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.

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 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.

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.

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.

Mito-Nuclear Communication by Mitochondrial Metabolites and Its Regulation by B-Vitamins.

Mitochondria are cellular organelles that control metabolic homeostasis and ATP generation, but also play an important role in other processes, like cell death decisions and immune signaling. Mitochondria produce a diverse array of metabolites that act in the mitochondria itself, but also function as signaling molecules to other parts of the cell. Communication of mitochondria with the nucleus by metabolites that are produced by the mitochondria provides the cells with a dynamic regulatory system that is able to respond to changing metabolic conditions. Dysregulation of the interplay between mitochondrial metabolites and the nucleus has been shown to play a role in disease etiology, such as cancer and type II diabetes. Multiple recent studies emphasize the crucial role of nutritional cofactors in regulating these metabolic networks. Since B-vitamins directly regulate mitochondrial metabolism, understanding the role of B-vitamins in mito-nuclear communication is relevant for therapeutic applications and optimal dietary lifestyle. In this review, we will highlight emerging concepts in mito-nuclear communication and will describe the role of B-vitamins in mitochondrial metabolite-mediated nuclear signaling.