A practical and robust method to evaluate metabolic fluxes in primary pancreatic islets.

OBJECTIVE: Evaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables, is expensive and poorly reproducible. Our aim was to establish a practical method to assess primary islet metabolic fluxes using standard commercial consumables. METHODS: Pancreatic islets were isolated from mice/rats, dispersed with trypsin, and adhered to pre-coated standard Seahorse or Resipher microplates. Oxygen consumption was evaluated using a Seahorse Extracellular Flux Analyzer or a Resipher Real-time Cell Analyzer. RESULTS: We provide a detailed protocol with all steps to optimize islet isolation with high yield and functionality. Our method requires a few islets per replicate; both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators and glucose. The technique was validated by other functional assays, which show these cells present conserved calcium influx and insulin secretion in response to glucose. We also show that our dispersed islets maintain robust basal respiration levels, in addition to maintaining up to 89% viability after five days in dispersed cultures. Furthermore, OCRs can be measured in Seahorse analyzers and in other plate respirometry systems, using standard materials. CONCLUSIONS: Overall, we established a practical and robust method to assess islet metabolic fluxes and oxidative phosphorylation, a valuable tool to uncover basic ß-cell metabolic mechanisms as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols.

How and when to measure mitochondrial inner membrane potentials.

The scientific literature on mitochondria has increased significantly over the years due to findings that these organelles have widespread roles in the onset and progression of pathological conditions such as metabolic disorders, neurodegenerative and cardiovascular diseases, inflammation, and cancer. Researchers have extensively explored how mitochondrial properties and functions are modified in different models, often using fluorescent inner mitochondrial membrane potential (ΔΨm) probes to assess functional mitochondrial aspects such as protonmotive force and oxidative phosphorylation. This review provides an overview of existing techniques to measure ΔpH and ΔΨm, highlighting their advantages, limitations, and applications. It discusses drawbacks of ΔΨm probes, especially when used without calibration, and conditions where alternative methods should replace ΔΨm measurements for the benefit of the specific scientific objectives entailed. Studies investigating mitochondria and their vast biological roles would be significantly advanced by the understanding of the correct applications as well as limitations of protonmotive force measurements and use of fluorescent ΔΨm probes, adopting more precise, artifact-free, sensitive, and quantitative measurements of mitochondrial functionality.

Increased glycolysis is an early consequence of palmitate lipotoxicity mediated by redox signaling.

Exposure to toxic levels of fatty acids (lipotoxicity) leads to cell damage and death and is involved in the pathogenesis of the metabolic syndrome. Since the metabolic consequences of lipotoxicity are still poorly understood, we studied the bioenergetic effects of the saturated fatty acid palmitate, quantifying changes in mitochondrial morphology, real-time oxygen consumption, ATP production sources, and extracellular acidification in hepatoma cells. Surprisingly, glycolysis was enhanced by the presence of palmitate as soon as 1 h after stimulus, while oxygen consumption and oxidative phosphorylation were unchanged, despite overt mitochondrial fragmentation. Palmitate only induced mitochondrial fragmentation if glucose and glutamine were available, while glycolytic enhancement did not require glutamine, showing it is independent of mitochondrial morphological changes. Redox state was altered by palmitate, as indicated by NAD(P)H quantification. Furthermore, the mitochondrial antioxidant mitoquinone, or a selective inhibitor of complex I electron leakage (S1QEL) further enhanced palmitate-induced glycolysis. Our results demonstrate that palmitate overload and lipotoxicity involves an unexpected and early increase in glycolytic flux, while, surprisingly, no changes in oxidative phosphorylation are observed. Interestingly, enhanced glycolysis involves signaling by mitochondrially-generated oxidants, uncovering a novel regulatory mechanism for this pathway.

Satellite cell self-renewal in endurance exercise is mediated by inhibition of mitochondrial oxygen consumption.

BACKGROUND: Skeletal muscle stem cells (satellite cells) are well known to participate in regeneration and maintenance of the tissue over time. Studies have shown increases in the number of satellite cells after exercise, but their functional role in endurance training remains unexplored. METHODS: Young adult mice were submitted to endurance exercise training and the function, differentiation, and metabolic characteristics of satellite cells were investigated in vivo and in vitro. RESULTS: We found that injured muscles from endurance-exercised mice display improved regenerative capacity, demonstrated through higher densities of newly formed myofibres compared with controls (evidenced by an increase in embryonic myosin heavy chain expression), as well as lower inflammation (evidenced by quantifying CD68-marked macrophages), and reduced fibrosis. Enhanced myogenic function was accompanied by an increased fraction of satellite cells expressing self-renewal markers, while control satellite cells had morphologies suggestive of early differentiation. The beneficial effects of endurance exercise were associated with satellite cell metabolic reprogramming, including reduced mitochondrial respiration (O2 consumption) under resting conditions (absence of muscle injury) and increased stemness. During proliferation or activated states (3 days after injury), O2 consumption was equal in control and exercised cells, while exercise enhanced myogenic colony formation. Surprisingly, inhibition of mitochondrial O2 consumption was sufficient to enhance muscle stem cell self-renewal characteristics in vitro. Moreover, transplanted muscle satellite cells from exercised mice or cells with reduced mitochondrial respiration promoted a significant reduction in inflammation compared with controls. CONCLUSIONS: Our results indicate that endurance exercise promotes self-renewal and inhibits differentiation in satellite cells, an effect promoted by metabolic reprogramming and respiratory inhibition, which is associated with a more favourable muscular response to injury.

Lifespan-extending interventions enhance lipid-supported mitochondrial respiration in Caenorhabditis elegans.

Dietary restriction and reduced reproduction have been linked to long lifespans in the vast majority of species tested. Although decreased mitochondrial mass and/or function are hallmarks of aging, little is known about the mechanisms by which these organelles contribute to physiological aging or to the effects of lifespan-extending interventions, particularly with respect to oxidative phosphorylation and energy production. Here, we employed the nematode Caenorhabditis elegans to examine the effects of inhibition of germline proliferation and dietary restriction, both of which extend the lifespan of C. elegans, on mitochondrial respiratory activity in whole animals and isolated organelles. We found that oxygen consumption rates and mitochondrial mass were reduced in wild-type (WT) C. elegans subjected to bacterial deprivation (BD) compared with animals fed ad libitum (AL). In contrast, BD decreased the rate of oxygen uptake but not mitochondrial mass in germline-less glp-1(e2144ts) mutants. Interestingly, mitochondria isolated from animals subjected to BD and/or inhibition of germline proliferation showed no differences in complex I-mediated respiratory activity compared to control mitochondria, whereas both interventions enhanced the efficiency with which mitochondria utilized lipids as respiratory substrates. Notably, the combination of BD and inhibition of germline proliferation further increased mitochondrial lipid oxidation compared to either intervention alone. We also detected a striking correlation between lifespan extension in response to BD and/or inhibition of germline proliferation and the capacity of C. elegans to generate ATP from lipids. Our results thus suggest that the ability to oxidize lipids may be determinant in enhanced longevity.

A selective inhibitor of mitofusin 1-ßIIPKC association improves heart failure outcome in rats.

We previously demonstrated that beta II protein kinase C (ßIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that ßIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. ßIIPKC siRNA or a ßIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-ßIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMßA, a rationally-designed peptide that selectively antagonizes Mfn1-ßIIPKC association. SAMßA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMßA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMßA may be a potential treatment for patients with heart failure.

Single Cell Oxygen Mapping (SCOM) by Scanning Electrochemical Microscopy Uncovers Heterogeneous Intracellular Oxygen Consumption.

We developed a highly sensitive oxygen consumption scanning microscopy system using platinized platinum disc microelectrodes. The system is capable of reliably detecting single-cell respiration, responding to classical regulators of mitochondrial oxygen consumption activity as expected. Comparisons with commercial multi-cell oxygen detection systems show that the system has comparable errors (if not smaller), with the advantage of being able to monitor inter and intra-cell heterogeneity in oxygen consumption characteristics. Our results uncover heterogeneous oxygen consumption characteristics between cells and within the same cell´s microenvironments. Single Cell Oxygen Mapping (SCOM) is thus capable of reliably studying mitochondrial oxygen consumption characteristics and heterogeneity at a single-cell level.

Caloric restriction protects livers from ischemia/reperfusion damage by preventing Ca2+-induced mitochondrial permeability transition.

Caloric restriction (CR) promotes lifespan extension and protects against many pathological conditions, including ischemia/reperfusion injury to the brain, heart and kidney. In the liver, ischemia/reperfusion damage is related to excessive mitochondrial Ca2+ accumulation, leading to the mitochondrial permeability transition. Indeed, liver mitochondria isolated from animals maintained on CR for 4 months were protected against permeability transition and capable of taking up Ca2+ at faster rates and in larger quantities. These changes were not related to modifications in mitochondrial respiratory activity, but rather to a higher proportion of ATP relative to ADP in CR liver mitochondria. Accordingly, both depletion of mitochondrial adenine nucleotides and loading mitochondria with exogenous ATP abolished the differences between CR and ad libitum (AL) fed groups. The prevention against permeability transition promoted by CR strongly protected against in vivo liver damage induced by ischemia/reperfusion. Overall, our results show that CR strongly protects the liver against ischemia/reperfusion and uncover a mechanism for this protection, through a yet undescribed diet-induced change in liver mitochondrial Ca2+ handling related to elevated intramitochondrial ATP.

Lack of XPC leads to a shift between respiratory complexes I and II but sensitizes cells to mitochondrial stress.

Genomic instability drives tumorigenesis and DNA repair defects are associated with elevated cancer. Metabolic alterations are also observed during tumorigenesis, although a causal relationship between these has not been clearly established. Xeroderma pigmentosum (XP) is a DNA repair disease characterized by early cancer. Cells with reduced expression of the XPC protein display a metabolic shift from OXPHOS to glycolysis, which was linked to accumulation of nuclear DNA damage and oxidants generation via NOX-1. Using XP-C cells, we show that mitochondrial respiratory complex I (CI) is impaired in the absence of XPC, while complex II (CII) is upregulated in XP-C cells. The CI/CII metabolic shift was dependent on XPC, as XPC complementation reverted the phenotype. We demonstrate that mitochondria are the primary source of H2O2 and glutathione peroxidase activity is compromised. Moreover, mtDNA is irreversibly damaged and accumulates deletions. XP-C cells were more sensitive to the mitochondrial inhibitor antimycin A, an effect also prevented in XPC-corrected cells. Our results show that XPC deficiency leads to alterations in mitochondrial redox balance with a CI/CII shift as a possible adaptation to lower CI activity, but at the cost of sensitizing XP-C cells to mitochondrial oxidative stress.

Soluble Uric Acid Activates the NLRP3 Inflammasome.

Uric acid is a damage-associated molecular pattern (DAMP), released from ischemic tissues and dying cells which, when crystalized, is able to activate the NLRP3 inflammasome. Soluble uric acid (sUA) is found in high concentrations in the serum of great apes, and even higher in some diseases, before the appearance of crystals. In the present study, we sought to investigate whether uric acid, in the soluble form, could also activate the NLRP3 inflammasome and induce the production of IL-1ß. We monitored ROS, mitochondrial area and respiratory parameters from macrophages following sUA stimulus. We observed that sUA is released in a hypoxic environment and is able to induce IL-1ß release. This process is followed by production of mitochondrial ROS, ASC speck formation and caspase-1 activation. Nlrp3-/- macrophages presented a protected redox state, increased maximum and reserve oxygen consumption ratio (OCR) and higher VDAC protein levels when compared to WT and Myd88-/- cells. Using a disease model characterized by increased sUA levels, we observed a correlation between sUA, inflammasome activation and fibrosis. These findings suggest sUA activates the NLRP3 inflammasome. We propose that future therapeutic strategies for renal fibrosis should include strategies that block sUA or inhibit its recognition by phagocytes.

Constitutive adipocyte mTORC1 activation enhances mitochondrial activity and reduces visceral adiposity in mice.

Mechanistic target of rapamycin complex 1 (mTORC1) loss of function reduces adiposity whereas partial mTORC1 inhibition enhances fat deposition. Herein we evaluated how constitutive mTORC1 activation in adipocytes modulates adiposity in vivo. Mice with constitutive mTORC1 activation in adipocytes induced by tuberous sclerosis complex (Tsc)1 deletion and littermate controls were evaluated for body mass, energy expenditure, glucose and fatty acid metabolism, mitochondrial function, mRNA and protein contents. Adipocyte-specific Tsc1 deletion reduced visceral, but not subcutaneous, fat mass, as well as adipocyte number and diameter, phenotypes that were associated with increased lipolysis, UCP-1 content (browning) and mRNA levels of pro-browning transcriptional factors C/EBPß and ERRα. Adipocyte Tsc1 deletion enhanced mitochondrial oxidative activity, fatty acid oxidation and the expression of PGC-1α and PPARα in both visceral and subcutaneous fat. In brown adipocytes, however, Tsc1 deletion did not affect UCP-1 content and basal respiration. Adipocyte Tsc1 deletion also reduced visceral adiposity and enhanced glucose tolerance, liver and muscle insulin signaling and adiponectin secretion in mice fed with purified low- or high-fat diet. In conclusion, adipocyte-specific Tsc1 deletion enhances mitochondrial activity, induces browning and reduces visceral adiposity in mice.

Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells.

The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.

Murine Mesenchymal Stem Cell Commitment to Differentiation Is Regulated by Mitochondrial Dynamics.

Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105(+) CD90(+) CD73(+) CD29(+) CD34(-) mesodermal precursors which, after in vitro induction, undergo chondro, adipo, and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro, and adipocytes and measuring changes in mass, morphology, dynamics, and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity, and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1, and 2, and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells.

H2O2 release from the very long chain acyl-CoA dehydrogenase.

Enhanced mitochondrial generation of oxidants, including hydrogen peroxide (H2O2), is related to a large number of pathological conditions, including diet-induced obesity and steatohepatosis. Indeed, we have previously shown that high fat diets increase the generation of H2O2 in liver mitochondria energized by activated fatty acids. Here, we further study fatty-acid induced H2O2 release in liver mitochondria, and determine the characteristics that regulate it. We find that this production of H2O2 is independent of mitochondrial inner membrane integrity and insensitive to purine nucleotides. On the other hand, palmitate-induced H2O2 production is strongly enhanced by high fat diets and is pH-sensitive, with a peak at a matrix pH of ~8.5. Using recombinantly expressed human very long chain acyl-CoA dehydrogenase, we are able to demonstrate that palmitate-induced H2O2 release may be ascribed to the activity of this enzyme alone, acting as an oxidase. Our results add to a number of findings indicating that sources outside of the electron transport chain can generate significant, physiopathologically relevant, amounts of oxidants in mitochondria.

Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: benefits of Alda-1.

BACKGROUND/OBJECTIVES: We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy are unknown and should be established to determine the optimal time window for drug treatment. METHODS: Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats. RESULTS: We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24h after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats. CONCLUSION: Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction cardiomyopathy.

Intermittent fasting induces hypothalamic modifications resulting in low feeding efficiency, low body mass and overeating.

Intermittent fasting (IF) is an often-used intervention to decrease body mass. In male Sprague-Dawley rats, 24 hour cycles of IF result in light caloric restriction, reduced body mass gain, and significant decreases in the efficiency of energy conversion. Here, we study the metabolic effects of IF in order to uncover mechanisms involved in this lower energy conversion efficiency. After 3 weeks, IF animals displayed overeating during fed periods and lower body mass, accompanied by alterations in energy-related tissue mass. The lower efficiency of energy use was not due to uncoupling of muscle mitochondria. Enhanced lipid oxidation was observed during fasting days, whereas fed days were accompanied by higher metabolic rates. Furthermore, an increased expression of orexigenic neurotransmitters AGRP and NPY in the hypothalamus of IF animals was found, even on feeding days, which could explain the overeating pattern. Together, these effects provide a mechanistic explanation for the lower efficiency of energy conversion observed. Overall, we find that IF promotes changes in hypothalamic function that explain differences in body mass and caloric intake.

Deletion of the transcriptional regulator opi1p decreases cardiolipin content and disrupts mitochondrial metabolism in Saccharomyces cerevisiae.

Cardiolipin, the main anionic phospholipid in the inner mitochondrial membrane, provides shape, charge and osmotic support to this membrane due to its biophysical properties. In addition, it helps form respiratory supercomplexes and provides functionality to mitochondrial proteins. Defects in the biosynthesis or remodeling of cardiolipin have been related to severe diseases, such as Barth syndrome. Opi1p, a transcriptional repressor for most enzymes in phospholipid biosynthesis found in Saccharomyces cerevisiae, has been demonstrated not to affect the biosynthesis of this mitochondrial phospholipid. However, we found that opi1 deletion compromises mitochondrial metabolism producing severe respiratory defects. The mechanism producing this phenotype was explored and found to be a mitochondrial cardiolipin depletion of almost 50%, resulting in low cytochrome content and high mitochondrial DNA instability. The origin of this low cardiolipin content strongly correlated with the overproduction of inositol, an intrinsic phenotype of this mutation. Overall, our results show that adequate regulation of phospholipid synthesis is essential for the maintenance of mitochondrial function.

Mitochondria as a source of reactive oxygen and nitrogen species: from molecular mechanisms to human health.

Mitochondrially generated reactive oxygen species are involved in a myriad of signaling and damaging pathways in different tissues. In addition, mitochondria are an important target of reactive oxygen and nitrogen species. Here, we discuss basic mechanisms of mitochondrial oxidant generation and removal and the main factors affecting mitochondrial redox balance. We also discuss the interaction between mitochondrial reactive oxygen and nitrogen species, and the involvement of these oxidants in mitochondrial diseases, cancer, neurological, and cardiovascular disorders.

Serum from calorie-restricted rats activates vascular cell eNOS through enhanced insulin signaling mediated by adiponectin.

eNOS activation resulting in mitochondrial biogenesis is believed to play a central role in life span extension promoted by calorie restriction (CR). We investigated the mechanism of this activation by treating vascular cells with serum from CR rats and found increased Akt and eNOS phosphorylation, in addition to enhanced nitrite release. Inhibiting Akt phosphorylation or immunoprecipitating adiponectin (found in high quantities in CR serum) completely prevented the increment in nitrite release and eNOS activation. Overall, we demonstrate that adiponectin in the serum from CR animals increases NO• signaling by activating the insulin pathway. These results suggest this hormone may be a determinant regulator of the beneficial effects of CR.