A whole blood approach improves speed and accuracy when measuring mitochondrial respiration in intact avian blood cells.

Understanding mitochondrial biology and pathology is key to understanding the evolution of animal form and function. However, mitochondrial measurement often involves invasive, or even terminal, sampling, which can be difficult to reconcile in wild models or longitudinal studies. Non-mammal vertebrates contain mitochondria in their red blood cells, which can be exploited for minimally invasive mitochondrial measurement. Several recent bird studies have measured mitochondrial function using isolated blood cells. Isolation adds time in the laboratory and might be associated with physiological complications. We developed and validated a protocol to measure mitochondrial respiration in bird whole blood. Endogenous respiration was comparable between isolated blood cells and whole blood. However, respiration towards oxidative phosphorylation was higher in whole blood, and whole blood mitochondria were better coupled and had higher maximum working capacity. Whole blood measurement was also more reproducible than measurement on isolated cells for all traits considered. Measurements were feasible over a 10-fold range of sample volumes, although both small and large volumes were associated with changes to respiratory traits. The protocol was compatible with long-term storage: after 24 h at 5°C without agitation, all respiration traits but maximum working capacity remained unchanged, the latter decreasing by 14%. Our study suggests that whole blood measurement provides faster, more reproducible, and more biologically and physiologically relevant (mitochondrial integrity) assessment of mitochondrial respiration. We recommend future studies to take a whole blood approach unless specific circumstances require the use of isolated blood cells.

Plasticity of mitochondrial function safeguards phosphorylating respiration during in vitro simulation of rest-phase hypothermia.

Many animals downregulate body temperature to save energy when resting (rest-phase hypothermia). Small birds that winter at high latitudes have comparatively limited capacity for hypothermia and so pay large energy costs for thermoregulation during cold nights. Available evidence suggests this process is fueled by adenosine triphosphate (ATP)-dependent mechanisms. Most ATP is produced by oxidative phosphorylation in the mitochondria, but mitochondrial respiration may be lower during hypothermia because of the temperature dependence of biological processes. This can create conflict between increased organismal ATP demand and a lower mitochondrial capacity to provide it. We studied this in blood cell mitochondria of wild great tits (Parus major) by simulating rest-phase hypothermia via a 6°C reduction in assay temperature in vitro. The birds had spent the night preceding the experiment in thermoneutrality or in temperatures representing mild or very cold winter nights, but night temperatures never affected mitochondrial respiration. However, across temperature groups, endogenous respiration was 14% lower in hypothermia. This did not reflect general thermal suppression of mitochondrial function because phosphorylating respiration was unaffected by thermal state. Instead, hypothermia was associated with a threefold reduction of leak respiration, from 17% in normothermia to 4% in hypothermia. Thus, the coupling of total respiration to ATP production was 96% in hypothermia, compared to 83% in normothermia. Our study shows that the thermal insensitivity of phosphorylation combined with short-term plasticity of leak respiration may safeguard ATP production when endogenous respiration is suppressed. This casts new light on the process by which small birds endure harsh winter cold and warrants future tests across tissues in vivo.

Acid-triggered controlled release and fluorescence-switchable phthalocyanine nanoassemblies combined with O2-economizer for tumor imaging and collaborative photodynamic antitumor therapy.

Photodynamic therapy (PDT) has emerged as a highly efficacious therapeutic modality for malignant tumors owing to its non-invasive property and minimal adverse effects. However, the pervasive hypoxic microenvironment within tumors significantly compromises the efficacy of oxygen-dependent PDT, posing a formidable challenge to the advancement of high-efficiency PDT. Here, we developed a nanostructured photosensitizer (PS) assembled by cationic and anionic zinc phthalocyanines to load oxygen-throttling drug atovaquone (ATO), which was subsequently coated with polydopamine to obtain the final product ATO/ZnPc-CA@DA. ATO/ZnPc-CA@DA exhibited excellent stability, particularly in the blood milieu. Interestingly, the acidic microenvironment can trigger drug release from ATO/ZnPc-CA@DA, leading to a significant enhancement in fluorescence and an augmented generation of reactive oxygen species (ROS). ATO/ZnPc-CA@DA can induce synergistic cytotoxicity of PS and ATO, and significantly enhance the killing ability against tumor cells under hypoxic conditions. The mechanism underlying cytotoxicity of ATO/ZnPc-CA@DA was demonstrated to be associated with augmented cell apoptosis, disruption of mitochondrial membrane potential, diminished ATP production, heightened intracellular ROS generation, and reduced intracellular oxygen consumption. The animal experiments indicated that ATO/ZnPc-CA@DA possessed enhanced tumor targeting capability, along with a reduction in PS distribution within normal organs. Furthermore, ATO/ZnPc-CA@DA exhibited enhanced inhibitory effect on tumor growth and caused aggravated damage to tumor tissue. The construction strategy of nanostructured PS and the synergistic antitumor principle of combined oxygen-throttling drugs can be applied to other PSs, thereby advancing the development of photodynamic antitumor therapy and promoting the clinical translation.

Deactivation blocks proton pathways in the mitochondrial complex I.

Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane-bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the "active"-to-"deactive" transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.

Susceptibility of the Different Oxygen-Sensing Probes to Interferences in Respirometric Bacterial Assays with Complex Media.

Respirometric microbial assays are gaining popularity, but their uptake is limited by the availability of optimal O2 sensing materials and the challenge of validating assays with complex real samples. We conducted a comparative evaluation of four different O2-sensing probes based on Pt-porphyrin phosphors in respirometric bacterial assays performed on standard time-resolved fluorescence reader. The macromolecular MitoXpress, nanoparticle NanO2 and small molecule PtGlc4 and PtPEG4 probes were assessed with E. coli cells in five growth media: nutrient broth (NB), McConkey (MC), Rapid Coliform ChromoSelect (RCC), M-Lauryl lauryl sulfate (MLS), and Minerals-Modified Glutamate (MMG) media. Respiration profiles of the cells were recorded and analyzed, along with densitometry profiles and quenching studies of individual media components. This revealed several limiting factors and interferences impacting assay performance, which include probe quenched lifetime, instrument temporal resolution, inner filter effects (mainly by indicator dyes), probe binding to lipophilic components, and dynamic and static quenching by media components. The study allowed for the ranking of the probes based on their ruggedness, resilience to interferences and overall performance in respirometric bacterial assays. The 'shielded' probe NanO2 outperformed the established MitoXpress probe and the small molecule probes PtGlc4 and PtPEG4.

Serum concentration impacts myosin heavy chain expression but not cellular respiration in human LHCN-M2 myoblasts undergoing differentiation.

NEW FINDINGS: What is the central question of this study? Does the concentration of human serum affect skeletal muscle differentiation and cellular respiration of LHCN-M2 myoblasts? What is the main finding and its importance? The concentration of serum used to differentiate LHCN-M2 skeletal muscle cells impacts the coverage of myosin heavy chain, a marker of terminally differentiated myotubes. Normalisation of mitochondrial function data to total protein negates the differences observed in absolute values, which differ as a result of increased protein content when differentiation occurs with increasing concentration of serum. ABSTRACT: The human LHCN-M2 myoblast cell line has the potential to be used to investigate skeletal muscle development and metabolism. Experiments were performed to determine how different concentrations of human serum affect myogenic differentiation and mitochondrial function of LHCN-M2 cells. LHCN-M2 myoblasts were differentiated in serum-free medium, 0.5% or 2% human serum for 5 and 10 days. Myotube formation was assessed by immunofluorescence staining of myosin heavy chain (MHC) and molecularly by mRNA expression of Myogenic differentiation 1 (MYOD1) and Myoregulatory factor 5 (MYF5). Following differentiation, mitochondrial function was assessed to establish the impact of serum concentration on mitochondrial function. Time in differentiation increased mRNA expression of MYOD1 (day 5, 6.58 ± 1.33-fold; and day 10, 4.28 ± 1.71-fold) (P = 0.012), while suppressing the expression of MYF5 (day 5, 0.21 ± 0.11-fold; and day 10, 0.06 ± 0.03-fold) (P = 0.001), regardless of the serum concentration. Higher serum concentrations increased MHC area (serum free, 11.92 ± 0.85%; 0.5%, 23.10 ± 5.82%; 2%, 43.94 ± 8.92%) (P = 0.001). Absolute basal respiration approached significance (P = 0.06) with significant differences in baseline oxygen consumption rate (P = 0.025) and proton leak (P = 0.006) when differentiated in 2% human serum, but these were not different between conditions when normalised to total protein. Our findings show that increasing concentrations of serum of LHCN-M2 skeletal muscle cells into multinucleated myotubes, but this does not affect relative mitochondrial function.

Bacterial approaches to sensing and responding to respiration and respiration metabolites.

Bacterial respiration of diverse substrates is a primary contributor to the diversity of life. Respiration also drives alterations in the geosphere and tethers ecological nodes together. It provides organisms with a means to dissipate reductants and generate potential energy in the form of an electrochemical gradient. Mechanisms have evolved to sense flux through respiratory pathways and sense the altered concentrations of respiration substrates or byproducts. These genetic regulatory systems promote efficient utilization of respiration substrates, as well as fine-tune metabolism to promote cellular fitness and negate the accumulation of toxic byproducts. Many bacteria can respire one or more chemicals, and these regulatory systems promote the prioritization of high-energy metabolites. Herein, we focus on regulatory paradigms and discuss systems that sense the concentrations of respiration substrates and flux through respiratory pathways. This is a broad field of study, and therefore we focus on key fundamental and recent developments and highlight specific systems that capture the diversity of sensing mechanisms.

Carrier Free O2 -Economizer for Photodynamic Therapy Against Hypoxic Tumor by Inhibiting Cell Respiration.

Abnormal tumor metabolism causes the hypoxic microenvironment, which greatly limits the efficacy of photodynamic therapy (PDT). In this work, a strategy of metabolic reprogramming is proposed to economize O2 for enhanced PDT against hypoxic tumors. The carrier-free O2 -economizer (designated as LonCe) is prepared based on the metabolic antitumor drug of Lonidamine (Lon) and the photosensitizer of chlorin e6 (Ce6). By virtue of intermolecular interactions, Lon and Ce6 self-assemble into nanosized LonCe with favorable stability and high drug contents. Compared with Ce6, LonCe exhibits an improved cellular uptake and photodynamic property for tumor treatment. Moreover, LonCe is capable of inhibiting cell metabolism and mitochondrial respiration to remit the tumor hypoxia, which would promote reactive oxygen species (ROS) production and elevate the PDT efficacy on tumor suppression. In vivo experiments indicate that intravenously injected LonCe prefers to accumulate at the tumor site for highly efficient PDT regardless of the hypoxic environment. Besides, the self-delivery LonCe is fabricated without any carriers, which avoids the excipients induced system toxicity and immunogenicity in vivo. This carrier-free nanomedicine with cell respiratory inhibition mechanism would expedite the development and clinical translation of photodynamic nanoplatforms in tumor treatment.

Dual effects of the tryptophan-derived bacterial metabolite indole on colonic epithelial cell metabolism and physiology: comparison with its co-metabolite indoxyl sulfate.

Indole, which is produced by the intestinal microbiota from L-tryptophan, is recovered at millimolar concentrations in the human feces. Indoxyl sulfate (IS), the main indole co-metabolite, can be synthesized by the host tissues. Although indole has been shown to restore intestinal barrier function in experimental colitis, little is known on the effects of indole and IS on colonic epithelial cell metabolism and physiology. In this study, we compared the effects of indole and IS on the human colonic epithelial HT-29 Glc-/+ and Caco-2 cell lines, exposed to these compounds for 1-48 h. Indole, but not IS, was cytotoxic at 5 mM, altering markedly colonocyte proliferation. Both molecules, used up to 2.5 mM, induced a transient oxidative stress in colonocytes, that was detected after 1 h, but not after 48 h exposure. This was associated with the induction after 24 h of the expression of glutathione reductase, heme oxygenase, and cytochrome P450 (CYP)1B1. Indole and IS used at 2.5 mM impaired colonocyte respiration by diminishing mitochondrial oxygen consumption and maximal respiratory capacity. Indole, but not IS, displayed a slight genotoxic effect on colonocytes. Indole, but not IS, increased transepithelial resistance in colonocyte monolayers. Indole and IS used at 2.5 mM, induced a secretion of the pro-inflammatory interleukin-8 after 3 h incubation, and an increase of tumor necrosis factor-α secretion after 48 h. Although our results suggest beneficial effect of indole on epithelial integrity, overall they indicate that indole and IS share adverse effects on colonocyte respiration and production of reactive oxygen species, in association with the induction of enzymes of the antioxidant defense system. This latter process can be viewed as an adaptive response toward oxidative stress. Both compounds increased the production of inflammatory cytokines from colonocytes. However, only indole, but not IS, affected DNA integrity in colonocytes. Since colonocytes little convert indole to IS, the deleterious effects of indole on colonocytes appear to be unrelated to its conversion to IS.

Mitochondrial Respiration in Drosophila Ovaries after a Full Cycle of Oogenesis under Simulated Microgravity.

Studies of the function of the female reproductive system in zero gravity are urgent for the future exploration of deep space. Female reproductive cells, oocytes, are rich in mitochondria, which allow oocytes to produce embryos. The rate of cellular respiration was determined to assess the functional state of the mitochondrial apparatus in Drosophila melanogaster ovaries in which the full cycle of oogenesis took place under simulated microgravity. Since cellular respiration depends on the state of the cytoskeleton, the contents of the main cytoskeletal proteins were determined by Western blotting. To modulate the structure of the cytoskeleton, essential phospholipids were administered per os at a dosage of 500 mg/kg in medium. The results of this study show that after a full cycle of oogenesis under simulated microgravity, the rate of cellular respiration in the fruit fly ovaries increases, apparently due to complex II of the respiratory chain. At the same time, we did not find any changes in the area of oocytes or in the content of proteins in the respiratory chain. However, changes were found in the relative contents of proteins of the actin cytoskeleton. There were no changes of essential phospholipids and no increase in the rate of cellular respiration of the ovaries after exposure to simulated microgravity. However, in the control, the administration of essential phospholipids led to a decrease in the efficiency of oxygen consumption in the flies' ovaries due to complexes IV-V.

Expanded role of the Cu-sensing transcription factor Mac1p in Candida albicans.

As part of the innate immune response, the host withholds metal micronutrients such as Cu from invading pathogens, and microbes respond through metal starvation stress responses. With the opportunistic fungal pathogen Candida albicans, the Cu-sensing transcription factor Mac1p governs the cellular response to Cu starvation by controlling Cu import. Mac1p additionally controls reactive oxygen species (ROS) homeostasis by repressing a Cu-containing superoxide dismutase (SOD1) and inducing Mn-containing SOD3 as a non-Cu alternative. We show here that C. albicans Mac1p is essential for virulence in a mouse model for disseminated candidiasis and that the cellular functions of Mac1p extend beyond Cu uptake and ROS homeostasis. Specifically, mac1∆/∆ mutants are profoundly deficient in mitochondrial respiration and Fe accumulation, both Cu-dependent processes. Surprisingly, these deficiencies are not simply the product of impaired Cu uptake; rather mac1∆/∆ mutants appear defective in Cu allocation. The respiratory defect of mac1∆/∆ mutants was greatly improved by a sod1∆/∆ mutation, demonstrating a role for SOD1 repression by Mac1p in preserving respiration. Mac1p downregulates the major Cu consumer SOD1 to spare Cu for respiration that is essential for virulence of this fungal pathogen. The implications for such Cu homeostasis control in other pathogenic fungi are discussed.

Redox-coupled quinone dynamics in the respiratory complex I.

Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.

State of Drosophila melanogaster Ovaries after a Full Cycle of Gametogenesis under Microgravity Modeling: Cellular Respiration and the Content of Cytoskeletal Proteins.

The effect of weightlessness on gametogenesis and the functional state of female germ cells are still poorly understood. We studied the ovaries of Drosophila melanogaster, the full development cycle of which (from zygote to sexually mature adults) passed under simulated microgravity by a random positioning machine. The rate of cellular respiration was studied by polarography as a parameter reflecting the functional state of mitochondria. The content of cytoskeletal proteins and histones was determined using Western blotting. The relative content of mRNA was determined using qRT-PCR. The results obtained indicated an increase in the rate of cellular respiration under simulated microgravity conditions during the full cycle of gametogenesis in Drosophila melanogaster due to complex I of the respiratory chain. In addition, an increase in the contents of actin cytoskeleton components was observed against the background of an increase in the mRNA content of the cytoskeleton's encoding genes. Moreover, we observed an increase in the relative content of histone H3 acetylated at Lys9 and Lys27, which may explain the increase in the expression of cytoskeletal genes. In conclusion, the formation of an adaptive pattern of functioning of the Drosophila melanogaster ovaries that developed under simulated microgravity includes structural and functional changes and epigenetic regulation.

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations.

Proton pumping A-type cytochrome c oxidase (CcO) terminates the respiratory chains of mitochondria and many bacteria. Three possible proton transfer pathways (D, K, and H channels) have been identified based on structural, functional, and mutational data. Whereas the D channel provides the route for all pumped protons in bacterial A-type CcOs, studies of bovine mitochondrial CcO have led to suggestions that its H channel instead provides this route. Here, we have studied H-channel function by performing atomistic molecular dynamics simulations on the entire, as well as core, structure of bovine CcO in a lipid-solvent environment. The majority of residues in the H channel do not undergo large conformational fluctuations. Its upper and middle regions have adequate hydration and H-bonding residues to form potential proton-conducting channels, and Asp51 exhibits conformational fluctuations that have been observed crystallographically. In contrast, throughout the simulations, we do not observe transient water networks that could support proton transfer from the N phase toward heme a via neutral His413, regardless of a labile H bond between Ser382 and the hydroxyethylfarnesyl group of heme a In fact, the region around His413 only became sufficiently hydrated when His413 was fixed in its protonated imidazolium state, but its calculated pKa is too low for this to provide the means to create a proton transfer pathway. Our simulations show that the electric dipole moment of residues around heme a changes with the redox state, hence suggesting that the H channel could play a more general role as a dielectric well.

Supramolecular Organization of Respiratory Complexes.

Since the discovery of the existence of superassemblies between mitochondrial respiratory complexes, such superassemblies have been the object of a passionate debate. It is accepted that respiratory supercomplexes are structures that occur in vivo, although which superstructures are naturally occurring and what could be their functional role remain open questions. The main difficulty is to make compatible the existence of superassemblies with the corpus of data that drove the field to abandon the early understanding of the physical arrangement of the mitochondrial respiratory chain as a compact physical entity (the solid model). This review provides a nonexhaustive overview of the evolution of our understanding of the structural organization of the electron transport chain from the original idea of a compact organization to a view of freely moving complexes connected by electron carriers. Today supercomplexes are viewed not as a revival of the old solid model but rather as a refined revision of the fluid model, which incorporates a new layer of structural and functional complexity.

Drosophila melanogaster Sperm under Simulated Microgravity and a Hypomagnetic Field: Motility and Cell Respiration.

The role of the Earth's gravitational and magnetic fields in the evolution and maintenance of normal processes of various animal species remains unclear. The aim of this work was to determine the effect of simulated microgravity and hypomagnetic conditions for 1, 3, and 6 h on the sperm motility of the fruit fly Drosophila melanogaster. In addition to the usual diet, the groups were administered oral essential phospholipids at a dosage of 500 mg/kg in medium. The speed of the sperm tails was determined by video recording and analysis of the obtained video files, protein content by western blotting, and cell respiration by polarography. The results indicated an increase in the speed of movement of the sperm tails after 6 h in simulated microgravity. The levels of proteins that form the axoneme of the sperm tail did not change, but cellular respiration was altered. A similar effect occurred with the administration of essential phospholipids. These results may be due to a change in the level of phosphorylation of motor proteins. Exposure to hypomagnetic conditions led to a decrease in motility after 6 h against a background of a decrease in the rate of cellular respiration due to complex I of the respiratory chain. This effect was not observed in the flies that received essential phospholipids. However, after 1 h under hypomagnetic conditions, the rate of cellular respiration also increased due to complex I, including that in the sperm of flies receiving essential phospholipids.

Sperm Motility of Mice under Simulated Microgravity and Hypergravity.

For deep space exploration, reproductive health must be maintained to preserve the species. However, the mechanisms underlying the effect of changes in gravity on male germ cells remain poorly understood. The aim of this study was to determine the effect of simulated micro- and hypergravity on mouse sperm motility and the mechanisms of this change. For 1, 3 and 6 h, mouse sperm samples isolated from the caudal epididymis were subjected to simulated microgravity using a random position machine and 2g hypergravity using a centrifuge. The experimental samples were compared with static and dynamic controls. The sperm motility and the percentage of motile sperm were determined using microscopy and video analysis, cell respiration was determined by polarography, the protein content was assessed by Western blotting and the mRNA levels were determined using qRT-PCR. The results indicated that hypergravity conditions led to more significant changes than simulated microgravity conditions: after 1 h, the speed of sperm movement decreased, and after 3 h, the number of motile cells began to decrease. Under the microgravity model, the speed of movement did not change, but the motile spermatozoa decreased after 6 h of exposure. These changes are likely associated with a change in the structure of the microtubule cytoskeleton, and changes in the energy supply are an adaptive reaction to changes in sperm motility.

Interplay of Nitric Oxide Synthase (NOS) and SrrAB in Modulation of Staphylococcus aureus Metabolism and Virulence.

Staphylococcus aureus nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of nos expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring nos mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a nos srrAB mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The nos srrAB mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the nos srrAB double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits S. aureus to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The nos, srrAB, and nos srrAB mutants showed comparable defects in endothelial intracellular survival, whereas the srrAB and nos srrAB mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant in vivo.

Calcium Deregulation and Mitochondrial Bioenergetics in GDAP1-Related CMT Disease.

The pathology of Charcot-Marie-Tooth (CMT), a disease arising from mutations in different genes, has been associated with an impairment of mitochondrial dynamics and axonal biology of mitochondria. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause several forms of CMT neuropathy, but the pathogenic mechanisms involved remain unclear. GDAP1 is an outer mitochondrial membrane protein highly expressed in neurons. It has been proposed to play a role in different aspects of mitochondrial physiology, including mitochondrial dynamics, oxidative stress processes, and mitochondrial transport along the axons. Disruption of the mitochondrial network in a neuroblastoma model of GDAP1-related CMT has been shown to decrease Ca2+ entry through the store-operated calcium entry (SOCE), which caused a failure in stimulation of mitochondrial respiration. In this review, we summarize the different functions proposed for GDAP1 and focus on the consequences for Ca2+ homeostasis and mitochondrial energy production linked to CMT disease caused by different GDAP1 mutations.

Metabolic activation-driven mitochondrial hyperpolarization predicts insulin secretion in human pancreatic beta-cells.

Mitochondrial metabolism plays a central role in insulin secretion in pancreatic beta-cells. Generation of protonmotive force and ATP synthesis from glucose-originated pyruvate are critical steps in the canonical pathway of glucose-stimulated insulin secretion. Mitochondrial metabolism is intertwined with pathways that are thought to amplify insulin secretion with mechanisms distinct from the canonical pathway, and the relative importance of these two pathways is controversial. Here I show that glucose-induced mitochondrial membrane potential (MMP) hyperpolarization is necessary for, and predicts, the rate of insulin secretion in primary cultured human beta-cells. When glucose concentration is elevated, increased metabolism results in a substantial MMP hyperpolarization, as well as in increased rates of ATP synthesis and turnover marked by faster cell respiration. Using modular kinetic analysis I explored what properties of cellular energy metabolism enable a large glucose-induced change in MMP in human beta-cells. I found that an ATP-dependent pathway activates glucose or substrate oxidation, acting as a positive feedback in energy metabolism. This activation mechanism is essential for concomitant fast respiration and high MMP, and for a high magnitude glucose-induced MMP hyperpolarization and therefore for insulin secretion.