Controlled power: how biology manages succinate-driven energy release.

Oxidation of succinate by mitochondria can generate a higher protonmotive force (pmf) than can oxidation of NADH-linked substrates. Fundamentally, this is because of differences in redox potentials and gearing. Biology adds kinetic constraints that tune the oxidation of NADH and succinate to ensure that the resulting mitochondrial pmf is suitable for meeting cellular needs without triggering pathology. Tuning within an optimal range is used, for example, to shift ATP consumption between different consumers. Conditions that overcome these constraints and allow succinate oxidation to drive pmf too high can cause pathological generation of reactive oxygen species. We discuss the thermodynamic properties that allow succinate oxidation to drive pmf higher than NADH oxidation, and discuss the evidence for kinetic tuning of ATP production and for pathologies resulting from substantial succinate oxidation in vivo.

Total Cellular ATP Production Changes With Primary Substrate in MCF7 Breast Cancer Cells.

Cancer growth is predicted to require substantial rates of substrate catabolism and ATP turnover to drive unrestricted biosynthesis and cell growth. While substrate limitation can dramatically alter cell behavior, the effects of substrate limitation on total cellular ATP production rate is poorly understood. Here, we show that MCF7 breast cancer cells, given different combinations of the common cell culture substrates glucose, glutamine, and pyruvate, display ATP production rates 1.6-fold higher than when cells are limited to each individual substrate. This increase occurred mainly through faster oxidative ATP production, with little to no increase in glycolytic ATP production. In comparison, non-transformed C2C12 myoblast cells show no change in ATP production rate when substrates are limited. In MCF7 cells, glutamine allows unexpected access to oxidative capacity that pyruvate, also a strictly oxidized substrate, does not. Pyruvate, when added with other exogenous substrates, increases substrate-driven oxidative ATP production, by increasing both ATP supply and demand. Overall, we find that MCF7 cells are highly flexible with respect to maintaining total cellular ATP production under different substrate-limited conditions, over an acute (within minutes) timeframe that is unlikely to result from more protracted (hours or more) transcription-driven changes to metabolic enzyme expression. The near-identical ATP production rates maintained by MCF7 and C2C12 cells given single substrates reveal a potential difficulty in using substrate limitation to selectively starve cancer cells of ATP. In contrast, the higher ATP production rate conferred by mixed substrates in MCF7 cells remains a potentially exploitable difference.

The Whys and Hows of Calculating Total Cellular ATP Production Rate.

Quantifying total cellular ATP production rate has become easier with recent technology and is essential to understanding energy metabolism in cells and tissues. We review fundamental concepts for determining total cellular ATP production rate from measurements of oxygen consumption and acidification rates and discuss their application to answering biological questions.

Plate-Based Measurement of Respiration by Isolated Mitochondria.

Measuring respiration rate can be a powerful way to assess energetic function in isolated mitochondria. Current, plate-based methods have several advantages over older, suspension-based systems, including greater throughput and the requirement of only µg quantities of material. In this chapter, we describe a plate-based method for measuring oxygen consumption by isolated adherent mitochondria.

Osteoblast-like MC3T3-E1 Cells Prefer Glycolysis for ATP Production but Adipocyte-like 3T3-L1 Cells Prefer Oxidative Phosphorylation.

Mesenchymal stromal cells (MSCs) are early progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes. We hypothesized that osteoblasts and adipocytes utilize distinct bioenergetic pathways during MSC differentiation. To test this hypothesis, we compared the bioenergetic profiles of preosteoblast MC3T3-E1 cells and calvarial osteoblasts with preadipocyte 3T3L1 cells, before and after differentiation. Differentiated MC3T3-E1 osteoblasts met adenosine triphosphate (ATP) demand mainly by glycolysis with minimal reserve glycolytic capacity, whereas nondifferentiated cells generated ATP through oxidative phosphorylation. A marked Crabtree effect (acute suppression of respiration by addition of glucose, observed in both MC3T3-E1 and calvarial osteoblasts) and smaller mitochondrial membrane potential in the differentiated osteoblasts, particularly those incubated at high glucose concentrations, indicated a suppression of oxidative phosphorylation compared with nondifferentiated osteoblasts. In contrast, both nondifferentiated and differentiated 3T3-L1 adipocytes met ATP demand primarily by oxidative phosphorylation despite a large unused reserve glycolytic capacity. In sum, we show that nondifferentiated precursor cells prefer to use oxidative phosphorylation to generate ATP; when they differentiate to osteoblasts, they gain a strong preference for glycolytic ATP generation, but when they differentiate to adipocytes, they retain the strong preference for oxidative phosphorylation. Unique metabolic programming in mesenchymal progenitor cells may influence cell fate and ultimately determine the degree of bone formation and/or the development of marrow adiposity. © 2018 American Society for Bone and Mineral Research.

Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements.

Partitioning of ATP generation between glycolysis and oxidative phosphorylation is central to cellular bioenergetics but cumbersome to measure. We describe here how rates of ATP generation by each pathway can be calculated from simultaneous measurements of extracellular acidification and oxygen consumption. We update theoretical maximum ATP yields by mitochondria and cells catabolizing different substrates. Mitochondrial P/O ratios (mol of ATP generated per mol of [O] consumed) are 2.73 for oxidation of pyruvate plus malate and 1.64 for oxidation of succinate. Complete oxidation of glucose by cells yields up to 33.45 ATP/glucose with a maximum P/O of 2.79. We introduce novel indices to quantify bioenergetic phenotypes. The glycolytic index reports the proportion of ATP production from glycolysis and identifies cells as primarily glycolytic (glycolytic index > 50%) or primarily oxidative. The Warburg effect is a chronic increase in glycolytic index, quantified by the Warburg index. Additional indices quantify the acute flexibility of ATP supply. The Crabtree index and Pasteur index quantify the responses of oxidative and glycolytic ATP production to alterations in glycolysis and oxidative reactions, respectively; the supply flexibility index quantifies overall flexibility of ATP supply; and the bioenergetic capacity quantifies the maximum rate of total ATP production. We illustrate the determination of these indices using C2C12 myoblasts. Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how extracellular fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics. We provide a simple spreadsheet to calculate glycolytic and oxidative ATP production rates from raw extracellular acidification and respiration data.

Positive Feedback Amplifies the Response of Mitochondrial Membrane Potential to Glucose Concentration in Clonal Pancreatic Beta Cells.

Analysis of the cellular mechanisms of metabolic disorders, including type 2 diabetes mellitus, is complicated by the large number of reactions and interactions in metabolic networks. Metabolic control analysis with appropriate modularization is a powerful method for simplifying and analyzing these networks. To analyze control of cellular energy metabolism in adherent cell cultures of the INS-1 832/13 pancreatic ß-cell model we adapted our microscopy assay of absolute mitochondrial membrane potential (ΔψM) to a fluorescence microplate reader format, and applied it in conjunction with cell respirometry. In these cells the sensitive response of ΔψM to extracellular glucose concentration drives glucose-stimulated insulin secretion. Using metabolic control analysis we identified the control properties that generate this sensitive response. Force-flux relationships between ΔψM and respiration were used to calculate kinetic responses to ΔψM of processes both upstream (glucose oxidation) and downstream (proton leak and ATP turnover) of ΔψM. The analysis revealed that glucose-evoked ΔψM hyperpolarization is amplified by increased glucose oxidation activity caused by factors downstream of ΔψM. At high glucose, the hyperpolarized ΔψM is stabilized almost completely by the action of glucose oxidation, whereas proton leak also contributes to the homeostatic control of ΔψM at low glucose. These findings suggest a strong positive feedback loop in the regulation of ß-cell energetics, and a possible regulatory role of proton leak in the fasting state. Analysis of islet bioenergetics from published cases of type 2 diabetes suggests that disruption of this feedback can explain the damaged bioenergetic response of ß-cells to glucose. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.

Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.

The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic ß-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic ß-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed ß-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with ß-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited ß-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.

Determining Maximum Glycolytic Capacity Using Extracellular Flux Measurements.

Measurements of glycolytic rate and maximum glycolytic capacity using extracellular flux analysis can give crucial information about cell status and phenotype during normal operation, development of pathology, differentiation, and malignant transformation. They are also of great use when assessing the effects of chemical or drug treatments. Here, we experimentally define maximum glycolytic capacity, demonstrate how it differs from glycolytic rate, and provide a protocol for determining the basal glycolytic rate and maximum glycolytic capacity in cells using extracellular flux measurements. The results illustrate the power of extracellular flux analysis to describe the energetics of adherent cells in culture in a fully quantitative way.

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis(1). Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate(2), the same is not true for extracellular acid production and its relationship to glycolytic rate (3-6). Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate(-) + H(+) and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3(-) + H(+) is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification (6). Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Role of sigma-1 receptors in neurodegenerative diseases.

Neurodegenerative diseases with distinct genetic etiologies and pathological phenotypes appear to share common mechanisms of neuronal cellular dysfunction, including excitotoxicity, calcium dysregulation, oxidative damage, ER stress and mitochondrial dysfunction. Glial cells, including microglia and astrocytes, play an increasingly recognized role in both the promotion and prevention of neurodegeneration. Sigma receptors, particularly the sigma-1 receptor subtype, which are expressed in both neurons and glia of multiple regions within the central nervous system, are a unique class of intracellular proteins that can modulate many biological mechanisms associated with neurodegeneration. These receptors therefore represent compelling putative targets for pharmacologically treating neurodegenerative disorders. In this review, we provide an overview of the biological mechanisms frequently associated with neurodegeneration, and discuss how sigma-1 receptors may alter these mechanisms to preserve or restore neuronal function. In addition, we speculate on their therapeutic potential in the treatment of various neurodegenerative disorders.

The contributions of respiration and glycolysis to extracellular acid production.

BACKGROUND: The rate at which cells acidify the extracellular medium is frequently used to report glycolytic rate, with the implicit assumption that conversion of uncharged glucose or glycogen to lactate(-)+H(+) is the only significant source of acidification. However, another potential source of extracellular protons is the production of CO2 during substrate oxidation: CO2 is hydrated to H2CO3, which then dissociates to HCO3(-)+H(+). METHODS: O2 consumption and pH were monitored in a popular platform for measuring extracellular acidification (the Seahorse XF Analyzer). RESULTS: We found that CO2 produced during respiration caused almost stoichiometric release of H(+) into the medium. With C2C12 myoblasts given glucose, respiration-derived CO2 contributed 34% of the total extracellular acidification. When glucose was omitted or replaced by palmitate or pyruvate, this value was 67-100%. Analysis of primary cells, cancer cell lines, stem cell lines, and isolated synaptosomes revealed contributions of CO2-produced acidification that were usually substantial, ranging from 3% to 100% of the total acidification rate. CONCLUSION: Measurement of glycolytic rate using extracellular acidification requires differentiation between respiratory and glycolytic acid production. GENERAL SIGNIFICANCE: The data presented here demonstrate the importance of this correction when extracellular acidification is used for quantitative measurement of glycolytic flux to lactate. We describe a simple way to correct the measured extracellular acidification rate for respiratory acid production, using simultaneous measurement of oxygen consumption rate. SUMMARY STATEMENT: Extracellular acidification is often assumed to result solely from glycolytic lactate production, but respiratory CO2 also contributes. We demonstrate that extracellular acidification by myoblasts given glucose is 66% glycolytic and 34% respiratory and describe a method to differentiate these sources.

Characteristics of the turnover of uncoupling protein 3 by the ubiquitin proteasome system in isolated mitochondria.

Uncoupling protein 3 (UCP3) is implicated in mild uncoupling and the regulation of mitochondrial ROS production. We previously showed that UCP3 turns over rapidly in C2C12 myoblasts, with a half-life of 0.5-4h, and that turnover can be reconstituted in vitro. We show here that rapid degradation of UCP3 in vitro in isolated brown adipose tissue mitochondria required the 26S proteasome, ubiquitin, ATP, succinate to generate a high membrane potential, and a pH of 7.4 or less. Ubiquitin containing lysine-48 was both necessary and sufficient to support UCP3 degradation, implying a requirement for polyubiquitylation at this residue. The 20S proteasome did not support degradation. UCP3 degradation was prevented by simultaneously blocking matrix ATP generation and import, showing that ATP in the mitochondrial matrix was required. Degradation did not appear to require a transmembrane pH gradient, but was very sensitive to membrane potential: degradation was halved when membrane potential decreased 10-20mV from its resting value, and was not significant below about 120mV. We propose that matrix ATP and a high membrane potential are needed for UCP3 to be polyubiquitylated through lysine-48 of ubiquitin and exported to the cytosolic 26S proteasome, where it is de-ubiquitylated and degraded.

Mitochondrial uncoupling and lifespan.

The quest to understand why we age has given rise to numerous lines of investigation that have gradually converged to include metabolic control by mitochondrial activity as a major player. That is, the ideal balance between nutrient uptake, its transduction into usable energy, and the mitigation of damaging byproducts can be regulated by mitochondrial respiration and output (ATP, reactive oxygen species (ROS), and heat). Mitochondrial inefficiency through proton leak, which uncouples substrate oxidation from ADP phosphorylation, can comprise as much as 30% of the basal metabolic rate. This uncoupling is hypothesized to protect cells from conditions that favor ROS production. Uncoupling can also occur through pharmacological induction of proton leak and activity of the uncoupling proteins. Mitochondrial uncoupling is implicated in lifespan extension through its effects on metabolic rate and ROS production. However, evidence to date does not suggest a consistent role for uncoupling in lifespan. The purpose of this review is to discuss recent work examining how mitochondrial uncoupling impacts lifespan.

Rapid turnover of mitochondrial uncoupling protein 3.

UCP3 (uncoupling protein 3) and its homologues UCP2 and UCP1 are regulators of mitochondrial function. UCP2 is known to have a short half-life of approx. 1 h, owing to its rapid degradation by the cytosolic 26S proteasome, whereas UCP1 is turned over much more slowly by mitochondrial autophagy. In the present study we investigate whether UCP3 also has a short half-life, and whether the proteasome is involved in UCP3 degradation. UCP3 half-life was examined in the mouse C2C12 myoblast cell line by inhibiting protein synthesis with cycloheximide and monitoring UCP3 protein levels by immunoblot analysis. We show that UCP3 has a short half-life of 0.5-4 h. Rapid degradation was prevented by a cocktail of proteasome inhibitors, supporting a proteasomal mechanism for turnover. In addition, this phenotype is recapitulated in vitro: UCP3 was degraded in mitochondria isolated from rat skeletal muscle or brown adipose tissue with a half-life of 0.5-4 h, but only in the presence of a purified 26S proteasomal fraction. This in vitro proteolysis was also sensitive to proteasome inhibition. This phenotype is in direct contrast with the related proteins UCP1 and the adenine nucleotide translocase, which have long half-lives. Therefore UCP3 is turned over rapidly in multiple cell types in a proteasome-dependent manner.

Overlapping contributions of Msh1p and putative recombination proteins Cce1p, Din7p, and Mhr1p in large-scale recombination and genome sorting events in the mitochondrial genome of Saccharomyces cerevisiae.

The mechanisms that govern mutation avoidance in the mitochondrial genome, though believed to be numerous, are poorly understood. The identification of individual genes has implicated mismatch repair and several recombination pathways in maintaining the fidelity and structural stability of mitochondrial DNA. However, the majority of genes in these pathways have not been identified and the interactions between different pathways have not been extensively studied. Additionally, the multicopy presence of the mitochondrial genome affects the occurrence and persistence of mutant phenotypes, making mitochondrial DNA transmission and sorting important factors affecting mutation accumulation. We present new evidence that the putative recombination genes CCE1, DIN7, and MHR1 have overlapping function with the mismatch repair homolog MSH1 in point mutation avoidance and suppression of aberrant recombination events. In addition, we demonstrate a novel role for Msh1p in mtDNA transmission, a role not predicted by studies of its nuclear homologs.

Analysis of the functional domains of the mismatch repair homologue Msh1p and its role in mitochondrial genome maintenance.

Mitochondrial DNA (mtDNA) repair occurs in all eukaryotic organisms and is essential for the maintenance of mitochondrial function. Evidence from both humans and yeast suggests that mismatch repair is one of the pathways that functions in overall mtDNA stability. In the mitochondria of the yeast Saccharomyces cerevisiae, the presence of a homologue to the bacterial MutS mismatch repair protein, MSH1, has long been known to be essential for mitochondrial function. The mechanisms for which it is essential are unclear, however. Here, we analyze the effects of two point mutations, msh1-F105A and msh1-G776D, both predicted to be defective in mismatch repair; and we show that they are both able to maintain partial mitochondrial function. Moreover, there are significant differences in the severity of mitochondrial disruption between the two mutants that suggest multiple roles for Msh1p in addition to mismatch repair. Our overall findings suggest that these additional predicted functions of Msh1p, including recombination surveillance and heteroduplex rejection, may be primarily responsible for its essential role in mtDNA stability.