SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology.

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.

Functional and molecular profiling of fasted piglets reveals decreased energy metabolic function and cell proliferation in the small intestine.

The small intestine requires energy to exert its important role in nutrient uptake and barrier function. Pigs are an important source of food and a model for humans. Young piglets and infants can suffer from periods of insufficient food intake. Whether this functionally affects the small intestinal epithelial cell (IEC) metabolic capacity and how this may be associated with an increased vulnerability to intestinal disease is unknown. We therefore performed a 48-h fasting intervention in young piglets. After feeding a standard weaning diet for 2 wk, 6-wk-old piglets (n = 16/group) were fasted for 48 h, and midjejunal IECs were collected upon euthanasia. Functional metabolism of isolated IECs was analyzed with the Seahorse XF analyzer and gene expression was assessed using RNA-sequencing. Fasting decreased the mitochondrial and glycolytic function of the IECs by 50% and 45%, respectively (P < 0.0001), signifying that overall metabolic function was decreased. The RNA-sequencing results corroborated our functional metabolic measurements, showing that particularly pathways related to mitochondrial energy production were decreased. Besides oxidative metabolic pathways, decreased cell-cycle progression pathways were most regulated in the fasted piglets, which were confirmed by 43% reduction of Ki67-stained cells (P < 0.05). Finally, the expression of barrier function genes was reduced upon fasting. In conclusion, we found that the decreased IEC energy metabolic function in response to fasting is supported by a decreased gene expression of mitochondrial pathways and is likely linked to the observed decreased intestinal cell proliferation and barrier function, providing insight into the vulnerability of piglets, and infants, to decreased food intake.NEW & NOTEWORTHY Fasting is identified as one of the underlying causes potentiating diarrhea development, both in piglets and humans. With this study, we demonstrate that fasting decreases the metabolism of intestinal epithelial cells, on a functional and transcriptional level. Transcriptional and histological data also show decreased intestinal cell proliferation. As such, fasting-induced intestinal energy shortage could contribute to intestinal dysfunction upon fasting.

Single and Joined Behaviour of Circulating Biomarkers and Metabolic Parameters in High-Fit and Low-Fit Healthy Females.

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.

FOXO3A-short is a novel regulator of non-oxidative glucose metabolism associated with human longevity.

Intronic single-nucleotide polymorphisms (SNPs) in FOXO3A are associated with human longevity. Currently, it is unclear how these SNPs alter FOXO3A functionality and human physiology, thereby influencing lifespan. Here, we identify a primate-specific FOXO3A transcriptional isoform, FOXO3A-Short (FOXO3A-S), encoding a major longevity-associated SNP, rs9400239 (C or T), within its 5' untranslated region. The FOXO3A-S mRNA is highly expressed in the skeletal muscle and has very limited expression in other tissues. We find that the rs9400239 variant influences the stability and functionality of the primarily nuclear protein(s) encoded by the FOXO3A-S mRNA. Assessment of the relationship between the FOXO3A-S polymorphism and peripheral glucose clearance during insulin infusion (Rd clamp) in a cohort of Danish twins revealed that longevity T-allele carriers have markedly faster peripheral glucose clearance rates than normal lifespan C-allele carriers. In vitro experiments in human myotube cultures utilizing overexpression of each allele showed that the C-allele represses glycolysis independently of PI3K signaling, while overexpression of the T-allele represses glycolysis only in a PI3K-inactive background. Supporting this finding inducible knockdown of the FOXO3A-S C-allele in cultured myotubes increases the glycolytic rate. We conclude that the rs9400239 polymorphism acts as a molecular switch which changes the identity of the FOXO3A-S-derived protein(s), which in turn alters the relationship between FOXO3A-S and insulin/PI3K signaling and glycolytic flux in the skeletal muscle. This critical difference endows carriers of the FOXO3A-S T-allele with consistently higher insulin-stimulated peripheral glucose clearance rates, which may contribute to their longer and healthier lifespans.

Mastering the game of Stratego with model-free multiagent reinforcement learning.

We introduce DeepNash, an autonomous agent that plays the imperfect information game Stratego at a human expert level. Stratego is one of the few iconic board games that artificial intelligence (AI) has not yet mastered. It is a game characterized by a twin challenge: It requires long-term strategic thinking as in chess, but it also requires dealing with imperfect information as in poker. The technique underpinning DeepNash uses a game-theoretic, model-free deep reinforcement learning method, without search, that learns to master Stratego through self-play from scratch. DeepNash beat existing state-of-the-art AI methods in Stratego and achieved a year-to-date (2022) and all-time top-three ranking on the Gravon games platform, competing with human expert players.

Extracellular flux analyses reveal differences in mitochondrial PBMC metabolism between high-fit and low-fit females.

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 (V̇o2peak ≥ 47 mL/kg/min, n = 15) and low-fit (V̇o2peak ≤ 37 mL/kg/min, n = 16) groups were isolated at baseline and overnight after a single bout of exercise (60 min, 70% V̇o2peak). 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 low-fit 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.NEW & NOTEWORTHY Mitochondrial metabolism was significantly higher in PBMCs from high-fit than from low-fit females. This 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.

The female mouse is resistant to mild vitamin B3 deficiency.

PURPOSE: Vitamin B3 provides nicotinamide adenine dinucleotide (NAD+), an essential coenzyme in oxidoreductase reactions. Severe vitamin B3 deficiency leads to the disease Pellagra, while mild vitamin B3 deficiency has been linked to age-related and metabolic diseases. Mild vitamin B3 deficiency is understudied, especially in females. Therefore, we examined how female mice responded to a diet that induced mild vitamin B3 deficiency in male mice. METHODS: Female C57BL/6RccHsd mice were subjected for 18 weeks to a diet without vitamin B3 and low but sufficient tryptophan (0.115%) (0NR) and were compared to control female mice on the same diet with the reference dose of vitamin B3 (30NR, 30 mg nicotinamide riboside/ kg diet). RESULTS: In the female mice, no differences between the two dietary groups were found in liver nicotinamide mononucleotide (NMN) levels, body composition, whole body energy and substrate metabolism measured by indirect calorimetry, or liver triacylglycerol metabolism. Expression of seven genes that previously were shown to respond to mild vitamin B3 deficiency in male white adipose tissue were not differentially expressed between the female dietary groups, neither was insulin sensitivity. CONCLUSION: We concluded that the female 0NR mice were not vitamin B3 deficient; the role of age, sex and health status is discussed. Demonstrated by clear differences between females and males, the latter showing mild deficiency under the same conditions, this study highlights the importance of studying both sexes.

The Effect of a Single Bout of Exercise on Vitamin B2 Status Is Not Different between High- and Low-Fit Females.

High-fitness individuals have been suggested to be at risk of a poor vitamin B2 (riboflavin) status due to a potentially higher vitamin B2 demand, as measured by the erythrocyte glutathione reductase (EGR) activation coefficient (EGRAC). Longer-term exercise interventions have been shown to result in a lower vitamin B2 status, but studies are contradictory. Short-term exercise effects potentially contribute to discrepancies between studies but have only been tested in limited study populations. This study investigated if vitamin B2 status, measured by EGRAC, is affected by a single exercise bout in females who differ in fitness levels, and that represents long-term physical activity. At baseline and overnight after a 60-min cycling bout at 70% V·O2peak, EGR activity and EGRAC were measured in 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) group. A single exercise bout significantly increased EGR activity in high-fit and low-fit females (Ptime = 0.006). This response was not affected by fitness level (Ptime*group = 0.256). The effect of exercise on EGRAC was not significant (Ptime = 0.079) and not influenced by EGR activity. The exercise response of EGRAC was not significantly different between high-fit and low-fit females (Ptime*group = 0.141). Thus, a single exercise bout increased EGR activity, but did not affect EGRAC, indicating that vitamin B2 status was not affected. The exercise response on EGRAC and EGR did not differ between high-fit and low-fit females.

OCRbayes: A Bayesian hierarchical modeling framework for Seahorse extracellular flux oxygen consumption rate data analysis.

BACKGROUND: Mitochondrial dysfunction is involved in many complex diseases. Efficient and accurate evaluation of mitochondrial functionality is crucial for understanding pathology as well as facilitating novel therapeutic developments. As a popular platform, Seahorse extracellular flux (XF) analyzer is widely used for measuring mitochondrial oxygen consumption rate (OCR) in living cells. A hidden feature of Seahorse XF OCR data is that it has a complex data structure, caused by nesting and crossing between measurement cycles, wells and plates. Surprisingly, statistical analysis of Seahorse XF data has not received sufficient attention, and current methods completely ignore the complex data structure, impairing the robustness of statistical inference. RESULTS: To rigorously incorporate the complex structure into data analysis, here we developed a Bayesian hierarchical modeling framework, OCRbayes, and demonstrated its applicability based on analysis of published data sets. CONCLUSIONS: We showed that OCRbayes can analyze Seahorse XF OCR experimental data derived from either single or multiple plates. Moreover, OCRbayes has potential to be used for diagnosing patients with mitochondrial diseases.

Matrisome, innervation and oxidative metabolism affected in older compared with younger males with similar physical activity.

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 kinase (MUSK) protein levels in the older group was observed (P = 0.08). CONCLUSIONS: There are clear differences in the transcriptome signatures of the vastus lateralis muscle of healthy older and young males with similar physical activity levels, including significant differences at the protein level. By disentangling physical activity and ageing, we appoint early skeletal muscle ageing processes that occur despite similar physical activity. Improved understanding of these processes will be key to design targeted anti-ageing therapies.

Propionate hampers differentiation and modifies histone propionylation and acetylation in skeletal muscle cells.

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.

Muscle mitochondrial capacity in high- and low-fitness females using near-infrared spectroscopy.

The recovery of muscle oxygen consumption (m V˙ 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 ( V˙ O2 peak ≥ 47 ml/kg/min) and relatively low aerobic fitness group ( V˙ O2 peak ≤ 37 ml/kg/min). m V˙ 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, V˙ O2 peak was significantly correlated to m V˙ 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 V˙ O2 peak. 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.

Novel standardized method for extracellular flux analysis of oxidative and glycolytic metabolism in peripheral blood mononuclear cells.

Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.

Increased protein propionylation contributes to mitochondrial dysfunction in liver cells and fibroblasts, but not in myotubes.

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.

An optimized desuccinylase activity assay reveals a difference in desuccinylation activity between proliferative and differentiated cells.

Succinylation is a novel post-translational modification identified on many proteins and is involved in multiple biological processes. Succinylation levels are dynamically regulated, balanced by succinylation and desuccinylation processes, and are closely connected to metabolic state in vivo. Sirtuins have been shown to possess NAD+-dependent desuccinylation activity in vitro and in vivo, among which the desuccinylation activity of SIRT5 is most extensively studied. Our understanding of the response of succinylation levels to different metabolic conditions, is hampered by the lack of a fast NAD+-dependent desuccinylation assay in a physiological context. In the present study, we therefore optimized and validated a fluorescence-based assay for measuring NAD+-dependent desuccinylation activity in cell lysates. Our results demonstrated that shorter and stricter reaction time was critical to approach the initial rate of NAD+-dependent desuccinylation activity in crude cell lysate systems, as compared to the desuccinylation reaction of purified His-SIRT5. Analysis of desuccinylation activity in SIRT5 knockout HEK293T cells confirmed the relevance of SIRT5 in cellular desuccinylation activity, as well as the presence of other NAD+-dependent desuccinylase activities. In addition, we were able to analyse desuccinylation and deacetylation activity in multiple cell lines using this assay. We showed a remarkably higher desuccinylase activity, but not deacetylase activity, in proliferative cultured muscle and adipose cells in comparison with their differentiated counterparts. Our results reveal an alteration in NAD+-dependent desuccinylation activity under different metabolic states.

Fish Macrophages Show Distinct Metabolic Signatures Upon Polarization.

Macrophages play important roles in conditions ranging from host immune defense to tissue regeneration and polarize their functional phenotype accordingly. Next to differences in the use of L-arginine and the production of different cytokines, inflammatory M1 macrophages and anti-inflammatory M2 macrophages are also metabolically distinct. In mammals, M1 macrophages show metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation to generate energy. The presence of polarized functional immune phenotypes conserved from mammals to fish led us to hypothesize that a similar metabolic reprogramming in polarized macrophages exists in carp. We studied mitochondrial function of M1 and M2 carp macrophages under basal and stressed conditions to determine oxidative capacity by real-time measurements of oxygen consumption and glycolytic capacity by measuring lactate-based acidification. In M1 macrophages, we found increased nitric oxide production and irg1 expression in addition to altered oxidative phosphorylation and glycolysis. In M2 macrophages, we found increased arginase activity, and both oxidative phosphorylation and glycolysis were similar to control macrophages. These results indicate that M1 and M2 carp macrophages show distinct metabolic signatures and indicate that metabolic reprogramming may occur in carp M1 macrophages. This immunometabolic reprogramming likely supports the inflammatory phenotype of polarized macrophages in teleost fish such as carp, similar to what has been shown in mammals.

In vivo assessment of mitochondrial capacity using NIRS in locomotor muscles of young and elderly males with similar physical activity levels.

Mitochondrial capacity is pivotal to skeletal muscle function and is suggested to decline with age. However, there is large heterogeneity in current data, possibly due to effect modifiers such as physical activity, sex and muscle group. Yet, few studies have compared multiple muscle groups in different age groups with comparable physical activity levels. Here, we newly used near-infrared spectroscopy (NIRS) to characterise mitochondrial capacity in three different locomotor muscles in young (19-25 year) and older (65-71 year), healthy males with similar physical activity levels. Mitochondrial capacity and reperfusion after arterial occlusion was measured in the vastus lateralis (VL), the gastrocnemius (GA) and the tibialis anterior (TA). Physical activity was verified using accelerometry and was not different between the age groups (404.3 ± 214.9 vs 494.9 ± 187.0 activity kcal per day, p = 0.16). Mitochondrial capacity was significantly lower in older males in the GA and VL, but not in the TA (p = 0.048, p = 0.036 and p = 0.64, respectively). Reperfusion rate was not significantly different for the GA (p = 0.55), but was significantly faster in the TA and VL in the young group compared to the older group (p = 0.0094 and p = 0.039, respectively). In conclusion, we identified distinct modes of mitochondrial ageing in different locomotor muscles in a young and older population with similar physical activity patterns. Furthermore, we show that NIRS is suitable for relatively easy application in ageing research and can reveal novel insights into mitochondrial functioning with age.

High Dose of Dietary Nicotinamide Riboside Induces Glucose Intolerance and White Adipose Tissue Dysfunction in Mice Fed a Mildly Obesogenic Diet.

Nicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD+) precursor vitamin. The scarce reports on the adverse effects on metabolic health of supplementation with high-dose NR warrant substantiation. Here, we aimed to examine the physiological responses to high-dose NR supplementation in the context of a mildly obesogenic diet and to substantiate this with molecular data. An 18-week dietary intervention was conducted in male C57BL/6JRccHsd mice, in which a diet with 9000 mg NR per kg diet (high NR) was compared to a diet with NR at the recommended vitamin B3 level (control NR). Both diets were mildly obesogenic (40 en% fat). Metabolic flexibility and glucose tolerance were analyzed and immunoblotting, qRT-PCR and histology of epididymal white adipose tissue (eWAT) were performed. Mice fed with high NR showed a reduced metabolic flexibility, a lower glucose clearance rate and aggravated systemic insulin resistance. This was consistent with molecular and morphological changes in eWAT, including sirtuin 1 (SIRT1)-mediated PPARγ (proliferator-activated receptor γ) repression, downregulated AKT/glucose transporter type 4 (GLUT4) signaling, an increased number of crown-like structures and macrophages, and an upregulation of pro-inflammatory gene markers. In conclusion, high-dose NR induces the onset of WAT dysfunction, which may in part explain the deterioration of metabolic health.

In vivo assessment of muscle mitochondrial function in healthy, young males in relation to parameters of aerobic fitness.

PURPOSE: The recovery of muscle oxygen consumption (m[Formula: see text]O2) after exercise provides a measure of skeletal muscle mitochondrial capacity, as more and better-functioning mitochondria will be able to restore m[Formula: see text]O2 faster to the pre-exercise state. The aim was to measure muscle mitochondrial capacity using near-infrared spectroscopy (NIRS) within a healthy, normally active population and relate this to parameters of aerobic fitness, investigating the applicability and relevance of using NIRS to assess muscle mitochondrial capacity non-invasively. METHODS: Mitochondrial capacity was analysed in the gastrocnemius and flexor digitorum superficialis (FDS) muscles of eight relatively high-aerobic fitness ([Formula: see text]O2peak ≥ 57 mL/kg/min) and eight relatively low-aerobic fitness male subjects ([Formula: see text]O2peak ≤ 47 mL/kg/min). Recovery of whole body [Formula: see text]O2, i.e. excess post-exercise oxygen consumption (EPOC) was analysed after a cycling protocol. RESULTS: Mitochondrial capacity, as analysed using NIRS, was significantly higher in high-fitness individuals compared to low-fitness individuals in the gastrocnemius, but not in the FDS (p = 0.0036 and p = 0.20, respectively). Mitochondrial capacity in the gastrocnemius was significantly correlated with [Formula: see text]O2peak (R2 = 0.57, p = 0.0019). Whole body [Formula: see text]O2 recovery was significantly faster in the high-fitness individuals (p = 0.0048), and correlated significantly with mitochondrial capacity in the gastrocnemius (R2 = 0.34, p = 0.028). CONCLUSION: NIRS measurements can be used to assess differences in mitochondrial muscle oxygen consumption within a relatively normal, healthy population. Furthermore, mitochondrial capacity correlated with parameters of aerobic fitness ([Formula: see text]O2peak and EPOC), emphasising the physiological relevance of the NIRS measurements.

Transcriptional Response of White Adipose Tissue to Withdrawal of Vitamin B3.

SCOPE: Distinct markers for mild vitamin B3 deficiency are lacking. To identify these, the molecular responses of white adipose tissue (WAT) to vitamin B3 withdrawal are examined. METHODS AND RESULTS: A dietary intervention is performed in male C57BL/6JRccHsd mice, in which a diet without nicotinamide riboside (NR) is compared to a diet with NR at the recommended vitamin B3 level. Both diets contain low but adequate level of tryptophan. Metabolic flexibility and systemic glucose tolerance are analyzed and global transcriptomics, qRT-PCR, and histology of epididymal WAT (eWAT) are performed. A decreased insulin sensitivity and a shift from carbohydrate to fatty acid oxidation in response to vitamin B3 withdrawal are observed. This is consistent with molecular changes in eWAT, including an activated MEK/ERK signaling, a lowering of glucose utilization markers, and an increase in makers of fatty acid catabolism, possibly related to the consistent lower expression of mitochondrial electron transport complexes. The synthesis pathway of tetrahydropteridine (BH4), an essential cofactor for neurotransmitter synthesis, is transcriptionally activated. Genes marking these processes are technically validated. CONCLUSION: The downregulation of Anp32a, Tnk2 and the upregulation of Mapk1, Map2k1, Qdpr, Mthfs, and Mthfsl are proposed as a WAT transcriptional signature marker for mild vitamin B3 deficiency.