Exercise training improves fat metabolism independent of total energy expenditure in sedentary overweight men, but does not restore lean metabolic phenotype.

BACKGROUND: Obesity is a dietary fat storage disease. Although exercise prevents weight gain, effects of chronic training on dietary fat oxidation remains understudied in overweight adults. OBJECTIVE: We tested whether 2 months of training at current guidelines increase dietary fat oxidation in sedentary overweight adults like in sedentary lean adults. DESIGN: Sedentary lean (n=10) and overweight (n=9) men trained on a cycle ergometer at 50% VO2peak, 1 h day-1, four times per week, for 2 months while energy balance was clamped. Metabolic fate of [d31]palmitate and [1-13C]oleate mixed in standard meals, total substrate use, total energy expenditure (TEE), activity energy expenditure (AEE) and key muscle proteins/enzymes were measured before and at the end of the intervention. RESULTS: Conversely to lean subjects, TEE and AEE did not increase in overweight participants due to a spontaneous decrease in non-training AEE. Despite this compensatory behavior, aerobic fitness, insulin sensitivity and fat oxidation were improved by exercise training. The latter was not explained by changes in dietary fat trafficking but more likely by a coordinated response at the muscle level enhancing fat uptake, acylation and oxidation (FABPpm, CD36, FATP1, ACSL1, CPT1, mtGPAT). ACSL1 fold change positively correlated with total fasting (R2=0.59, P<0.0001) and post-prandial (R2=0.49, P=0.0006) fat oxidation whereas mtGPAT fold change negatively correlated with dietary palmitate oxidation (R2=0.40, P=0.009), suggesting modified fat trafficking between oxidation and storage within the muscle. However, for most of the measured parameters the post-training values observed in overweight adults remained lower than the pre-training values observed in the lean subjects. CONCLUSION: Independent of energy balance and TEE, exercise training at current recommendations improved fitness and fat oxidation in overweight adults. However the improved metabolic phenotype of overweight adults was not as healthy as the one of their lean counterparts before the 2-month training, likely due to the spontaneous reduction in non-training AEE.

A socio-ecological approach promoting physical activity and limiting sedentary behavior in adolescence showed weight benefits maintained 2.5 years after intervention cessation.

BACKGROUND: Obesity in youth remains a major public health issue. Yet no effective long-term preventive strategy exists. We previously showed that a school-based socio-ecological approach targeting behavior and social/environmental influences on physical activity (PA) prevented 4-year excessive weight gain in 12-year olds. In this study, we investigated if this efficacy persists 30 months after intervention cessation. METHODS AND FINDINGS: The program targeted students, family, school and the living environment to promote/support PA and prevent sedentary behavior (SB). A total of 732 students from eight randomized middle schools completed the 4-year trial. At the 30-month post-trial follow-up, body mass index (BMI), fat mass index (FMI), leisure PA (LPA), home/school/workplace active commuting, TV/video time (TVT), and attitudes toward PA were measured in 531 adolescents. The beneficial effects of the intervention on the excess BMI increase (+0.01 vs +0.34 kg m(-2) in the intervention and control groups, respectively) and on the overweight incidence in initially non-overweight students (4.3% vs 8.6%; odds ratio=0.48 (95% confidence interval: 0.23-1.01)) were maintained at the post-trial follow-up. LPA was not maintained at the level achieved during the trial. However, we still observed a prevention of the age-related decrease of the adolescents' percentage reporting regular LPA (-14.4% vs -26.5%) and a higher intention to exercise in the intervention group. The intervention promoted lower TVT (-14.0 vs +13.6 min per day) and higher active commuting changes (+11.7% vs -4.8%). Trends in higher BMI reduction in students with high initial TVT and in the least wealthy group were noted. TVT changes throughout the follow-up predicted excess BMI and FMI changes. CONCLUSIONS: Long-term multilevel approach targeting PA and SB prevents excessive weight gain up to 30 months after intervention cessation. The efficacy may be higher in the most sedentary and least wealthy adolescents. Healthy PA-related behavior inducing long-lasting weight effects can be promoted in youth providing that an ecological approach is introduced in the prevention strategy.

Validity of combining heart rate and uniaxial acceleration to measure free-living physical activity energy expenditure in young men.

Combining accelerometry (ACC) with heart rate (HR) monitoring is thought to improve activity energy expenditure (AEE) estimations compared with ACC alone to evaluate the validity of ACC and HR used alone or combined. The purpose of this study was to estimate AEE in free-living conditions compared with doubly labeled water (DLW). Ten-day free-living AEE was measured by a DLW protocol in 35 18- to 55-yr-old men (11 lean active; 12 lean sedentary; 12 overweight sedentary) wearing an Actiheart (combining ACC and HR) and a RT3 accelerometer. AEE was estimated using group or individual calibration of the HR/AEE relationship, based on an exercise-tolerance test. In a subset (n = 21), AEE changes (ΔAEE) were measured after 1 mo of detraining (active subjects) or an 8-wk training (sedentary subjects). Actiheart-combined ACC/HR estimates were more accurate than estimates from HR or ACC alone. Accuracy of the Actiheart group-calibrated ACC/HR estimates was modest [intraclass correlation coefficient (ICC) = 0.62], with no bias but high root mean square error (RMSE) and limits of agreement (LOA). The mean bias of the estimates was reduced by one-third, like RMSE and LOA, by individual calibration (ICC = 0.81). Contrasting with group-calibrated estimates, the Actiheart individual-calibrated ACC/HR estimates explained 40% of the variance of the DLW-ΔAEE (ICC = 0.63). This study supports a good level of agreement between the Actiheart ACC/HR estimates and DLW-measured AEE in lean and overweight men with varying fitness levels. Individual calibration of the HR/AEE relationship is necessary for AEE estimations at an individual level rather than at group scale and for ΔAEE evaluation.

Microarray analysis of genes with impaired insulin regulation in the skeletal muscle of type 2 diabetic patients indicates the involvement of basic helix-loop-helix domain-containing, class B, 2 protein (BHLHB2).

AIMS/HYPOTHESIS: One of the major processes by which insulin exerts its multiple biological actions is through gene expression regulation. Thus, the identification of transcription factors affected by insulin in target tissues represents an important challenge. The aim of the present study was to gain a greater insight into this issue through the identification of transcription factor genes with insulin-regulated expression in human skeletal muscle. METHODS: Using microarray analysis, we defined the sets of genes modulated during a 3 h hyperinsulinaemic-euglycaemic clamp (2 mU min(-1) kg(-1)) in the skeletal muscle of insulin-sensitive control volunteers and in moderately obese insulin-resistant type 2 diabetic patients. RESULTS: Of the 1,529 and 1,499 genes regulated during the clamp in control and diabetic volunteers, respectively, we identified 30 transcription factors with impaired insulin-regulation in type 2 diabetic patients. Analysis of the promoters of the genes encoding these factors revealed a possible contribution of the transcriptional repressor basic helix-loop-helix domain-containing, class B, 2 protein (BHLHB2), insulin regulation of which is strongly altered in the muscle of diabetic patients. Gene ontology analysis of BHLHB2 target genes, identified after BHLHB2 overexpression in human primary myotubes, demonstrated that about 10% of the genes regulated in vivo during hyperinsulinaemia are potentially under the control of this repressor. The data also suggested that BHLHB2 is situated at the crossroads of a complex transcriptional network that is able to modulate major metabolic and biological pathways in skeletal muscle, including the regulation of a cluster of genes involved in muscle development and contraction. CONCLUSIONS/INTERPRETATION: We have identified BHLHB2 as a potential novel mediator of insulin transcriptional action in human skeletal muscle.

Activation of liver X receptors promotes lipid accumulation but does not alter insulin action in human skeletal muscle cells.

AIMS/HYPOTHESIS: The aim of this study was to investigate the effects of liver X receptor (LXR) activation on lipid metabolism and insulin action in human skeletal muscle cells prepared from control subjects and from patients with type 2 diabetes. SUBJECTS AND METHODS: Cultured myotubes were obtained from muscle biopsies of 11 lean, healthy control subjects and ten patients with type 2 diabetes. The mRNA levels of LXR isoforms and lipogenic genes were estimated by RT-quantitative PCR, and the effects of LXR agonists on insulin action were evaluated by assays of protein kinase B serine 473 phosphorylation and glycogen synthesis. RESULTS: Both LXRalpha and LXRbeta were expressed in human skeletal muscle and adipose tissue and there was no difference in their mRNA abundance in tissues from patients with type 2 diabetes compared with control subjects. In cultured muscle cells, LXR activation by T0901317 strongly increased expression of the genes encoding lipogenic enzymes, including sterol regulatory element binding protein 1c, fatty acid synthase and stearoyl-CoA desaturase 1, and also promoted triglyceride accumulation in the presence of a high glucose concentration. Importantly, these effects on lipid metabolism did not affect protein kinase B activation by insulin. Furthermore, LXR agonists did not modify insulin action in muscle cells from patients with type 2 diabetes. CONCLUSIONS/INTERPRETATION: These data suggest that LXR agonists may lead to increased utilisation of lipids and glucose in muscle cells without affecting the mechanism of action of insulin. However, the long-term consequences of triglyceride accumulation in muscle should be evaluated before the development of effective LXR-based therapeutic agents.

Expression of the splice variants of the p85alpha regulatory subunit of phosphoinositide 3-kinase in muscle and adipose tissue of healthy subjects and type 2 diabetic patients.

The regulation by insulin of the expression of the p85alpha regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase) is impaired in skeletal muscle and adipose tissue of type 2 diabetic patients. The gene encoding p85alpha (named grb-1) can generate several variants by alternative splicing, all being able to activate the p110 catalytic subunits of PI 3-kinase. Our aims were (i) to determine the mRNA expression profiles of these variants in human skeletal muscle and adipose tissue; (ii) to investigate the effect of insulin on their expression in vivo and in vitro in muscle and (iii) to verify whether this regulation is defective in type 2 diabetes. We determined the human genomic organization of grb-1 and set up reverse transcriptase competitive PCR assays for the quantification of each mRNA variant. In muscle, p85alpha and p50alpha mRNAs were the most abundant, and p55alpha represented less than 20% of all grb-1-derived mRNAs. In adipose tissue, p85alpha was expressed predominantly and p55alpha mRNA was not detectable. These expression profiles were not different in type 2 diabetics. During a 3 h hyperinsulinaemic clamp, insulin increased the mRNA expression of the three variants in muscle of control subjects. In diabetic patients, the effect of insulin on p85alpha and p50alpha mRNAs was blunted, and largely reduced on p55alpha transcripts. In cultured human myotubes, up-regulation of p85alpha, p55alpha and p50alpha mRNAs by insulin was abolished by LY294002 (10 microM) and by rapamycin (50 nM), suggesting that the PI 3-kinase/protein kinase B/p70 S6 kinase pathway could be involved in the stimulation of grb-1 gene expression by insulin in human muscle cells.

Overexpression of the catalytic subunit of DNA polymerase gamma results in depletion of mitochondrial DNA in Drosophila melanogaster.

The mechanisms involved in the regulation of mitochondrial DNA (mtDNA) replication, a process that is crucial for mitochondrial biogenesis, are not well understood. In this study, we evaluate the role of DNA polymerase gamma (pol gamma), the key enzyme in mtDNA replication, in both Drosophila cell culture and in developing flies. We report that overexpression of the pol gamma catalytic subunit (pol gamma-alpha) in cultured Schneider cells does not alter either the amount of mtDNA or the growth rate of the culture. The polypeptide is properly targeted to mitochondria, yet the large excess of pol gamma-alpha does not interfere with mtDNA replication under these conditions where the endogenous polypeptide is apparently present in amounts that exceed of the demand for its function in the cell. In striking contrast, overexpression of pol gamma-alpha at the same level in transgenic flies interferes with the mtDNA replication process, presumably by altering the mechanism of DNA synthesis, suggesting differential requirements for, and/or regulation of, mtDNA replication in Drosophila cell culture versus the developing organism. Overexpression of pol gamma-alpha in transgenic flies produces a significant depletion of mtDNA that causes a broad variety of phenotypic effects. These alterations range from pupal lethality to moderate morphological abnormalities in adults. depending on the level and temporal pattern of overexpression. Our results demonstrate that although cells may tolerate a variable amount of the pol gamma catalytic subunit under some conditions, its level may be critical in the context of the whole organism.

Differential regulation of the catalytic and accessory subunit genes of Drosophila mitochondrial DNA polymerase.

The developmental pattern of expression of the genes encoding the catalytic (alpha) and accessory (beta) subunits of mitochondrial DNA polymerase (pol gamma) has been examined in Drosophila melanogaster. The steady-state level of pol gamma-beta mRNA increases during the first hours of development, reaching its maximum value at the start of mtDNA replication in Drosophila embryos. In contrast, the steady-state level of pol gamma-alpha mRNA decreases as development proceeds and is low in stages of active mtDNA replication. This difference in mRNA abundance results at least in part from differences in the rates of mRNA synthesis. The pol gamma genes are located in a compact cluster of five genes that contains three promoter regions (P1-P3). The P1 region directs divergent transcription of the pol gamma-beta gene and the adjacent rpII33 gene. P1 contains a DNA replication-related element (DRE) that is essential for pol gamma-beta promoter activity, but not for rpII33 promoter activity in Schneider's cells. A second divergent promoter region (P2) controls the expression of the orc5 and sop2 genes. The P2 region contains two DREs that are essential for orc5 promoter activity, but not for sop2 promoter activity. The expression of the pol gamma-alpha gene is directed by P3, a weak promoter that does not contain DREs. Electrophoretic mobility shift experiments demonstrate that the DRE-binding factor (DREF) regulatory protein binds to the DREs in P1 and P2. DREF regulates the expression of several genes encoding key factors involved in nuclear DNA replication. Its role in controlling the expression of the pol gamma-beta and orc5 genes establishes a common regulatory mechanism linking nuclear and mitochondrial DNA replication. Overall, our results suggest that the accessory subunit of mtDNA polymerase plays an important role in the control of mtDNA replication in Drosophila.

The highly compact structure of the mitochondrial DNA polymerase genomic region of Drosophila melanogaster: functional and evolutionary implications.

The structure of a Drosophila melanogaster genomic region containing five tightly clustered genes has been determined and evaluated with regard to its functional and evolutionary relationships. In addition to the genes encoding the two subunits (alpha and beta) of the DNA polymerase gamma holoenzyme, the key enzyme for mitochondrial DNA replication, other genes contained in the cluster may be also involved in the cellular distribution of mitochondria and in the coordination of mitochondrial and nuclear DNA replication. The gene cluster is extremely compact, with very little intergenic space. It contains two bidirectional promoter regions, and particularly notable is the 5' end overlap detected in two of its genes, an exceptional situation in both prokaryotic and eukaryotic genome organization.

Regulation of mitochondrial single-stranded DNA-binding protein gene expression links nuclear and mitochondrial DNA replication in drosophila.

The structural organization of the Drosophila melanogaster gene encoding mitochondrial single-stranded DNA-binding protein (mtSSB) has been determined and its pattern of expression evaluated during Drosophila development. The D. melanogaster mtSSB gene contains four exons and three small introns. The transcriptional initiation site is located 22 nucleotides upstream from the initiator translation codon in adults, whereas several initiation sites are found in embryos. No consensus TATA or CAAT sequences are located at canonical positions, although an AT-rich sequence was identified flanking the major transcriptional initiation site. Northern analyses indicated that the mtSSB transcript is present at variable levels throughout development. In situ hybridization analysis shows that maternally deposited mtSSB mRNA is distributed homogeneously in the early embryo, whereas de novo transcript is produced specifically at an elevated level in the developing midgut. Transfection assays in cultured Schneider cells with promoter region deletion constructs revealed that the proximal 230 nucleotides contain cis-acting elements required for efficient gene expression. Putative transcription factor binding sites clustered within this region include two Drosophila DNA replication-related elements (DRE) and a single putative E2F binding site. Deletion and base substitution mutagenesis of the DRE sites demonstrated that they are required for efficient promoter activity, and gel electrophoretic mobility shift analyses showed that DRE binding factor (DREF) binds to these sites. Our data suggest strongly that the Drosophila mtSSB gene is regulated by the DRE/DREF system. This finding represents a first link between nuclear and mitochondrial DNA replication.

Quantitative decrease of human cytochrome c oxidase during development: evidences for a post-transcriptional regulation.

In an earlier study, we showed that cytochrome c oxidase activity, measured in mitochondria isolated from human muscular biopsies, decreased steadily and substantially between the age of four years and adulthood (P < 0.05), whereas complexes I and III activity remained constant. The present study investigates a number of possible causes for this change in activity: although there is a drop in the apparent Vmax, neither the apparent enzyme Km, nor the cellular mtDNA concentration shows any variations over the studied period. Steady-state concentrations of mitochondrial gene transcripts (CO I. CO II, CO III, but also 12S, cytochrome b, or ND4) increase within this age group, indicating an overall increase in mitochondrial genome expression. Concentrations of transcripts of nuclear genes CO IV, CO Vb, and CO VIaH likewise show an increase, albeit less marked. On the other hand, heme aa3 levels and concentrations of mitochondrial (CO II) or nuclear (CO IV, CO VIIaH) subunits, estimated using specific antibodies, correlate closely with enzymatic activity and show a parallel decrease between 4 and 20 years. The observed decrease in complex IV activity is thus quantitative, and subject to post-transcriptional and/or post-translational regulation.

Enzymatic activities of mitochondrial respiratory complexes from children muscular biopsies. Age-related evolutions.

Measurements were performed to determine maximum enzymatic activities of citrate synthetase and respiratory complexes I, III, and IV of mitochondria obtained from muscular biopsies in control children. The significant number of determinations carried out (43 different biopsies in controls aged 3.8 to 19.1 years) permits the formulation of a table of statistically validated reference values for these activities. These values are independent of sex of the controls, and of the studied muscles. Citrate synthetase activity, which remains stable in this age range, thus constitutes a good internal indicator of mitochondrial activity. Complexes I and III manifest activity which does not vary with age. On the other hand, cytochrome oxidase activity shows a highly significant decrease in this age group. This decrease may be correlated with qualitative changes (subunits VIa and VIIa) in composition of this complex.