneficial effects on glucose metabolism were associated with increased AMP kinase and hexokinase activities, as well as elevated levels of GLUT4 and myocyte enhancer factor-2 proteins A and D in skeletal muscle. These results suggest that UCP-H mice have a mitochondrial myopathy due to depleted energy stores sufficient to compromise growth and impair muscle function. Enhanced skeletal muscle glucose transport in this setting suggests that excess intramyocellular lipid and mitochondrial dysfunction are not sufficient to cause insulin resistance in mice.

The people afflicted with obesity and its consequences need a menu of low-cost programmes to mitigate the over-nutrition and under-activity that underlie their condition. This editorial commentary points out the multiple merits (and shortcomings) of the Nordic walking - with ski poles - presented in an accompanying article. The Nordic exercise programme is then examined more broadly in the context of the general principles and limitations of exercise programmes - with and without calorie control - as a guide to future innovations.

To determine the mechanisms underlying increased aerobic power in response to exercise training in octogenarians, we studied mildly frail elderly men and women randomly assigned to an exercise group ( n = 22) who participated in a training program of 6 mo of physical therapy, strength training, and walking followed by 3 mo of more intense endurance exercise at 78% of peak heart rate or a control sedentary group ( n = 24). Peak O2 consumption (V̇o2 peak) increased 14% in the exercise group ( P < 0.0001) but decreased slightly in controls. Training induced 14% increase ( P = 0.027) in peak exercise cardiac output (Q̇), determined via acetylene re-breathing, and no change in arteriovenous O2 content difference. The increase in Q̇ was mediated by increases in heart rate ( P = 0.009) and probably stroke volume ( P = 0.096). Left ventricular stroke work also increased significantly. In the men, the increase in V̇o2 peak was exclusively due to a large increase in peak Q̇ (22%). In the women, the gain in V̇o2 peak was due to small increases in Q̇ and O2 extraction from skeletal muscles. Pulse pressure normalized for stroke volume and arterial elastance during peak effort did not change with training. Controls showed no changes. The results suggest that, although frail octogenarians have a diminished capacity for improvement in aerobic power in response to exercise training, this adaptation is mediated mostly by an increase in Q̇ during peak effort. Furthermore, Q̇ likely plays a greater role in the adaptive increase in V̇o2 peak in old men than old women.

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator of nuclear genes that encode a range of mitochondrial proteins including cytochrome c, various other respiratory chain subunits, and delta-aminolevulinate synthase. Activation of NRF-1 in fibroblasts has been shown to induce increases in cytochrome c expression and mitochondrial respiratory capacity. To further evaluate the role of NRF-1 in the regulation of mitochondrial biogenesis and respiratory capacity, we generated transgenic mice overexpressing NRF-1 in skeletal muscle. Cytochrome c expression was increased approximately twofold and delta-aminolevulinate synthase was increased approximately 50% in NRF-1 transgenic muscle. The levels of some mitochondrial proteins were increased 50-60%, while others were unchanged. Muscle respiratory capacity was not increased in the NRF-1 transgenic mice. A finding that provides new insight regarding the role of NRF-1 was that expression of MEF2A and GLUT4 was increased in NRF-1 transgenic muscle. The increase in GLUT4 was associated with a proportional increase in insulin-stimulated glucose transport. These results show that an isolated increase in NRF-1 is not sufficient to bring about a coordinated increase in expression of all of the proteins necessary for assembly of functional mitochondria. They also provide the new information that NRF-1 overexpression results in increased expression of GLUT4.

BACKGROUND Caloric restriction (CR), energy intake reduced below ad libitum (AL) intake, increases life span in many species. The implications for humans can be clarified by randomized controlled trials of CR. METHODS To determine CR's feasibility, safety, and effects on predictors of longevity, disease risk factors, and quality of life in nonobese humans aged 21-51 years, 218 persons were randomized to a 2-year intervention designed to achieve 25% CR or to AL diet. Outcomes were change from baseline resting metabolic rate adjusted for weight change ("RMR residual") and core temperature (primary); plasma triiodothyronine (T3) and tumor necrosis factor-α (secondary); and exploratory physiological and psychological measures. RESULTS Body mass index averaged 25.1 (range: 21.9-28.0 kg/m(2)). Eighty-two percent of CR and 95% of AL participants completed the protocol. The CR group achieved 11.7±0.7 %CR (mean ± standard error) and maintained 10.4±0.4% weight loss. Weight change in AL was negligible. RMR residual decreased significantly more in CR than AL at 12 months (p = .04) but not 24 months (M24). Core temperature change differed little between groups. T3 decreased more in CR at M12 and M24 (p < .001), while tumor necrosis factor-α decreased significantly more only at M24 (p = .02). CR had larger decreases in cardiometabolic risk factors and in daily energy expenditure adjusted for weight change, without adverse effects on quality of life. CONCLUSIONS Sustained CR is feasible in nonobese humans. The effects of the achieved CR on correlates of human survival and disease risk factors suggest potential benefits for aging-related outcomes that could be elucidated by further human studies.

Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria.

Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+can stimulate mitochondrial biogenesis. Continuous exposure to 1 μM ionomycin, a Ca2+ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an ∼40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+stimulates mitochondrial biogenesis in muscle cells.

There is evidence suggesting that adaptive increases in GLUT4 and mitochondria in skeletal muscle occur in parallel. It has been reported that raising cytosolic Ca2+in myocytes induces increases in mitochondrial enzymes. In this study, we tested the hypothesis that an increase in cytosolic Ca2+induces an increase in GLUT4. We found that raising cytosolic Ca2+by exposing L6 myotubes to 5 mM caffeine for 3 h/day for 5 days induced increases in GLUT4 protein and in myocyte enhancer factor (MEF)2A and MEF2D, which are transcription factors involved in regulating GLUT4 expression. The caffeine-induced increases in GLUT4 and MEF2A and MEF2D were partially blocked by dantrolene, an inhibitor of sarcoplasmic reticulum Ca2+release, and completely blocked by KN93, an inhibitor of Ca2+-calmodulin-dependent protein kinase (CAMK). Caffeine also induced increases in MEF2A, MEF2D, and GLUT4 in rat epitrochlearis muscles incubated with caffeine in culture medium. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), which activates AMP-activated protein kinase (AMPK), also induced approximately twofold increases in GLUT4, MEF2A, and MEF2D in L6 myocytes. Our results provide evidence that increases in cytosolic Ca2+and activation of AMPK, both of which occur in exercising muscle, increase GLUT4 protein in myocytes and skeletal muscle. The data suggest that this effect of Ca2+is mediated by activation of CAMK and indicate that MEF2A and MEF2D are involved in this adaptive response.

Evidence has accumulated that activation of AMP kinase (AMPK) mediates the acute increase in glucose transport induced by exercise. As the exercise-induced, insulin-independent increase in glucose transport wears off, it is followed by an increase in muscle insulin sensitivity. The major purpose of this study was to determine whether hypoxia and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), which also activate AMPK and stimulate glucose transport, also induce an increase in insulin sensitivity. We found that the increase in glucose transport in response to 30 μU/ml insulin was about twofold greater in rat epitrochlearis muscles that had been made hypoxic or treated with AICAR 3.5 h previously than in untreated control muscles. This increase in insulin sensitivity was similar to that induced by a 2-h bout of swimming or 10 min of in vitro electrically stimulated contractions. Neither phosphatidylinositol 3-kinase activity nor protein kinase B (PKB) phosphorylation in response to 30 μU/ml insulin was enhanced by prior exercise or AICAR treatment that increased insulin sensitivity of glucose transport. Inhibition of protein synthesis by inclusion of cycloheximide in the incubation medium for 3.5 h after exercise did not prevent the increase in insulin sensitivity. Contractions, hypoxia, and treatment with AICAR all caused a two- to three-fold increase in AMPK activity over the resting level. These results provide evidence that the increase in insulin sensitivity of muscle glucose transport that follows exercise is mediated by activation of AMPK and involves a step beyond PKB in the pathway by which insulin stimulates glucose transport.

Long-term endurance exercise training results in a reduction in the rates of muscle glycogen depletion and lactic acid accumulation during submaximal exercise; this adaptation is mediated by an increase in muscle mitochondria. There is evidence suggesting that short-term training induces adaptations that downregulate glycogenolysis before there is an increase in functional mitochondria. We discovered that a single long bout of exercise induces decreases in expression of glycogenolytic and glycolytic enzymes in rat skeletal muscle; this adaptation results in slower rates of glycogenolysis and lactic acid accumulation in muscle during contractile activity. Two additional days of training amplified the adaptive response, which appears to be mediated by PGC-1α; this adaptation is biologically significant, because glycogen depletion and lactic acid accumulation are major causes of muscle fatigue.Endurance exercise training can increase the ability to perform prolonged strenuous exercise. The major adaptation responsible for this increase in endurance is an increase in muscle mitochondria. This adaptation occurs too slowly to provide a survival advantage when there is a sudden change in environment that necessitates prolonged exercise. In the present study, we discovered another, more rapid adaptation, a downregulation of expression of the glycogenolytic and glycolytic enzymes in muscle that mediates a slowing of muscle glycogen depletion and lactic acid accumulation. This adaptation, which appears to be mediated by PGC-1α, occurs in response to a single exercise bout and is further enhanced by two additional daily exercise bouts. It is biologically significant, because glycogen depletion and lactic acid accumulation are two of the major causes of muscle fatigue and exhaustion.

Calorie restriction (CR) is shown to improve inflammatory and immune responses and extend life in animals; however, the effect of prolonged CR in humans has not been studied. CALERIE is a multi-cen...

Objective: Previous studies have demonstrated the benefit of short-term diets on glucose tolerance in obese individuals. The purpose of this study was to evaluate the effectiveness of modest lifestyle changes in maintaining improvements in glucose tolerance induced by short-term energy restriction in obese African Americans with impaired glucose tolerance or type 2 diabetes mellitus. Research Methods and Procedures: An intervention group (n = 45; 47 ± 1 year [mean ± SE]), 105 ± 4 kg; body mass index: 39 ± 1 kg/m2) received an energy-restricted diet (943 ± 26 kcal/d) for 1 week, followed by a lifestyle program of reduced dietary fat (−125 kcal/d) and increased physical activity (+125 kcal/d) for 1 year. Body weight and plasma concentrations of glucose, insulin, and C-peptide during an oral glucose tolerance test were measured at baseline, 1-week, and 4-month intervals. A control group (n = 24; 48 ± 1 year; 110 ± 5 kg; body mass index: 41 ± 2 kg/m2) underwent these measurements at 4-month intervals. Results: No changes in weight or glucose tolerance were observed in the control group. The intervention group had significant (p < 0.05) improvements in body weight and glucose tolerance in response to the 1-week diet, which persisted for 4 months (p < 0.001 vs. control for change in weight). A total of 19 subjects (42%) continued the intervention program for 1 year, with sustained improvements (weight: −4.6 ± 1.0 kg; p < 0.001 vs. control; oral glucose tolerance test glucose area: −103 ± 44 mM · min; p < 0.05 vs. control). Discussion: A modest lifestyle program facilitates weight loss and enables improvements in glucose tolerance to be maintained in obese individuals with abnormal glucose tolerance. However, attrition was high, despite the mild nature of the program.

In many patients with human immunodeficiency virus (HIV) treated with HIV protease inhibitors, a complication develops that resembles abdominal obesity syndrome, with insulin resistance and glucose intolerance that, in some cases, progresses to diabetes. In this study, we tested the hypothesis that indinavir, an HIV-protease inhibitor, directly induces insulin resistance of glucose transport in skeletal muscle. Rat epitrochlearis muscles were incubated with a maximally effective insulin concentration (12 nmol/l) and 0, 1, 5, 20, or 40 micromol/l indinavir for 4 h. In control muscles, insulin increased 3-O-[(3)H]methyl-D-glucose (3MG) transport from 0.15 +/- 0.03 to 1.10 +/- 0.05 micromol. ml(-)(1). 10 min(-)(1). Incubation of muscles with 5 micromol/l indinavir reduced the insulin-stimulated increase in 3MG transport by 40%, whereas 20 micromol/l indinavir reduced the insulin-stimulated increase in 3MG transport by 58%. Indinavir induced a similar reduction in maximally insulin-stimulated 3MG transport in the soleus muscle. The increase in glucose transport activity induced by stimulating epitrochlearis muscles to contract was also markedly reduced by indinavir. The insulin-stimulated increase in cell-surface GLUT4, assessed using the 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-[2-(3)H] (D-mannose-4-yloxy)-2-propylamine exofacial photolabeling technique, was reduced by approximately 70% in the presence of 20 micromol/l indinavir. Insulin stimulation of phosphatidylinositol 3-kinase activity and phosphorylation of protein kinase B were not decreased by indinavir. These results provide evidence that indinavir inhibits the translocation or intrinsic activity of GLUT4 rather than insulin signaling.

It has been variously hypothesized that the insulin resistance induced in rodents by a high-fat diet is due to increased visceral fat accumulation, to an increase in muscle triglyceride (TG) content, or to an effect of diet composition. In this study we used a number of interventions: fish oil, leptin, caloric restriction, and shorter duration of fat feeding, to try to disassociate an increase in visceral fat from muscle insulin resistance. Substituting fish oil (18% of calories) for corn oil in the high-fat diet partially protected against both the increase in visceral fat and muscle insulin resistance without affecting muscle TG content. Injections of leptin during the last 4 days of a 4-wk period on the high-fat diet partially reversed the increase in visceral fat and the muscle insulin resistance, while completely normalizing muscle TG. Restricting intake of the high-fat diet to 75% of ad libitum completely prevented the increase in visceral fat and muscle insulin resistance. Maximally insulin-stimulated glucose transport was negatively correlated with visceral fat mass ( P < 0.001) in both the soleus and epitrochlearis muscles and with muscle TG concentration in the soleus ( P < 0.05) but not in the epitrochlearis. Thus we were unable to dissociate the increase in visceral fat from muscle insulin resistance using a variety of approaches. These results support the hypothesis that an increase in visceral fat is associated with development of muscle insulin resistance.

Exercise training induces an increase in GLUT-4 in muscle. We previously found that feeding rats a high-carbohydrate diet after exercise, with muscle glycogen supercompensation, results in a decrease in insulin responsiveness so severe that it masks the effect of a training-induced twofold increase in GLUT-4 on insulin-stimulated muscle glucose transport. One purpose of this study was to determine whether insulin signaling is impaired. Maximally insulin-stimulated phosphatidylinositol (PI) 3-kinase activity was not significantly reduced, whereas protein kinase B (PKB) phosphorylation was approximately 50% lower (P0.01) in muscles of chow-fed, than in those of fasted, exercise-trained rats. Our second purpose was to determine whether contraction-stimulated glucose transport is also impaired. The stimulation of glucose transport and the increase in cell surface GLUT-4 induced by contractions were both decreased by approximately 65% in glycogen-supercompensated muscles of trained rats. The contraction-stimulated increase in AMP kinase activity, which has been implicated in the activation of glucose transport by contractions, was approximately 80% lower in the muscles of the fed compared with the fasted rats 18 h after exercise. These results show that both the insulin- and contraction-stimulated pathways for muscle glucose transport activation are impaired in glycogen-supercompensated muscles and provide insight regarding possible mechanisms.