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Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7–3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4–3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5β (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP.

Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours. A positron emission tomography imaging tracer is developed to image mitochondrial function in vivo, and application of this tracer to a mouse model of lung cancer identifies distinct functional mitochondrial heterogeneity between tumour cells.

Glucose is a major source of energy for most living organisms, and its aberrant uptake is linked to many pathological conditions. However, our understanding of disease-associated glucose flux is limited owing to the lack of robust tools. To date, positron-emission tomography imaging remains the gold standard for measuring glucose uptake, and no optical tools exist for non-invasive longitudinal imaging of this important metabolite in in vivo settings. Here, we report the development of a bioluminescent glucose-uptake probe for real-time, non-invasive longitudinal imaging of glucose absorption both in vitro and in vivo. In addition, we demonstrate that the sensitivity of our method is comparable with that of commonly used 18F-FDG-positron-emission-tomography tracers and validate the bioluminescent glucose-uptake probe as a tool for the identification of new glucose transport inhibitors. The new imaging reagent enables a wide range of applications in the fields of metabolism and drug development. A bioluminescent glucose-uptake probe enables accurate, real-time, non-invasive longitudinal imaging of d-glucose absorption both in vitro and in vivo.

Combined fatty acid esterification and lipolysis, termed lipid cycling, is an ATP‐consuming process that contributes to energy expenditure. Therefore, interventions that stimulate energy expenditure through lipid cycling are of great interest. Here we find that pharmacological and genetic inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes activates lipid cycling and energy expenditure, even in the absence of adrenergic stimulation. We show that the resulting increase in ATP demand elevates mitochondrial respiration coupled to ATP synthesis and fueled by lipid oxidation. We identify that glutamine consumption and the Malate‐Aspartate Shuttle are required for the increase in Energy Expenditure induced by MPC inhibition in Brown Adipocytes (MAShEEBA). We thus demonstrate that energy expenditure through enhanced lipid cycling can be activated in brown adipocytes by decreasing mitochondrial pyruvate availability. We present a new mechanism to increase energy expenditure and fat oxidation in brown adipocytes, which does not require adrenergic stimulation of mitochondrial uncoupling., Inhibition of mitochondrial pyruvate import in brown adipocytes activates lipid cycling and increases energy expenditure even in the absence of adrenergic stimulation. In the absence of mitochondrial pyruvate entry, the TCA cycle receives carbons from glutamine to support beta‐oxidation.

Assessing mitochondrial function in cell-based systems is a central component of metabolism research. However, the selection of an initial measurement technique may be complicated given the range of parameters that can be studied as well as the need to define the mitochondrial (dys)function of interest. This methods-focused review compares and contrasts the use of mitochondrial membrane potential measurements, plate-based respirometry, and metabolomics and stable isotope tracing. We demonstrate how measurements of (i) cellular substrate preference, (ii) respiratory chain activity, (iii) cell activation, and (iv) mitochondrial biogenesis are enriched by integrating information from multiple methods. This manuscript is meant to serve as a perspective to help choose which technique might be an appropriate initial method to answer a given question, as well as provide a broad 'roadmap' for designing follow-up assays to enrich datasets or resolve ambiguous results.

Futile lipid cycling is an ATP-wasting process proposed to participate in energy expenditure of mature fat-storing white adipocytes, given their inability to oxidize fat. The hallmark of activated brown adipocytes is to increase fat oxidation by uncoupling respiration from ATP synthesis. Whether ATP-consuming lipid cycling can contribute to BAT energy expenditure has been largely unexplored. Here we find that pharmacological inhibition of the mitochondrial pyruvate carrier (MPC) in brown adipocytes is sufficient to increase ATP-synthesis fueled by fatty acid oxidation, even in the absence of adrenergic stimulation. We find that elevated ATP-demand induced by MPC inhibition results from activation of futile lipid cycling. Furthermore, we identify that glutamine consumption and theMalate-AspartateShuttle are required for the increase inEnergyExpenditure induced by MPC inhibition inBrownAdipocytes (MAShEEBA). These data demonstrate that futile energy expenditure through lipid cycling can be activated in BAT by altering fuel availability to mitochondria. Therefore, we identify a new mechanism to increase fat oxidation and energy expenditure in BAT that bypasses the need for adrenergic stimulation of mitochondrial uncoupling.

Mitochondria are metabolic organelles essential not only for energy transduction, but also a range of other functions such as biosynthesis, ion and metal homeostasis, maintenance of redox balance, and cell signaling. A hallmark example of how mitochondria can rebalance these processes to adjust cell function is observed in macrophages. These innate immune cells are responsible for a remarkable breadth of processes including pathogen elimination, antigen presentation, debris clearance, and wound healing. These diverse, polarized functions often include similarly disparate alterations in the metabolic phenotype associated with their execution. In this chapter, mitochondrial bioenergetics and signaling are viewed through the lens of macrophage polarization: both classical, pro-inflammatory activation and alternative, anti-inflammatory activation are associated with substantive changes to mitochondrial metabolism. Emphasis is placed on recent evidence that aims to clarify the essential - rather than associative - mitochondrial alterations, as well as accumulating data suggesting a degree of plasticity within the metabolic phenotypes that can support pro- and anti-inflammatory functions.

A sharp increase in mitochondrial Ca2+marks the activation of the brown adipose tissue (BAT) thermogenesis, yet the mechanisms preventing Ca2+deleterious effects are poorly understood. Here, we show that adrenergic stimulation of BAT activates a PKA-dependent mitochondrial Ca2+extrusion via the mitochondrial Na+/Ca2+exchanger, NCLX. Adrenergic stimulation of NCLX-ablated brown adipocytes (BA) induces a profound mitochondrial Ca2+overload and impaired uncoupled respiration. Core body temperature, PET imaging and VO2 measurements confirm a BAT specific thermogenic defect in NCLX-null mice.We show that mitochondrial Ca2+overload induced by adrenergic stimulation of NCLX-null BAT, triggers the opening of the mitochondrial permeability transition pore (mPTP), leading to remarkable mitochondrial swelling, Cytochromecrelease and cell death in BAT. However, treatment with mPTP inhibitors rescue mitochondrial respiratory function and thermogenesis in NCLX-null BA,in vitroandin vivo.Our findings identify a novel pathway enabling non-lethal mitochondrial Ca2+elevation during adrenergic stimulation of uncoupled respiration. Deletion of NCLX transforms the adrenergic pathway responsible for the stimulation of thermogenesis into a death pathway.