Discordant gene expression in subcutaneous adipose and skeletal muscle tissues in response to exercise training.

Exercise has different effects on different tissues in the body, the sum of which may determine the response to exercise and the health benefits. In the present study, we aimed to investigate whether physical training regulates transcriptional network communites common to both skeletal muscle (SM) and subcutaneous adipose tissue (SAT). Eight such shared transcriptional communities were found in both tissues. Eighteen young overweight adults voluntarily participated in 7 weeks of combined strength and endurance training (five training sessions per week). Biopsies were taken from SM and SAT before and after training. Five of the network communities were regulated by training in SM but showed no change in SAT. One community involved in insulin- AMPK signaling and glucose utilization was upregulated in SM but downregulated in SAT. This diverging exercise regulation was confirmed in two independent studies and was also associated with BMI and diabetes in an independent cohort. Thus, the current finding is consistent with the differential responses of different tissues and suggests that body composition may influence the observed individual whole-body metabolic response to exercise training and help explain the observed attenuated whole-body insulin sensitivity after exercise training, even if it has significant effects on the exercising muscle.

Effects of Seven Weeks of Combined Physical Training on High-Density Lipoprotein Functionality in Overweight/Obese Subjects.

Our study aimed to investigate the effects of exercise on HDL composition and functional properties in overweight/obese subjects. Eighteen overweight/obese subjects (nine F and nine M, BMI = 30.3 ± 3 kg/m2) attended supervised training for 7 weeks. The protocol included combined resistance and conditioning training four to five times each week. The activity of the antioxidant enzyme paraoxonase-1 (PON1) associated with HDL was evaluated in all subjects before and after the training intervention. Moreover, myeloperoxidase (MPO) levels and oxidative stress markers (ox-LDLs and total antioxidant capacity) were studied in the serums of the subjects. At the end of the intervention, the activity of PON1 was increased (p < 0.0001), and MPO levels and the MPO/PON1 ratio were decreased (p < 0.0001). In addition, a significant improvement in muscle strength and maximal oxygen uptake (VO2max) (p < 0.0001) and a significant reduction in total and visceral adipose tissue mass (p < 0.001) and waist circumference (p < 0.008), without any significant decrease in body weight, were observed. A significant correlation was established between serum MPO/PON ratios, HDL redox activity and ox-LDLs. In conclusion, our results demonstrate that exercise training, without modifications of dietary habits, improved HDL functionality in overweight/obese adults, without any significant reduction in BMI or modifications of glucose and lipid biochemical parameters.

Improved Peripheral and Hepatic Insulin Sensitivity after Lifestyle Interventions in Type 2 Diabetes Is Associated with Specific Metabolomic and Lipidomic Signatures in Skeletal Muscle and Plasma.

Lifestyle interventions with weight loss can improve insulin sensitivity in type 2 diabetes (T2D), but mechanisms are unclear. We explored circulating and skeletal muscle metabolite signatures of altered peripheral (pIS) and hepatic insulin sensitivity (hIS) in overweight and obese T2D individuals that were randomly assigned a 12-week Paleolithic-type diet with (diet-ex, n = 13) or without (diet, n = 13) supervised exercise. Baseline and post-intervention measures included: mass spectrometry-based metabolomics and lipidomics of skeletal muscle and plasma; pIS and hIS; ectopic lipid deposits in the liver and skeletal muscle; and skeletal muscle fat oxidation rate. Both groups lowered BMI and total % fat mass and increased their pIS. Only the diet-group improved hIS and reduced ectopic lipids in the liver and muscle. The combined improvement in pIS and hIS in the diet-group were associated with decreases in muscle and circulating branched-chain amino acid (BCAA) metabolites, specifically valine. Improved pIS with diet-ex was instead linked to increased diacylglycerol (34:2) and triacylglycerol (56:0) and decreased phosphatidylcholine (34:3) in muscle coupled with improved muscle fat oxidation rate. This suggests a tissue crosstalk involving BCAA-metabolites after diet intervention with improved pIS and hIS, reflecting reduced lipid influx. Increased skeletal muscle lipid utilization with exercise may prevent specific lipid accumulation at sites that perturb insulin signaling.

Potential Physiological and Cellular Mechanisms of Exercise That Decrease the Risk of Severe Complications and Mortality Following SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unmasked mankind's vulnerability to biological threats. Although higher age is a major risk factor for disease severity in COVID-19, several predisposing risk factors for mortality are related to low cardiorespiratory and metabolic fitness, including obesity, cardiovascular disease, diabetes, and hypertension. Reaching physical activity (PA) guideline goals contribute to protect against numerous immune and inflammatory disorders, in addition to multi-morbidities and mortality. Elevated levels of cardiorespiratory fitness, being non-obese, and regular PA improves immunological function, mitigating sustained low-grade systemic inflammation and age-related deterioration of the immune system, or immunosenescence. Regular PA and being non-obese also improve the antibody response to vaccination. In this review, we highlight potential physiological, cellular, and molecular mechanisms that are affected by regular PA, increase the host antiviral defense, and may determine the course and outcome of COVID-19. Not only are the immune system and regular PA in relation to COVID-19 discussed, but also the cardiovascular, respiratory, renal, and hormonal systems, as well as skeletal muscle, epigenetics, and mitochondrial function.

The liver-alpha-cell axis after a mixed meal and during weight loss in type 2 diabetes.

OBJECTIVE: Glucagon and amino acids may be regulated in a feedback loop called the liver-alpha-cell axis with alanine or glutamine as suggested signal molecules. We assessed this concept in individuals with type 2 diabetes in the fasting state, after ingestion of a protein-rich meal, and during weight loss. Moreover, we investigated if postprandial glucagon secretion and hepatic insulin sensitivity were related. METHODS: This is a secondary analysis of a 12-week weight-loss trial (Paleolithic diet ± exercise) in 29 individuals with type 2 diabetes. Before and after the intervention, plasma glucagon and amino acids were measured in the fasting state and during 180 min after a protein-rich mixed meal. Hepatic insulin sensitivity was measured using the hyperinsulinemic-euglycemic clamp with [6,6-2H2]glucose as a tracer. RESULTS: The postprandial increase of plasma glucagon was associated with the postprandial increase of alanine and several other amino acids but not glutamine. In the fasted state and after the meal, glucagon levels were negatively correlated with hepatic insulin sensitivity (rS = -0.51/r = -0.58, respectively; both P < 0.05). Improved hepatic insulin sensitivity with weight loss was correlated with decreased postprandial glucagon response (r = -0.78; P < 0.001). CONCLUSIONS: Several amino acids, notably alanine, but not glutamine could be key signals to the alpha cell to increase glucagon secretion. Amino acids may be part of a feedback mechanism as glucagon increases endogenous glucose production and ureagenesis in the liver. Moreover, postprandial glucagon secretion seems to be tightly related to hepatic insulin sensitivity.

The Effect of Lactobacillus plantarum 299v on Iron Status and Physical Performance in Female Iron-Deficient Athletes: A Randomized Controlled Trial.

Iron is an essential micronutrient for oxygen transport and mitochondrial metabolism and is critical for physical performance. Compromised iron stores are more commonly found among athletes, and females are especially at risk. Iron deficiency is generally treated using oral iron supplements. However, only a small proportion of ingested iron is absorbed, necessitating higher intakes, which may result in adverse side effects, reduced compliance, and inefficient repletion of iron stores. The probiotic strain Lactobacillus plantarum 299v (Lp299v) significantly increases intestinal iron absorption in meal studies. The present study was conducted to explore the effects of 20 mg of iron with or without Lp299v on iron status, mood state, and physical performance. Fifty-three healthy non-anemic female athletes with low iron stores (ferritin < 30 µg/L) were randomized, and 39 completed the study. Intake of Lp299v with iron for four weeks increased ferritin levels more than iron alone (13.6 vs. 8.2 µg/L), but the difference between the groups was not significant (p = 0.056). The mean reticulocyte hemoglobin content increased after intake of Lp299v compared to control (1.5 vs. 0.82 pg) after 12 weeks, but the difference between the group was not significant (p = 0.083). The Profile of Mood States (POMS) questionnaire showed increased vigor with Lp299v vs. iron alone after 12 weeks (3.5 vs. 0.1, p = 0.015). No conclusive effects on physical performance were observed. In conclusion, Lp299v, together with 20 mg of iron, could result in a more substantial and rapid improvement in iron status and improved vigor compared to 20 mg of iron alone. A larger clinical trial is needed to further explore these findings as well as the impact of Lp299v on physical performance.

Effects of a Ketogenic Diet on Muscle Fatigue in Healthy, Young, Normal-Weight Women: A Randomized Controlled Feeding Trial.

Ketogenic low-carbohydrate high-fat (LCHF) diets are increasingly popular in broad sections of the population. The main objective of this study was to evaluate the effects of a non-energy-restricted ketogenic LCHF diet on muscle fatigue in healthy, young, and normal-weight women. Twenty-four women were randomly allocated to a 4-week ketogenic LCHF diet followed by a 4-week control diet (a National Food Agency recommended diet), or the reverse sequence due to the crossover design. Treatment periods were separated by a 15 week washout period. Seventeen women completed the study and were included in the analyses. Treatment effects were evaluated using mixed models. The ketogenic LCHF diet had no effect on grip strength or time to fatigue, measured with handgrip test (day 24-26). However, cycling time to fatigue decreased with almost two minutes (-1.85 min 95% CI:[-2.30;-1.40]; p < 0.001) during incremental cycling (day 25-27), accommodated with higher ratings of perceived exertion using the Borg scale (p < 0.01). Participants' own diary notes revealed experiences of muscle fatigue during daily life activities, as well as during exercise. We conclude that in young and healthy women, a ketogenic LCHF diet has an unfavorable effect on muscle fatigue and might affect perceived exertion during daily life activities.

Exercise Training Adds Cardiometabolic Benefits of a Paleolithic Diet in Type 2 Diabetes Mellitus.

Background The accumulation of myocardial triglycerides and remodeling of the left ventricle are common features in type 2 diabetes mellitus and represent potential risk factors for the development of diastolic and systolic dysfunction. A few studies have investigated the separate effects of diet and exercise training on cardiac function, but none have investigated myocardial changes in response to a combined diet and exercise intervention. This 12-week randomized study assessed the effects of a Paleolithic diet, with and without additional supervised exercise training, on cardiac fat, structure, and function. Methods and Results Twenty-two overweight and obese subjects with type 2 diabetes mellitus were randomized to either a Paleolithic diet and standard-care exercise recommendations ( PD ) or to a Paleolithic diet plus supervised exercise training 3 hours per week ( PD - EX ). This study includes secondary end points related to cardiac structure and function, ie, myocardial triglycerides levels, cardiac morphology, and strain were measured using cardiovascular magnetic resonance, including proton spectroscopy, at baseline and after 12 weeks. Both groups showed major favorable metabolic changes. The PD - EX group showed significant decreases in myocardial triglycerides levels (-45%, P=0.038) and left ventricle mass to end-diastolic volume ratio (-13%, P=0.008) while the left ventricle end-diastolic volume and stroke volume increased significantly (+14%, P=0.004 and +17%, P=0.008, respectively). These variables were unchanged in the PD group. Conclusions Exercise training plus a Paleolithic diet reduced myocardial triglycerides levels and improved left ventricle remodeling in overweight/obese subjects with type 2 diabetes mellitus. Clinical Trial Registration URL : http://www.clinicaltrials.gov . Unique identifier: NCT 01513798.

A heterogeneous response of liver and skeletal muscle fat to the combination of a Paleolithic diet and exercise in obese individuals with type 2 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: The aim of the study was to investigate ectopic fat deposition and insulin sensitivity, in a parallel single-blinded randomised controlled trial, comparing Paleolithic diet alone with the combination of Paleolithic diet and exercise in individuals with type 2 diabetes. METHODS: Thirty-two individuals with type 2 diabetes with BMI 25-40 kg/m2 and 30-70 years of age followed a Paleolithic diet ad libitum for 12 weeks. In addition, study participants were randomised by computer program to either supervised combined exercise training (PD-EX group) or standard care exercise recommendations (PD group). Staff performing examinations and assessing outcomes were blinded to group assignment. Thirteen participants were analysed in each group: hepatic and peripheral insulin sensitivity were measured using the hyperinsulinaemic-euglycaemic clamp technique combined with [6,6-2H2]glucose infusion, and liver fat was assessed by proton magnetic resonance spectroscopy; both analyses were secondary endpoints. Intramyocellular lipid (IMCL) content was measured by magnetic resonance spectroscopy as a secondary analysis. All examinations were performed at Umeå University Hospital, Umeå, Sweden. RESULTS: Both study groups showed a median body weight loss of 7 kg. Fat mass decreased by 5.7 kg in the PD group and by 6.5 kg in the PD-EX group. Maximum oxygen uptake increased in the PD-EX group only. Liver fat showed a consistent reduction (74% decrease) in the PD group, while the response in the PD-EX group was heterogeneous (p < 0.05 for the difference between groups). IMCL content of the soleus muscle decreased by 40% in the PD group and by 22% in the PD-EX group (p < 0.05 for the difference between groups). Both groups improved their peripheral and adipose tissue insulin sensitivity, but not their hepatic insulin sensitivity. Plasma fetuin-A decreased by 11% in the PD group (p < 0.05) and remained unchanged in the PD-EX group. Liver fat changes during the intervention were correlated with changes in fetuin-A (rS = 0.63, p < 0.01). Participants did not report any important adverse events caused by the intervention. CONCLUSIONS/INTERPRETATION: A Paleolithic diet reduced liver fat and IMCL content, while there was a tissue-specific heterogeneous response to added exercise training. TRIAL REGISTRATION: ClinicalTrials.gov NCT01513798 FUNDING: Swedish Diabetes Research Foundation, County Council of Västerbotten, Swedish Heart and Lung Foundation, King Gustav V and Queen Victoria's Foundation.

Benefits of a Paleolithic diet with and without supervised exercise on fat mass, insulin sensitivity, and glycemic control: a randomized controlled trial in individuals with type 2 diabetes.

BACKGROUND: Means to reduce future risk for cardiovascular disease in subjects with type 2 diabetes are urgently needed. METHODS: Thirty-two patients with type 2 diabetes (age 59 ± 8 years) followed a Paleolithic diet for 12 weeks. Participants were randomized to either standard care exercise recommendations (PD) or 1-h supervised exercise sessions (aerobic exercise and resistance training) three times per week (PD-EX). RESULTS: For the within group analyses, fat mass decreased by 5.7 kg (IQR: -6.6, -4.1; p < 0.001) in the PD group and by 6.7 kg (-8.2, -5.3; p < 0.001) in the PD-EX group. Insulin sensitivity (HOMA-IR) improved by 45% in the PD (p < 0.001) and PD-EX (p < 0.001) groups. HbA1c decreased by 0.9% (-1.2, -0.6; p < 0.001) in the PD group and 1.1% (-1.7, -0.7; p < 0.01) in the PD-EX group. Leptin decreased by 62% (p < 0.001) in the PD group and 42% (p < 0.001) in the PD-EX group. Maximum oxygen uptake increased by 0.2 L/min (0.0, 0.3) in the PD-EX group, and remained unchanged in the PD group (p < 0.01 for the difference between intervention groups). Male participants decreased lean mass by 2.6 kg (-3.6, -1.3) in the PD group and by 1.2 kg (-1.3, 1.0) in the PD-EX group (p < 0.05 for the difference between intervention groups). CONCLUSIONS: A Paleolithic diet improves fat mass and metabolic balance including insulin sensitivity, glycemic control, and leptin in subjects with type 2 diabetes. Supervised exercise training may not enhance the effects on these outcomes, but preserves lean mass in men and increases cardiovascular fitness. Copyright © 2016 John Wiley & Sons, Ltd.

Vitamin D in relation to bone health and muscle function in young female soccer players.

The present work investigated serum vitamin D (25(OH)D) status in relation to bone and muscle qualities and functions in 19 female soccer players (13-16 years) resident at northern latitude with very low sun exposure (∼32-36 h/month) during winter season (late January to early March). Serum 25(OH)D, parathyroid hormone and bone turnover markers osteocalcin (OC) and beta carboxy-terminal collagen cross-links (ß-Ctx), as well as body composition and muscle performance were examined. Hormones were tested using routine laboratory methods. Fat mass, lean mass, and bone mineral density in whole body, as well as femur and lumbar spine were evaluated with dual-energy X-ray absorptiometry. Muscle performance was assessed through isokinetic knee extension and flexion, countermovement jump, and sprint running. 25(OH)D was low (50.5 ± 12.8 nmol l-1), whereas the values of bone turnover markers were markedly high (OC: 59.4 ± 18.6 µg l-1; ß-Ctx: 1075 ± 408 ng l-1). All bone and muscle measurements were normal or above normal. 25(OH)D was not significantly correlated with most of the parameters of bone and muscle quality or function, except the knee extension time to peak torque (r = -0.50, p = .03). In conclusion, the level of vitamin D is markedly low in adolescent female soccer players during the winter in Sweden. However, vitamin D levels did not significantly correlate with measures of bone and muscle except a moderate correlation in time to peak torque in the knee extensors. The practical implication of low vitamin D levels in young growing female athletes remains unclear.

Gene expression and fiber type variations in repeated vastus lateralis biopsies.

INTRODUCTION: Muscle sample collection can introduce variation in any measured variable due to inter- and intramuscle variation. We investigated the variation in gene expression and fiber type composition after repeated biopsy sampling from the vastus lateralis muscle. METHODS: Six subjects donated 3 tissue samples each. One hour after baseline sampling from 1 vastus lateralis muscle, samples from both vastus lateralis muscles were obtained. RESULTS: The fiber type composition differed between biopsies taken from the same leg. There were no within-subject differences in gene expression between the 3 biopsies. Multivariate analysis supports a model in which gene expression differs significantly between individuals but is not affected by repeated muscle biopsy sampling from the same subject. CONCLUSION: One vastus lateralis muscle sample per subject is sufficient to establish a reliable baseline for comparing gene expression representing selected pathways over time within the same individual.

Life style intervention in moderately overweight individuals is associated with decreased levels of cathepsins L and S in plasma.

BACKGROUND: Adipose tissue cells produce cathepsins L and S, which have proatherogenic effects. Obesity is strongly linked to atherogenesis, cardiovascular morbidity, and mortality. OBJECTIVE: The aim of the present study was to see if life style interventions/weight reduction could decrease cathepsin L and S levels in blood plasma. METHOD: Study subjects (n=31) were recruited to a life style intervention program aiming at increased physical activity, more healthy eating habits, and weight reduction for most of the participants. Blood samples were collected at inclusion and after 4 and 8 weeks. RESULTS: Cathepsin L was significantly reduced at 4 weeks (p<0.0001) and 8 weeks (p=0.0004). A similar reduction was also seen for cathepsin S at 4 weeks (p=0.03) and 8 weeks (p=0.008). No significant change in fractalkine values was observed at 4 weeks (p=0.58), but a significant increase was apparent at 8 weeks (p=0.0002). CONCLUSION: The intervention program resulted in significant reductions of cathepsin L and S levels in plasma after 4 and 8 weeks of intervention.

Metabolic adaptations in skeletal muscle, adipose tissue, and whole-body oxidative capacity in response to resistance training.

PURPOSE: The effects of resistance training on mitochondrial biogenesis and oxidative capacity in skeletal muscle are not fully characterized, and even less is known about alterations in adipose tissue. We aimed to investigate adaptations in oxidative metabolism in skeletal muscle and adipose tissue after 8 weeks of heavy resistance training in apparently healthy young men. METHODS: Expression of genes linked to oxidative metabolism in the skeletal muscle and adipose tissue was assessed before and after the training program. Body composition, peak oxygen uptake (VO2 peak), fat oxidation, activity of mitochondrial enzyme in muscle, and serum adiponectin levels were also determined before and after resistance training. RESULTS: In muscle, the expression of the genes AdipoR1 and COX4 increased after resistance training (9 and 13 %, respectively), whereas the expression levels of the genes PGC-1α, SIRT1, TFAM, CPT1b, and FNDC5 did not change. In adipose tissue, the expression of the genes SIRT1 and CPT1b decreased after training (20 and 23 %, respectively). There was an increase in lean mass (from 59.7 ± 6.1 to 61.9 ± 6.2 kg), VO2 peak (from 49.7 ± 5.5 to 56.3 ± 5.0 ml/kg/min), and fat oxidation (from 6.8 ± 2.1 to 9.1 ± 2.7 mg/kg fat-free mass/min) after training, whereas serum adiponectin levels decreased significantly and enzyme activity of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase did not change. CONCLUSION: Despite significant increases in VO2 peak, fat oxidation, and lean mass following resistance training, the total effect on gene expression and enzyme activity linked to oxidative metabolism was moderate.

Physical fitness level is reflected by alterations in the human plasma metabolome.

An excessive energy intake combined with a low level of physical activity induces detrimental processes involved in disease development, e.g. type 2 diabetes and cardiovascular disease. However the underlying mechanisms for regulation of metabolic capacity and fitness status remain unclear. Metabolomics involves global studies of the metabolic reactions in an organism or cell. Thus hypotheses regarding biochemical events can be generated to increase the understanding of disease development and thereby aid in the development of novel treatments or preventions. We present the first standardized intervention study focusing on characterizing the human metabolome in relation to moderate differences in cardiorespiratory fitness. Gas chromatography-time of flight/mass spectrometry (GC-TOF/MS) was used to characterize 460 plasma samples from 27 individuals divided into two groups based on physical fitness level (VO(2)max). Multi- and univariate between group comparisons based on 197 metabolites were carried out in samples collected at rest prior to any intervention, over time following a nutritional load or a standardized exercise scheme, with and without nutritional load. We detected decreased levels of gamma-tocopherol (GT), a vitamin E isomer, in response to a high fitness level, whereas the opposite was seen for the alpha isomer (AT). In addition, the high fitness level was associated with elevated ω3-PUFA (DHA, 22 : 6ω3) and a decrease in ω6-PUFA (18 : 2ω6) as well as in saturated (16 : 0, 18 : 0), monounsaturated (18 : 1) and trans (16 : 1) fatty acids. We thus hypothesize that high fitness status induces an increased cardiorespiratory inflammatory and antioxidant defense system, more prone to deal with the inflammatory response following exercise and nutrition intake.

Validated and predictive processing of gas chromatography-mass spectrometry based metabolomics data for large scale screening studies, diagnostics and metabolite pattern verification.

The suggested approach makes it feasible to screen large metabolomics data, sample sets with retained data quality or to retrieve significant metabolic information from small sample sets that can be verified over multiple studies. Hierarchical multivariate curve resolution (H-MCR), followed by orthogonal partial least squares discriminant analysis (OPLS-DA) was used for processing and classification of gas chromatography/time of flight mass spectrometry (GC/TOFMS) data characterizing human serum samples collected in a study of strenuous physical exercise. The efficiency of predictive H-MCR processing of representative sample subsets, selected by chemometric approaches, for generating high quality data was proven. Extensive model validation by means of cross-validation and external predictions verified the robustness of the extracted metabolite patterns in the data. Comparisons of extracted metabolite patterns between models emphasized the reliability of the methodology in a biological information context. Furthermore, the high predictive power in longitudinal data provided proof for the potential use in clinical diagnosis. Finally, the predictive metabolite pattern was interpreted physiologically, highlighting the biological relevance of the diagnostic pattern.

Predictive metabolomics evaluation of nutrition-modulated metabolic stress responses in human blood serum during the early recovery phase of strenuous physical exercise.

We have investigated whether postexercise ingestion of carbohydrates in combination with proteins generates a different systemic metabolic response, as compared to the sole ingestion of carbohydrate or water, in the early recovery phase following exercise. In addition, metabolic patterns related to fitness level were studied together with individual responses to nutritional modulation. Twenty-four male subjects were exposed to 90 min of ergometer-cycling. Each participant was subject to four identical test-sessions, including ingestion of one of four beverages (water, low-carbohydrate beverage, high-carbohydrate beverage, and low-carbohydrate-protein beverage (LCHO-P)) immediately after cycling. Blood was collected at six time points, one pre- and five postexercise. Extracted blood serum was subject to metabolomic characterization by gas chromatography/time-of-flight mass spectrometry (GC-TOF MS). Data was processed using hierarchical multivariate curve resolution (HMCR), and multivariate statistical analysis was carried out using orthogonal partial least-squares (OPLS). Predictive metabolomics, including predictive HMCR and OPLS classification, was applied to ensure efficient sample processing and validation of detected metabolic patterns. Separation of subjects in relation to ingested beverage was detected and interpreted. Pseudouridine was suggested as a novel marker for pro-anabolic effect following LCHO-P ingestion, which was supported by the detected decrease of the catabolic marker 3-methylhistidine. Separation of subjects according to fitness level was achieved, and nutritional modulation by LCHO-P was shown to improve the metabolic status of less fit subjects in the recovery phase. In addition, the potential of the methodology for detection of early signs of insulin resistance was also demonstrated.

Evaluation of 2-D DIGE for skeletal muscle: protocol and repeatability.

Proteomic analysis has the potential to yield vast amounts of data. The available proteomic methods have been hampered by methodological errors in quantification due to large gel-to-gel variations. The inclusion of an internal standard greatly reduces this variation, and therefore the purpose of this investigation was: 1) to develop a sample preparation protocol for human skeletal muscle for two-dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one particular system, the Ettan DIGE. To test repeatability, nine aliquots from the same homogenate were labelled with three different CyDye(trade mark) dyes (Cy2, Cy3, Cy5). Samples were run on 18 x 24 cm gels, scanned with a Typhoon 9410 laser scanner and analysed in the DeCyder software. When selecting spots appearing only in triplicate (n = 1314), the mean error was 1.7 % (SD: 10.5 %; 95 % CI: 1.1-2.4 %). When setting the significance level to 99 %, no false-positive changes in protein volume ratios were detected. In the protocol presented here, only 0.5 mg tissue was used and separation of >2500 distinct protein spots in the pH range 3-11 and MW 10-200 kDa. Changes in protein abundance of <20 % could be detected. The method is especially useful when comparing muscle proteins between different conditions; for example, healthy and diseased tissue, before and after treatment or different exercise protocols.

A multivariate screening strategy for investigating metabolic effects of strenuous physical exercise in human serum.

A novel hypothesis-free multivariate screening methodology for the study of human exercise metabolism in blood serum is presented. Serum gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) data was processed using hierarchical multivariate curve resolution (H-MCR), and orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to model the systematic variation related to the acute effect of strenuous exercise. Potential metabolic biomarkers were identified using data base comparisons. Extensive validation was carried out including predictive H-MCR, 7-fold full cross-validation, and predictions for the OPLS-DA model, variable permutation for highlighting interesting metabolites, and pairwise t tests for examining the significance of metabolites. The concentration changes of potential biomarkers were verified in the raw GC/TOFMS data. In total, 420 potential metabolites were resolved in the serum samples. On the basis of the relative concentrations of the 420 resolved metabolites, a valid multivariate model for the difference between pre- and post-exercise subjects was obtained. A total of 34 metabolites were highlighted as potential biomarkers, all statistically significant (p < 8.1E-05). As an example, two potential markers were identified as glycerol and asparagine. The concentration changes for these two metabolites were also verified in the raw GC/TOFMS data. The strategy was shown to facilitate interpretation and validation of metabolic interactions in human serum as well as revealing the identity of potential markers for known or novel mechanisms of human exercise physiology. The multivariate way of addressing metabolism studies can help to increase the understanding of the integrative biology behind, as well as unravel new mechanistic explanations in relation to, exercise physiology.