Adipose tissues, particularly beige and brown adipose tissue, play crucial roles in energy metabolism. Brown adipose tissues\' thermogenic capacity and the appearance of beige cells within white adipose tissue have spurred interest in their metabolic impact and therapeutic potential. Brown and beige fat cells, activated by environmental factors like cold exposure or by pharmacology, share metabolic mechanisms that drive non-shivering thermogenesis. Understanding these two cell types requires advanced, yet broadly applicable in vitro models that reflect the complex microenvironment and vasculature of adipose tissues. Here we present mouse vascularized adipose spheroids of the stromal vascular microenvironment from inguinal white adipose tissue, a tissue with \'beiging\' capacity in mice and humans. We show that adding a scaffold improves vascular sprouting, enhances spheroid growth, and upregulates adipogenic markers, thus reflecting increased adipocyte maturity. Transcriptional profiling via RNA sequencing revealed distinct metabolic pathways upregulated in our vascularized adipose spheroids, with increased expression of genes involved in glucose metabolism, lipid metabolism, and thermogenesis. Functional assessment demonstrated increased oxygen consumption in vascularized adipose spheroids compared to classical 2D cultures, which was enhanced by β-adrenergic receptor stimulation correlating with elevated β-adrenergic receptor expression. Moreover, stimulation with the naturally occurring adipokine, FGF21, induced Ucp1 mRNA expression in the vascularized adipose spheroids. In conclusion, vascularized inguinal white adipose tissue spheroids provide a physiologically relevant platform to study how the stromal vascular microenvironment shapes adipocyte responses and influence activated thermogenesis in beige adipocytes.

Acute aerobic exercise increases the number and proportions of circulating peripheral blood mononuclear cells (PMBC) and can alter PBMC mitochondrial bioenergetics. In this study, we aimed to examine the impact of a maximal exercise bout on immune cell metabolism in collegiate swimmers. Eleven (7 M/4F) collegiate swimmers completed a maximal exercise test to measure anaerobic power and capacity. Pre- and postexercise PBMCs were isolated to measure the immune cell phenotypes and mitochondrial bioenergetics using flow cytometry and high-resolution respirometry. The maximal exercise bout increased circulating levels of PBMCs, particularly in central memory (KLRG1+/CD57-) and senescent (KLRG1+/CD57+) CD8+ T cells, whether measured as a % of PMBCs or as absolute concentrations (all p < 0.05). At the cellularlevel, the routine oxygen flow (IO2 [pmol·s-1 ·106 PBMCs-1 ]) increased following maximal exercise (p = 0.042); however, there were no effects of exercise on the IO2 measured under the LEAK, oxidative phosphorylation (OXPHOS), or electron transfer (ET) capacities. There were exercise-induced increases in the tissue-level oxygen flow (IO2-tissue [pmol·s-1 ·mL blood-1 ]) for all respiratory states (all p < 0.01), except for the LEAK state, after accounting for the mobilization of PBMCs. Future subtype-specific studies are needed to characterize further maximal exercise\'s true impact on immune cell bioenergetics.

Berries and other anthocyanin-rich foods have demonstrated anti-obesity effects in rodents and humans. However, the bioactive components of these foods and their mechanisms of action are unclear. We conducted an intervention study with overweight and obese adults to isolate the effects of different berry components on bioenergetics. Subjects consumed whole mixed berries (high anthocyanin, high fiber), pressed berry juice (high anthocyanin, low fiber), berry-flavored gelatin (low anthocyanin, low fiber), or fiber-enriched gelatin (low anthocyanin, high fiber) for one week prior to a meal challenge with the same treatment food as the pre-feed period. Peripheral blood mononuclear cells were collected 2 h after the meal challenge, and cellular respiration was assessed via high-resolution respirometry. The high-anthocyanin, low-fiber treatment (berry juice) and the low-anthocyanin, high-fiber treatment (fiber-enriched gelatin) had opposite effects on cellular respiration. In the fasted state, berry juice resulted in the highest oxygen-consumption rate (OCR), while fiber-enriched gelatin resulted in the highest OCR in the fed state. Differences were observed in multiple respiration states (basal, state 3, state 4, uncoupled), with the greatest differences being between the pressed berry juice and the fiber-enriched gelatin. Different components of berries, specifically anthocyanins/flavonoids and fiber, appear to have differential effects on cellular respiration.

Physiological investigations of fish gills have traditionally centred on the two principal functions of the gills: gas exchange and ion regulation. Mitochondrion-rich cells (MRCs) are primarily found within the gill filaments of fish, and are thought to proliferate in order to increase the ionoregulatory capacity of the gill in response to environmentally induced osmotic challenges. However, surprisingly little attention has been paid to the metabolic function of mitochondria within fish gills. Here, we describe and validate a simple protocol for the permeabilization of fish gills and subsequent measurement of mitochondrial respiration rates in vitro Our protocol requires only small tissue samples (8 mg), exploits the natural structure of fish gills, does not require mechanical separation of the gill tissue (so is relatively quick to perform), and yields accurate and highly reproducible measurements of respiration rates. It offers great potential for the study of mitochondrial function in gills over a wide range of fish sizes and species.

In this chapter, we describe detailed protocols for measuring high-resolution respirometry on mitochondria extracted from adult whole mouse heart using the Oroboros 2k-Oxygraph system. The method provides detailed procedures for the preparation of mitochondria and measurement of high-resolution respirometry in response to various respiration inhibitions. The method described in this chapter could discern the different respiration rate on mitochondria extracted from two spatially distinct mitochondrial subpopulations, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IFM). These approaches can easily be translated to other cells and tissues.

OBJECTIVE: The purpose of this study was to identify an effective lipid oxidation initiator which could predict, within 1 month, the long-term oxidative stability of a prototype skincare formulation. The main purpose was to find a potential initiator not to assess oxidation stability of the formulations.
METHODS: Four initiators (below) were examined in three steps: Reaction kinetics using a Clark electrode (Oxygraph); Effect of adding an initiator on the product\'s physical and oxidative stability in prototype skincare formulations by visual observation, peroxide value and headspace GC-MS determination of volatile oxidation products; and Ability to differentiate unstable vs. stable prototype creams by initiator addition. The four initiators explored were: FeCl2 /H2 O2 , FeCl3 /ascorbic acid, 2,2\'-Azobis(2,4-dimethylvaleronitrile) (AMVN) and 2,2\'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) RESULTS: In Oxygraph, the initiator systems FeCl2 /H2 O2 and FeCl3 /ascorbic acid were good accelerators of oxygen consumption. The addition of FeCl2 /H2 O2 to prototype formulations did not affect the physical stability. However, the addition of FeCl3 /ascorbic acid to prototype formulations resulted in phase separation and FeCl3 /ascorbic acid was therefore deemed unusable. Moreover, the addition of AAPH or AMVN resulted in an increased and decreased viscosity respectively. In the oxidation stability study, peroxide value increased significantly when AMVN was added. However, the peroxide value remained low for the other initiators and the control (no initiator). The secondary volatile oxidation product, butanal, increased most with the FeCl2 /H2 O2 addition. Three out of the four initiators did not have the ability to rank the stable and unstable formulations in accordance with the result obtained for volatile oxidation products after 42 days of storage at 20°C of formulations without initiator. Only, FeCl2 /H2 O2 was able to rank the formulations in accordance with the oxidative stability observed for volatile oxidation products after 42 days of storage.
CONCLUSIONS: FeCl2 /H2 O2 showed potential as an initiator to predict the oxidative stability of skincare formulations, but more studies are needed to confirm the result in a broader range of products over a longer time.
OBJECTIVE: Le but de cette étude était d\'identifier un initiateur efficace de l\'oxydation des lipides qui pourrait prédire, dans un délai d\'1 mois, la stabilité oxydative à long terme d\'une formulation prototype de soins pour la peau. Le principal objectif était de trouver un initiateur potentiel et non pas d’évaluer la stabilité à l\'oxydation des formulations. MÉTHODES: Quatre initiateurs (ci-dessous) ont été évalués au cours de trois étapes: Cinétique de réaction à l\'aide d\'une électrode de Clark (Oxygraphe); Effet de l\'addition d\'un initiateur sur la stabilité physique et oxydative du produit dans des prototypes de formulations par observation visuelle de l\'indice de peroxyde et détermination des produits d\'oxydation volatiles par GC-MS - espace de tête; et Capacité à différencier les prototypes de crèmes instables des prototypes de crèmes stables par addition d\'un initiateur. Les quatre initiateurs étudiés étaient : FeCl2 /H2 O2 , FeCl3 /acide ascorbique, 2,2′-azobis (2,4-diméthylvaléronitrile) (AMVN) et 2,2′-azobis (2-méthylpropionamidine), dichlorhydrate (AAPH) RÉSULTATS: Dans l\'oxygraphe, les systèmes initiateurs FeCl2 /H2 O2 et FeCl3 /acide ascorbique étaient de bons accélérateurs de la consommation en oxygène. L\'addition de FeCl2 /H2 O2 aux prototypes de formulations n\'a pas eu d\'impact sur la stabilité physique. Cependant, l\'addition de FeCl3 /acide ascorbique aux prototypes de formulations a entraîné une séparation des phases et le système FeCl3 /acide ascorbique a donc été jugé inutilisable. De plus, l\'addition d\'AAPH ou d\'AMVN a entraîné une augmentation et une diminution de la viscosité, respectivement. Dans l’étude de stabilité oxydative, l\'indice de peroxyde a significativement augmenté lorsque l\'AMVN a été ajouté. Cependant, l\'indice de peroxyde est resté faible pour les autres initiateurs et le témoin (sans initiateur). L\'augmentation la plus importante du produit d\'oxydation volatile secondaire, le butanal, a été observée avec l\'addition de FeCl2 /H2 O2 . Trois des quatre initiateurs n\'ont pas permis de classer les formulations stables et instables d\'après le résultat obtenu pour les produits d\'oxydation volatiles après 42 jours de conservation à 20 °C des formulations sans initiateur. Seul FeCl2 /H2 O2 a été en mesure de classer les formulations conformément à la stabilité oxydative observée pour les produits d\'oxydation volatiles après 42 jours de conservation.
CONCLUSIONS: FeCl2 /H2 O2 a montré un potentiel comme initiateur pour prédire la stabilité oxydative de formulations de soins pour la peau, mais des études supplémentaires sont nécessaires pour confirmer le résultat sur une plus large gamme de produits et sur une plus longue période.

Mitochondrial oxidative phosphorylation is central for generating ATP and maintaining energy homeostasis in most eukaryotic cells. The ex vivo measurement of mitochondrial oxygen consumption rates in intact cells or isolated organelles is a valuable approach to assess mitochondrial bioenergetics in various experimental conditions. In this chapter, we describe several step-by-step protocols for measuring mitochondrial respiration in intact cells, permeabilized cells (in situ mitochondria), and isolated organelles using both Clark-type polarographic oxygen electrode devices and the newly developed oxygen-sensing fluorophore-based Seahorse technology.

The use of tissue homogenate has greatly aided the study of the functioning of mitochondria. However, the amount of ATP produced per oxygen molecule consumed, that is, the effective P/O ratio, has never been measured directly in tissue homogenate. Here we combine and refine existing methods previously used in permeabilized cells and isolated mitochondria to simultaneously measure mitochondrial ATP production (JATP) and oxygen consumption (JO2) in tissue homogenate. A major improvement over existing methods is in the control of ATPases that otherwise interfere with the ATP assay: our modified technique facilitates simultaneous measurement of the rates of \"uncorrected\" ATP synthesis and of ATP hydrolysis, thus minimizing the amount of tissue and time needed. Finally, we develop a novel method of calculating effective P/O ratios which corrects measurements of JATP and JO2 for rates of nonmitochondrial ATP hydrolysis and respiration, respectively. Measurements of JATP and JO2 in liver homogenates from brown trout (Salmo trutta) were highly reproducible, although activity declined once homogenates were 2 h old. We compared mitochondrial properties from fed and food-deprived animals to demonstrate that the method can detect mitochondrial flexibility in P/O ratios in response to nutritional state. This method simplifies studies examining the mitochondrial bioenergetics of tissue homogenates, obviating the need for differential centrifugation or chemical permeabilization and avoiding the use of nonmitochondrial ATPase inhibitors. We conclude that our approach for characterizing effective P/O ratio opens up new possibilities in the study of mitochondrial function in very small samples, where the use of other methods is limited.