Low ΔΨ Research Articles

Oxaloacetate regulates mitochondrial complex II respiration in brown fat: dependence on UCP1 expression

Objective: Oxaloacetate (OAA) is a long-known potent inhibitor of succinate dehydrogenase (SDH), a phenomenon that has, however, received only limited attention. We previously found that skeletal muscle mitochondria incubated at low membrane potential (Δψ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein Δψ is intrinsically low, accumulate OAA in amounts sufficient to inhibit SDH (complex II) respiration. We proposed a mechanism wherein low Δψ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance we carried out studies of mice with inherently different levels of IBAT mitochodnrial inner membrane potential. Hypothesis: Mice with differential expression of UCP1 would manifest Δψ-dependent differences in OAA-regulated mitochondrial respiration. Methodology: We examined several parameters in complex II and complex I energized IBAT mitochondria isolated from obesity prone C57BL/6J compared to obesity resistant 129SVE mice and from UCP1 knock-out (UCP1KO) compared to wildtype (WT) mice. Data were analyzed by unpaired, 2-tailed t-test and expressed as mean ± SE. Data: Isolated complex II (succinate)-energized IBAT mitochondria from 129SVE mice versus C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower Δψ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower Δψ, exhibited much lower respiration (AUC of respiration versus time, 19963 ± 1764 vs 36015 ± 2527, p = 0.0004, n = 6), greater OAA concentrations (22.9 ± 1.8 vs 12.1 ± 3.3, p = 0.016, n = 6), much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared to the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater Δψ, ROS, and NADH, but equal or greater O2 flux compared to WT mitochondria. GDP addition, compared to no GDP, increased Δψ and complex II respiration in WT mice associated with much less OAA. Respiration on complex I substrates for mitochondria of both strains of mice and for WT and UCP1KO mice followed the more classical dynamics of greater O2 flux at lower Δψ. Summary: These findings support the above-mentioned mechanism for OAA- and Δψ-dependent complex II respiration and support its physiologically relevance. Conclusions: Oxaloacetate regulates mitochondrial complex II dynamics in a manner dependent on inner membrane potential as intrinsically determined by mouse strain or genetic manipulation. NIH award, 1 R01 DK123043-01A1, VA Merit Review Award 2 I01 BX000285-06, and the Iowa Fraternal Order of the Eagles This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression.

We previously found that skeletal muscle mitochondria incubated at low membrane potential (ΔΨ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low, accumulate oxaloacetate (OAA) in amounts sufficient to inhibit complex II respiration. We proposed a mechanism wherein low ΔΨ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance, we carried out studies of mice with inherently different levels of IBAT mitochondrial inner membrane potential. Isolated complex II (succinate)-energized IBAT mitochondria from obesity-resistant 129SVE mice compared with obesity-prone C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower ΔΨ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower ΔΨ, exhibited much lower respiration, twofold greater OAA concentrations, much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared with the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater ΔΨ, ROS, and NADH, but equal or greater O2 flux compared with WT mitochondria. GDP addition, compared with no GDP, increased ΔΨ and complex II respiration in wild-type (WT) mice associated with much less OAA. Respiration on complex I substrates followed the more classical dynamics of greater respiration at lower ΔΨ. These findings support the abovementioned mechanism for OAA- and ΔΨ-dependent complex II respiration and support its physiological relevance.NEW & NOTEWORTHY We examined mitochondrial respiration initiated at mitochondrial complex II in mice with varying degrees of brown adipose tissue UCP1 expression. We show that, by affecting inner membrane potential, UCP1 expression determines reverse electron transport from complex II to complex I and, consequently, the NADH/NAD+ ratio. Accordingly, this regulates the level of oxaloacetate accumulation and the extent of oxaloacetate inhibition of complex II.

Metabolic clearance of oxaloacetate and mitochondrial complex II respiration: Divergent control in skeletal muscle and brown adipose tissue

At low inner mitochondrial membrane potential (ΔΨ) oxaloacetate (OAA) accumulates in the organelles concurrently with decreased complex II-energized respiration. This is consistent with ΔΨ-dependent OAA inhibition of succinate dehydrogenase. To assess the metabolic importance of this process, we tested the hypothesis that perturbing metabolic clearance of OAA in complex II-energized mitochondria would alter O2 flux and, further, that this would occur in both ΔΨ and tissue-dependent fashion. We carried out respiratory and metabolite studies in skeletal muscle and interscapular brown adipose tissue (IBAT) directed at the effect of OAA transamination to aspartate (catalyzed by the mitochondrial form of glutamic-oxaloacetic transaminase, Got2) on complex II-energized respiration. Addition of low amounts of glutamate to succinate-energized mitochondria at low ΔΨ increased complex II (succinate)-energized respiration in muscle but had little effect in IBAT mitochondria. The transaminase inhibitor, aminooxyacetic acid, increased OAA concentrations and impaired succinate-energized respiration in muscle but not IBAT mitochondria at low but not high ΔΨ. Immunoblotting revealed that Got2 expression was far greater in muscle than IBAT mitochondria. Because we incidentally observed metabolism of OAA to pyruvate in IBAT mitochondria, more so than in muscle mitochondria, we also examined the expression of mitochondrial oxaloacetate decarboxylase (ODX). ODX was detected only in IBAT mitochondria. In summary, at low but not high ΔΨ, mitochondrial transamination clears OAA preventing loss of complex II respiration: a process far more active in muscle than IBAT mitochondria. We also provide evidence that OAA decarboxylation clears OAA to pyruvate in IBAT mitochondria.

Membrane potential-dependent regulation of mitochondrial complex II by oxaloacetate in interscapular brown adipose tissue.

Classically, mitochondrial respiration responds to decreased membrane potential (ΔΨ) by increasing respiration. However, we found that for succinate‐energized complex II respiration in skeletal muscle mitochondria (unencumbered by rotenone), low ΔΨ impairs respiration by a mechanism culminating in oxaloacetate (OAA) inhibition of succinate dehydrogenase (SDH). Here, we investigated whether this phenomenon extends to far different mitochondria of a tissue wherein ΔΨ is intrinsically low, i.e., interscapular brown adipose tissue (IBAT). Also, to advance our knowledge of the mechanism, we performed isotopomer studies of metabolite flux not done in our previous muscle studies. In additional novel work, we addressed possible ways ADP might affect the mechanism in IBAT mitochondria. UCP1 activity, and consequently ΔΨ, were perturbed both by GDP, a well‐recognized potent inhibitor of UCP1 and by the chemical uncoupler carbonyl cyanide m‐chlorophenyl hydrazone (FCCP). In succinate‐energized mitochondria, GDP increased ΔΨ but also increased rather than decreased (as classically predicted under low ΔΨ) O2 flux. In GDP‐treated mitochondria, FCCP reduced potential but also decreased respiration. Metabolite studies by NMR and flux analyses by LC‐MS support a mechanism, wherein ΔΨ effects on the production of reactive oxygen alters the NADH/NAD+ ratio affecting OAA accumulation and, hence, OAA inhibition of SDH. We also found that ADP‐altered complex II respiration in complex fashion probably involving decreased ΔΨ due to ATP synthesis, a GDP‐like nucleotide inhibition of UCP1, and allosteric enzyme action. In summary, complex II respiration in IBAT mitochondria is regulated by UCP1‐dependent ΔΨ altering substrate flow through OAA and OAA inhibition of SDH.

SSADH Variants Increase Susceptibility of U87 Cells to Mitochondrial Pro-Oxidant Insult

Succinate semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme, encoded by ALDH5A1, mainly involved in γ-aminobutyric acid (GABA) catabolism and energy supply of neuronal cells, possibly contributing to antioxidant defense. This study aimed to further investigate the antioxidant role of SSADH, and to verify if common SNPs of ALDH5A1 may affect SSADH activity, stability, and mitochondrial function. In this study, we used U87 glioblastoma cells as they represent a glial cell line. These cells were transiently transfected with a cDNA construct simultaneously harboring three SNPs encoding for a triple mutant (TM) SSADH protein (p.G36R/p.H180Y/p.P182L) or with wild type (WT) cDNA. SSADH activity and protein level were measured. Cell viability, lipid peroxidation, mitochondrial morphology, membrane potential (ΔΨ), and protein markers of mitochondrial stress were evaluated upon Paraquat treatment, in TM and WT transfected cells. TM transfected cells show lower SSADH protein content and activity, fragmented mitochondria, higher levels of peroxidized lipids, and altered ΔΨ than WT transfected cells. Upon Paraquat treatment, TM cells show higher cell death, lipid peroxidation, 4-HNE protein adducts, and lower ΔΨ, than WT transfected cells. These results reinforce the hypothesis that SSADH contributes to cellular antioxidant defense; furthermore, common SNPs may produce unstable, less active SSADH, which could per se negatively affect mitochondrial function and, under oxidative stress conditions, fail to protect mitochondria.

Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.

We recently reported that membrane potential (ΔΨ) primarily determines the relationship of complex II-supported respiration by isolated skeletal muscle mitochondria to ADP concentrations. We observed that O2 flux peaked at low ADP concentration ([ADP]) (high ΔΨ) before declining at higher [ADP] (low ΔΨ). The decline resulted from oxaloacetate (OAA) accumulation and inhibition of succinate dehydrogenase. This prompted us to question the effect of incremental [ADP] on respiration in interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low because of uncoupling protein 1 (UCP1). We found that succinate-energized IBAT mitochondria, even in the absence of ADP, accumulate OAA and manifest limited respiration, similar to muscle mitochondria at high [ADP]. This could be prevented by guanosine 5'-diphosphate inhibition of UCP1. NAD+ cycling with NADH requires complex I electron flow and is needed to form OAA. Therefore, to assess the role of electron transit, we perturbed flow using a small molecule, N1-(3-acetamidophenyl)-N2-(2-(4-methyl-2-(p-tolyl)thiazol-5-yl)ethyl)oxalamide. We observed decreased OAA, increased NADH/NAD+, and increased succinate-supported mitochondrial respiration under conditions of low ΔΨ (IBAT) but not high ΔΨ (heart). In summary, complex II-energized respiration in IBAT mitochondria is tempered by complex I-derived OAA in a manner dependent on UCP1. These dynamics depend on electron transit in complex I.-Fink, B. D., Yu, L., Sivitz, W. I. Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow.

Adaptations of Vancomycin-Intermediate Sequence Type 72 Methicillin-Resistant Staphylococcus aureus for Daptomycin Nonsusceptibility.

In Korea, the major clonal type of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is sequence type 72 (ST72) with staphylococcal cassette chromosome mec (SCCmec) type IV (ST72-MRSA-IV). In this study, we used a previously well-characterized isogenic pair of ST72 vancomycin (VAN) susceptible-and VAN intermediate-MRSA strains (VSSA303 and VISA072) and several VSSA strains complemented with plasmids expressing single-point mutated genes (dprAG196C, femAF92C, vraRE127K, and vraSRE127K) identified in the VISA strain. Using the strain set, we assessed the (1) susceptibilities to daptomycin (DAP) and cationic antimicrobial peptides (CAMPs), (2) alterations in cell envelope phenotypes, such as cell wall autolysis, surface positive charge, and membrane potential (ΔΨ), (3) transcriptional expression profiles of genes involved in surface charge regulation and changes of ΔΨ, and (4) cytokine stimulation profiles in murine macrophages. The vraRE127K mutation could enhance surface positive charge through mprF- and dltABCD-independent mechanisms with thickened cell wall. However, none of the single-point mutated genes increased DAP resistance. The DAP nonsusceptible (DAP-NS) phenotype observed in VISA072 strain likely resulted from the combined effects of low ΔΨ and increased positive surface charge. These results suggest that physicochemical alterations in cell envelope are involved in the survival response of DAP-NS VISA072 in sites of infections.

Effects of rotenone and pyridaben on complex I electron transfer and on mitochondrial nitric oxide synthase functional activity

Rotenone and pyridaben were tested on activities and properties of rat brain mitochondria determining Ki (inhibitor concentration at half maximal inhibition) and Imax (% of inhibition at maximal inhibitor concentration). The assayed activities were complexes I, II and IV, respiration in states 3, 3u (uncoupled) and 4, biochemical and functional activities of mitochondrial nitric oxide synthase (mtNOS), and inner membrane potential. Selective inhibitions of complex I activity, mitochondrial respiration and membrane potential with malate-glutamate as substrate were observed, with a Ki of 0.28-0.36nmol inhibitor/mg of mitochondrial protein. Functional mtNOS activity was half-inhibited at 0.70-0.74nmol inhibitor/mg protein in state 3 mitochondria and at 2.52-2.98nmol inhibitor/mg protein in state 3u mitochondria. This fact is interpreted as an indication of mtNOS being structurally adjacent to complex I with an intermolecular mtNOS-complex I hydrophobic bonding that is stronger at high Δψ and weaker at low Δψ.

Comparative evaluation of hypoxic–ischemic brain injury by flow cytometric analysis of mitochondrial membrane potential with JC-1 in neonatal rats

We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) with a fluorescent probe, JC-1 (5,5′,6,6′–tetrachloro-1,1′,3,3′-tetraethyl benzimidazolo-carbocyanine iodide), for the quantitative evaluation of neonatal hypoxic–ischemic brain injury. Seven-day-old rat pups were subjected to 2h of 8% oxygen following unilateral carotid artery ligation. Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI), and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI), terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c. Flow cytograms of JC-1 showed two distinct sub-populations with different ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V+/PI+ necrotic cells, TUNEL-positive cells, TTC infarct area and western blot of cytosolic cytochrome c, and negative correlation with annexin V−/PI− live cells. In summary, low ΔΨ measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI. Therefore, fluorocytometric analysis of ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.

The Carboxyl-terminal Third of the Dicarboxylate Carrier Is Crucial for Productive Association with the Inner Membrane Twin-pore Translocase

The carrier proteins of the mitochondrial inner membrane consist of three structurally related tandem repeats (modules). Several different, and in some cases contradictory, views exist on the role individual modules play in carrier transport across the mitochondrial membranes and how they promote protein insertion into the inner membrane. Thus, by use of specific translocation intermediates, we performed a detailed analysis of carrier biogenesis and assessed the physical association of carrier modules with the inner membrane translocation machinery. Here we have reported that each module of the dicarboxylate carrier contains sufficient targeting information for its transport across the outer mitochondrial membrane. The carboxyl-terminal module possesses major targeting information to facilitate the direct binding of the carrier protein to the inner membrane twin-pore translocase and subsequent insertion into the inner membrane in a membrane potential-dependent manner. We concluded that, in this case, a single structural repeat can drive inner membrane insertion, whereas all three related units contribute targeting information for outer membrane translocation.

Alterations in mitochondrial membrane potential (δϕ) and oxidative stress in men with male infertility.

Objective: Oxidative stress due to excessive production of reactive oxygen species (ROS) is known to impair male fertility. Sperm mitochondria possess a distinct genome (mtDNA) which is more susceptible to ROS-induced damage than nuclear DNA. Fragmentation of genomic DNA is one of the hallmarks of apoptosis. Functional integrity of sperm mitochondria is indicative of apoptosis. Alterations in mitochondrial membrane potential (δψ) may occur during early stages of apoptosis. The ability to discriminate between mitochondria exhibiting high membrane potential from those having low δψ provides a rigorous estimate to metabolic function. The purpose of this study was to determine the effect of ROS on mitochondrial membrane potential and to evaluate its correlation with conventional semen parameter.

Intrinsic uncoupling of cytochrome c oxidase may cause the maternally inherited mitochondrial diseases MELAS and LHON

Mutations in the human mtDNA gene encoding subunit III of cytochrome c oxidase (CO) have been reported to cause MELAS and LHON. Paracoccus denitrificans cells expressing substitutions homologous to these MELAS- and LHON-causing mutations had lower growth yield than wild type cells and lower efficiency of proton pumping by CO (e.g. lower H +/e − ratio and lower ΔΨ), but had similar CO activity. These results indicate that both substitutions (F263L>A212T) cause intrinsic uncoupling, which may be the direct cause of the diseases. These results also suggest that subunit III is involved in proton pumping.

Membrane potential‐dependent calcium transport in right‐side‐out plasma membrane vesicles from Zea mays L. roots

SummaryRight‐side‐out plasma membrane vesicles isolated from Zea mays roots were used to study membrane potential (ΔΨ)‐dependent Ca2+ transport. Membrane potentials were imposed on the vesicles using either K+ concentration gradients and valinomycin or SCN concentration gradients, and the size of the imposed ΔΨ was measured with [14C]tetraphenylphosphonium. Uptake of 45Ca2+ into the vesicles was stimulated by inside‐negative ΔΨ. The rate of transport increased to a maximum at a ΔΨ of about ‐80 mV and then declined at more negative ΔΨ. When extravesicular Ca2+ concentration was varied, uptake was maximal in the range 100–200 μM Ca2+. Neither dihydropyridine nor phenylalkylamine Ca2+ channel blockers had any effect on Ca2+ uptake but 30 μM ruthenium red was completely inhibitory with half maximal inhibition at 10–15 μM ruthenium red. Calcium transport was also inhibited by inorganic cations. Zn2+, Gd3+ and Mg2+ inhibited by a maximum of 30% while La3+, Nd3+ and Mn2+ inhibited by 70%. The inhibitory effects of La3+ and Gd3+ were additive. Lanthanum‐insensitive Ca2+ five Ca2+ transport was totally inhibited by 80 μM Gd3+ and showed maximum activity at a ΔΨ of ‐60 mV, with less uptake at both higher and lower ΔΨ. Lanthanum and Gd3+ also inhibited Ca2+ uptake into protoplasts isolated from Zea roots and their individual and combined effects were similar in extent to those observed with plasma membrane vesicles. It is concluded that maize root plasma membrane contains two Ca2+‐permeable channels that can be distinguished by their susceptibility to inhibition by La3+ and Gd3+. Both are inhibited by ruthenium red but not by other organic Ca2+ channel blockers.

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they adsorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (Δψ). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low Δψ due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high Δψ. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.

The role of the proton-motive force and electron flow in light-driven solute transport in Rhodopseudomonas sphaeroides.

The initial rate of uptake of alanine was studied in whole cells of Rhodopseudomonas sphaeroides under conditions such that the proton-motive force generated by light-induced cyclic electron transfer, was composed of the membrane potential only. The proton-motive force was varied by varying the light intensity or by pretreatment of the cells with the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone. At constant light intensity a threshold delta psi is required before alanine uptake occurs. Above this threshold value the rate of alanine uptake increases with the delta psi. The threshold value of the delta psi is higher the lower the light intensity. Under conditions of constant delta psi the rate of alanine uptake increases linearly with the light intensity. At low delta psi values a threshold light intensity is required which is higher the lower the delta psi. These results demonstrate that both a proton-motive force and electron transfer are required for alanine uptake.

Safranine as membrane potential probe in rat liver mitochondria

The absorbance changes accompanying safranine uptake due either to respiration or to K + diffusion potential are maximal at a point of equivalence between number of dye molecules and binding sites (“saturation” point) and decrease both below and above this point. This behavior is in accord with a model where safranine uptake is followed by stacking on adjacent membrane sites below saturation and destacking on nonadjacent sites above saturation. The values of the endpoint titrations are similar for the respiration- and K + diffusion potentialinduced safranine uptakes only at protein concentrations above saturation. Calibration of the safranine response with K + diffusion potentials indicates a linear region at low Δψ and a deviation from linearity at high Δψ. The region of linear safranine response depends on the dye/protein ratio. Increase of dye/protein ratio leads to expansion of the linear range. However calibration at high dye/protein ratios is limited by the loss of matrix K +. At constant dye/protein ratios increase of dye concentration shifts the calibration curve at lower Δψ values. The half-time of the safranine response increases hyperbolically with the decrease of protein at constant dye concentration and with the decrease of dye concentration at constant dye/protein ratios. Uptake and binding of safranine result in mitochondrial damage with respect to degree of energy coupling, rate of electron transport, and ADP-stimulated respiration. Oxygen pulses to anaerobic mitochondria result in an electrical field which may be followed with the safranine response. The minimal amount of oxygen capable of inducing a maximal rate of safranine uptake is 0.5 natom × mg protein −1. Since the rate of safranine uptake is linear with Δψ, this amount of oxygen corresponds to the extent of charge separation required for a steady state Δψ.