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Muscle-aging drives sarcopenia and is a major public health issue. Mice are frequently used as a model for human muscle-aging, however, research investigating their translational value is limited. In addition, mechanisms underlying muscle-aging may have sex-specific features in humans, but it is not yet assessed whether these are recapitulated in mice. Here, we studied the effects of aging on a functional, histological and transcriptional level at multiple timepoints in male and female mice (4, 17, 21 and 25 months), with particular emphasis on sex-differences. The effects of natural aging on the transcriptome of quadriceps muscle were compared to humans on pathway level. Significant loss of muscle mass occurred late, at 25 months, in both male (-17%, quadriceps) and female mice (-10%, quadriceps) compared to young control mice. Concomitantly, we found in female, but not male mice, a slower movement speed in the aged groups compared to the young mice (P < 0.001). Consistently, weighted gene co-expression network analysis revealed a stronger association between the aging-related reduction of movement and aging-related changes in muscle transcriptome of female compared to male mice (P < 0.001). In male, but not female mice, major distinctive aging-related changes occurred in the last age group (25 months), which highlights the necessity for careful selection of age using mice as a muscle-aging model. Furthermore, contrasting to humans, more aging-related changes were found in the muscle transcriptome of male mice compared to female mice (4090 vs. 2285 differentially expressed genes at 25 months, respectively). Subsequently, male mice recapitulated more muscle-aging related pathways characteristic for both male and female humans. In conclusion, our data show that sex has a critical effect on the mouse muscle-aging trajectory, although these do not necessarily reflect sex differences observed in the human muscle-aging trajectory.

The prevalence of sarcopenia is increasing while it is often challenging, expensive and time-consuming to test the effectiveness of interventions against sarcopenia. Translational mouse models that adequately mimic underlying physiological pathways could accelerate research but are scarce. Here, we investigated the translational value of three potential mouse models for sarcopenia, namely partial immobilized (to mimic sedentary lifestyle), caloric restricted (CR; to mimic malnutrition) and a combination (immobilized & CR) model. C57BL/6J mice were calorically restricted (-40%) and/or one hindleg was immobilized for two weeks to induce loss of muscle mass and function. Muscle parameters were compared to those of young control (4 months) and old reference mice (21 months). Transcriptome analysis of quadriceps muscle was performed to identify underlying pathways and were compared with those being expressed in aged human vastus lateralis muscle-biopsies using a meta-analysis of five different human studies. Caloric restriction induced overall loss of lean body mass (-15%, p<0.001), whereas immobilization decreased muscle strength (-28%, p<0.001) and muscle mass of hindleg muscles specifically (on average -25%, p<0.001). The proportion of slow myofibers increased with aging in mice (+5%, p<0.05), and this was not recapitulated by the CR and/or immobilization models. The diameter of fast myofibers decreased with aging (-7%, p<0.05), and this was mimicked by all models. Transcriptome analysis revealed that the combination of CR and immobilization recapitulated more pathways characteristic for human muscle-aging (73%) than naturally aged (21 months old) mice (45%). In conclusion, the combination model exhibits loss of both muscle mass (due to CR) and function (due to immobilization) and has a remarkable similarity with pathways underlying human sarcopenia. These findings underline that external factors such as sedentary behavior and malnutrition are key elements of a transl

Background: Physical weakness is a key component of frailty, and is highly prevalent in older adults. While females have a higher prevalence and earlier onset, sex differences in the development of frailty-related physical weakness are hardly studied. Therefore, we investigated the intramuscular changes that differentiate between fit and weak older adults for each sex separately. Methods: Male (n = 28) and female (n = 26) older adults (75 + years) were grouped on the basis of their ranks according to three frailty-related physical performance criteria. Muscle biopsies taken from vastus lateralis muscle were used for transcriptome and histological examination. Pairwise comparisons were made between the fittest and weakest groups for each sex separately, and potential sex-specific effects were assessed. Results: Weak females were characterized by a higher expression of inflammatory pathways and infiltration of NOX2-expressing immune cells, concomitant with a higher VCAM1 expression. Weak males were characterized by a smaller diameter of type 2 (fast) myofibers and lower expression of PRKN. In addition, weakness-associated transcriptome changes in the muscle were distinct from aging, suggesting that the pathophysiology of frailty-associated physical weakness does not necessarily depend on aging. Conclusions: We conclude that physical weakness-associated changes in muscle are sex-specific and recommend that sex differences are taken into account in research on frailty, as these differences may have a large impact on the development of (pharmaceutical) interventions against frailty. Trial registration number: The FITAAL study was registered in the Dutch Trial Register, with registration code NTR6124 on 14-11-2016 (https://trialsearch.who.int/Trial2.aspx?TrialID=NTR6124). Highlights: • In female, but not male older adults, physical weakness was associated with a higher expression of intramuscular markers for inflammation. • In male, but not female older adults, physical w

Biomarkers are important in the assessment of health and disease, but are poorly studied in still healthy individuals with a (potential) different risk for metabolic disease. This study investigated, first, how single biomarkers and metabolic parameters, functional biomarker and metabolic parameter categories, and total biomarker and metabolic parameter profiles behave in young healthy female adults of different aerobic fitness and, second, how these biomarkers and metabolic parameters are affected by recent exercise in these healthy individuals. A total of 102 biomarkers and metabolic parameters were analysed in serum or plasma samples from 30 young, healthy, female adults divided into a high-fit (V̇O2peak ≥ 47 mL/kg/min, N = 15) and a low-fit (V̇O2peak ≤ 37 mL/kg/min, N = 15) group, at baseline and overnight after a single bout of exercise (60 min, 70% V̇O2peak). Our results show that total biomarker and metabolic parameter profiles were similar between high-fit and low-fit females. Recent exercise significantly affected several single biomarkers and metabolic parameters, mostly related to inflammation and lipid metabolism. Furthermore, functional biomarker and metabolic parameter categories corresponded to biomarker and metabolic parameter clusters generated via hierarchical clustering models. In conclusion, this study provides insight into the single and joined behavior of circulating biomarkers and metabolic parameters in healthy females, and identified functional biomarker and metabolic parameter categories that may be used for the characterisation of human health physiology.

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) can possibly serve as a cellular metabolic read-out for lifestyle factors and lifestyle interventions. However, the impact of PBMC composition on PBMC metabolism is not yet clear, neither is the differential impact of a longer-term lifestyle factor versus a short-term lifestyle intervention. We investigated the effect of aerobic fitness level and a recent exercise bout on PBMC metabolism in females. PBMCs from 31 young female adults divided into a high-fit (V˙O2peak ≥ 47 mL/kg/min, n = 15) and low-fit (V˙O2peak ≤ 37 mL/kg/min, n = 16) groups were isolated at baseline and overnight after a single bout of exercise (60 min, 70% V˙O2peak). Oxygen consumption rate (OCR) and glycolytic rate (GR) were measured using extracellular flux (XF) assays and PBMC subsets were characterized using fluorescence-activated cell sorting (FACS). Basal OCR, FCCP-induced OCR, spare respiratory capacity, ATP-linked OCR, and proton leak were significantly higher in high-fit than in low-fit females (all P < 0.01), whereas no significant differences in glycolytic rate (GR) were found (all P > 0.05). A recent exercise bout did not significantly affect GR or OCR parameters (all P > 0.05). The overall PBMC composition was similar between high-fit and low-fit females. Mitochondrial PBMC function was significantly higher in PBMCs from high-fit than from lowfit females, which was unrelated to PBMC composition and not impacted by a recent bout of exercise. Our study reveals a link between PBMC metabolism and levels of aerobic fitness, increasing the relevance of PBMC metabolism as a marker to study the impact of lifestyle factors on human health.

Protein acylation via metabolic acyl-CoA intermediates provides a link between cellular metabolism and protein functionality. A process in which acetyl-CoA and acetylation are fine-tuned is during myogenic differentiation. However, the roles of other protein acylations remain unknown. Protein propionylation could be functionally relevant because propionyl-CoA can be derived from the catabolism of amino acids and fatty acids and was shown to decrease during muscle differentiation. We aimed to explore the potential role of protein propionylation in muscle differentiation, by mimicking a pathophysiological situation with high extracellular propionate which increases propionyl-CoA and protein propionylation, rendering it a model to study increased protein propionylation. Exposure to extracellular propionate, but not acetate, impaired myogenic differentiation in C2C12 cells and propionate exposure impaired myogenic differentiation in primary human muscle cells. Impaired differentiation was accompanied by an increase in histone propionylation as well as histone acetylation. Furthermore, chromatin immunoprecipitation showed increased histone propionylation at specific regulatory myogenic differentiation sites of the Myod gene. Intramuscular propionylcarnitine levels are higher in old compared to young males and females, possibly indicating increased propionyl-CoA levels with age. The findings suggest a role for propionylation and propionyl-CoA in regulation of muscle cell differentiation and ageing, possibly via alterations in histone acylation.

Scope: Infant formula (IF) uses besides vegetable fats also bovine milk fat, which differs in triacylglycerol (TAG) structure. Furthermore, it differs in fatty acid (FA) composition. Whether changing fat source in IF affects postprandial energy metabolism, lipemic response, and blood lipid profile is unknown. Methods and Results: A proof-of-principle study, with a randomized controlled double-blind cross-over design, is conducted. Twenty healthy male adults consumed drinks with either 100% vegetable fat (VEG) or 67% bovine milk fat and 33% vegetable fat (BOV), on 2 separate days. For a detailed insight in the postprandial responses, indirect calorimetry is performed continuously, and venous blood samples are taken every 30 min, until 5 h postprandially. No differences in postprandial energy metabolism, serum lipids, lipoprotein, or chylomicron concentrations are observed between drinks. After consumption of VEG-drink, C18:2n-6 in serum increased. Observed differences in chylomicron FA profile reflect differences in initial FA profile of test drinks. Serum ketone bodies concentrations increase following consumption of BOV-drink. Conclusions: The use of bovine milk fat in IF does neither affect postprandial energy metabolism nor lipemic response in healthy adults, but alters postprandial FA profiles and ketone metabolism. Whether the exact same effects occur in infants requires experimental verification.

The recovery of muscle oxygen consumption (m (Formula presented.) O2) after exercise measured using near-infrared spectroscopy (NIRS) provides a measure of skeletal muscle mitochondrial capacity. Nevertheless, due to sex differences in factors that can influence scattering and thus penetration depth of the NIRS signal in the tissue, e.g., subcutaneous adipose tissue thickness and intramuscular myoglobin and hemoglobin, it is unknown whether results in males can be extrapolated to a female population. Therefore, the aim of this study was to measure skeletal muscle mitochondrial capacity in females at different levels of aerobic fitness to test whether NIRS can measure relevant differences in mitochondrial capacity. Mitochondrial capacity was analyzed in the gastrocnemius muscle and the wrist flexors of 32 young female adults, equally divided in relatively high ((Formula presented.) O2peak ≥ 47 ml/kg/min) and relatively low aerobic fitness group ((Formula presented.) O2peak ≤ 37 ml/kg/min). m (Formula presented.) O2 recovery was significantly faster in the high- compared to the low-fitness group in the gastrocnemius, but not in the wrist flexors (p = 0.009 and p = 0.0528, respectively). Furthermore, (Formula presented.) O2peak was significantly correlated to m (Formula presented.) O2 recovery in both gastrocnemius (R2 = 0.27, p = 0.0051) and wrist flexors (R2 = 0.13, p = 0.0393). In conclusion, NIRS measurements can be used to assess differences in mitochondrial capacity within a female population and is correlated to (Formula presented.) O2peak. This further supports NIRS assessment of muscle mitochondrial capacity providing additional evidence for NIRS as a promising approach to monitor mitochondrial capacity, also in an exclusively female population.

Background: Due to the interaction between skeletal muscle ageing and lifestyle factors, it is often challenging to attribute the decline in muscle mass and quality to either changes in lifestyle or to advancing age itself. Because many of the physiological factors affecting muscle mass and quality are modulated by physical activity and physical activity declines with age, the aim of this study is to better understand the effects of early ageing on muscle function by comparing a population of healthy older and young males with similar physical activity patterns. Methods: Eighteen older (69 ± 2.0 years) and 20 young (22 ± 2.0 years) males were recruited based on similar self-reported physical activity, which was verified using accelerometry measurements. Gene expression profiles of vastus lateralis biopsies obtained by RNA sequencing were compared, and key results were validated using quantitative polymerase chain reaction and western blot. Results: Total physical activity energy expenditure was similar between the young and old group (404 ± 215 vs. 411 ± 189 kcal/day, P = 0.11). Three thousand seven hundred ninety-seven differentially expressed coding genes (DEGs) were identified (adjusted P-value cut-off of <0.05), of which 1891 were higher and 1906 were lower expressed in the older muscle. The matrisome, innervation and inflammation were the main upregulated processes, and oxidative metabolism was the main downregulated process in old compared with young muscle. Lower protein levels of mitochondrial transcription factor A (TFAM, P = 0.030) and mitochondrial respiratory Complexes IV and II (P = 0.011 and P = 0.0009, respectively) were observed, whereas a trend was observed for Complex I (P = 0.062), in older compared with young muscle. Protein expression of Complexes I and IV was significantly correlated to mitochondrial capacity in the vastus lateralis as measured in vivo (P = 0.017, R2 = 0.42 and P = 0.030, R2 = 0.36). A trend for higher muscle-specific receptor

Post-translational protein modifications derived from metabolic intermediates, such as acyl-CoAs, have been shown to regulate mitochondrial function. Patients with a genetic defect in the propionyl-CoA carboxylase (PCC) gene clinically present symptoms related to mitochondrial disorders and are characterised by decreased mitochondrial respiration. Since propionyl-CoA accumulates in PCC deficient patients and protein propionylation can be driven by the level of propionyl-CoA, we hypothesised that protein propionylation could play a role in the pathology of the disease. Indeed, we identified increased protein propionylation due to pathologic propionyl-CoA accumulation in patient-derived fibroblasts and this was accompanied by defective mitochondrial respiration, as was shown by a decrease in complex I-driven respiration. To mimic pathological protein propionylation levels, we exposed cultured fibroblasts, Fao liver cells and C2C12 muscle myotubes to propionate levels that are typically found in these patients. This induced a global increase in protein propionylation and histone protein propionylation and was also accompanied by a decrease in mitochondrial respiration in liver and fibroblasts. However, in C2C12 myotubes propionate exposure did not decrease mitochondrial respiration, possibly due to differences in propionyl-CoA metabolism as compared to the liver. Therefore, protein propionylation could contribute to the pathology in these patients, especially in the liver, and could therefore be an interesting target to pursue in the treatment of this metabolic disease.

Background: A decreased intramuscular carnitine status with aging may contribute to frailty and fatigue, but studies are often confounded by sex-heterogeneity and lack of control groups. We hypothesize that muscle carnitine deficiency is associated with (pre-)frailty, diminished physical performance and altered mitochondrial function, possibly in a sex-dependent manner. This was tested in well-matched age groups, allowing gender-specific analyses. Methods: A cross-sectional study was performed in well-matched fit (n = 15) and (pre-)frail (n = 13) old males as well as in fit (n = 15) and (pre-)frail (n = 11) old females, using young males (n = 13) and females (n = 13) as healthy controls. In the elderly, frailty was assessed according to the Fried criteria and physical performance was determined by 400-m walk test, short physical performance battery and handgrip strength. Acylcarnitine status was assessed in muscle biopsies and blood plasma using liquid chromatography-tandem mass spectrometry. Muscle mitochondrial gene expression was analyzed. Results: Intramuscular total carnitine levels and short chain acylcarnitine levels were lower in (pre-)frail old females compared to fit old females and young females, whereas no differences were observed in males. Intramuscular short chain acylcarnitine levels were associated with low physical performance in females, even after correction for muscle mass (%), whereas in males no such association was found. The decline in short chain acylcarnitine levels in (pre-)frail old females was associated with low expression of genes involved in mitochondrial energy production and mitochondrial functionality. Conclusions: In (pre-)frail old females, intramuscular total carnitine levels and short chain acylcarnitine levels are decreased and this decrease is accompanied by reduced physical performance and low expression of a wide range of genes involved in mitochondrial function. Overall design: A cross-sectional study was performe, Background: A decreased intramuscular carnitine status with aging may contribute to frailty and fatigue, but studies are often confounded by sex-heterogeneity and lack of control groups. We hypothesize that muscle carnitine deficiency is associated with (pre-)frailty, diminished physical performance and altered mitochondrial function, possibly in a sex-dependent manner. This was tested in well-matched age groups, allowing gender-specific analyses. Methods: A cross-sectional study was performed in well-matched fit (n = 15) and (pre-)frail (n = 13) old males as well as in fit (n = 15) and (pre-)frail (n = 11) old females, using young males (n = 13) and females (n = 13) as healthy controls. In the elderly, frailty was assessed according to the Fried criteria and physical performance was determined by 400-m walk test, short physical performance battery and handgrip strength. Acylcarnitine status was assessed in muscle biopsies and blood plasma using liquid chromatography-tandem mass spectrometry. Muscle mitochondrial gene expression was analyzed. Results: Intramuscular total carnitine levels and short chain acylcarnitine levels were lower in (pre-)frail old females compared to fit old females and young females, whereas no differences were observed in males. Intramuscular short chain acylcarnitine levels were associated with low physical performance in females, even after correction for muscle mass (%), whereas in males no such association was found. The decline in short chain acylcarnitine levels in (pre-)frail old females was associated with low expression of genes involved in mitochondrial energy production and mitochondrial functionality. Conclusions: In (pre-)frail old females, intramuscular total carnitine levels and short chain acylcarnitine levels are decreased and this decrease is accompanied by reduced physical performance and low expression of a wide range of genes involved in mitochondrial function. Overall design: A cross-sectional study was performe

Fructose consumption has been linked to obesity and increased hepatic de novo lipogenesis (DNL). Excessive caloric intake often confounds the results of fructose studies, and experimental diets are generally low-fat diets, not representative for westernized diets. Here, we compared the effects of dietary fructose with those of dietary glucose, in adult male and female mice on a starch-containing moderate high-fat (HF) diet. After 5 weeks fattening on a HF high-glucose (HF-G) diet, mice were stratified per sex and assigned to one of the three intervention diets for 6 weeks: HF high fructose (HF-F), HF with equimolar glucose and fructose (HF-GF), or HF-G. Bodyweight (BW) and food intake were measured weekly. Indirect calorimetry was performed on week 5; animals were sacrificed in food-deprived state on week 6. Data were analyzed within sex. BW gain was similar among animals on the HF-G, HF-GF, and HF-F diets. Cumulative food intake was slightly lower in HF-F animals (both sexes). However, energy expenditure was not affected, or were circulating insulin and glucose concentrations, and hepatic triglyceride levels at endpoint. Hepatic gene expression analysis showed only minor alterations in hexokinase and glycolysis-related expression in males, and no alterations in sugar transporters, or DNL-related enzymes. In females, no consistent alterations in hepatic or small intestine gene expression were seen. Concluding, partial or complete replacement of dietary glucose with fructose does not increase caloric intake, and does not affect BW, hepatic triglyceride levels, or insulin concentrations in male and female mice on a moderate high-fat diet.

This study tested the hypothesis that in human aging, a decreased intramuscular acylcarnitine status is associated with (pre-)frailty, reduced physical performance, and altered mitochondrial function. We used a cross-sectional study design with well-matched fit and (pre-)frail old males and females, using young males and females as healthy controls. Frailty was assessed according to the Fried criteria and physical performance was determined by 400 m walk test, short physical performance battery and handgrip strength. Muscle and plasma acylcarnitine status, and muscle mitochondrial gene expression was analyzed. Results showed that intramuscular total carnitine levels and short-chain acylcarnitine levels were lower in (pre-)frail old females compared to fit old females and young females, whereas no differences were observed in males. The low intramuscular short-chain acylcarnitine levels in females correlated with low physical performance, even after correction for muscle mass (%), and were accompanied with lowered expression of genes involved in mitochondrial energy production and functionality. It is, therefore, concluded that in (pre-)frail old females, intramuscular total carnitine levels and short-chain acylcarnitine levels are decreased, and this decrease is associated with reduced physical performance and low expression of a wide range of genes critical for mitochondrial function. The results stress the importance of taking sex differences into account in aging research.