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Background: Mitochondrial dynamics (e.g. fission/fusion) play an important role in controlling chemoresistance in representative gynecologic malignancies, ovarian and cervical cancer. Processing the long form of Optic atrophy (L-Opa)1 is a distinctive character of mitochondrial fragmentation, associated with chemosensitivity. Here, we examined the role of prohibitin (Phb)1 in increasing L-Opa1 processing via the regulating mitochondrial protease, Oma1 and its direct interaction with p-p53 (ser15) and pro-apoptotic Bcl-2 antagonist/killer (Bak) 1 in the signaling axis and if this phenomenon is associated with prognosis of patients.Methods: We compared Cisplatin (CDDP)-induced response of mitochondrial dynamics, molecular interaction among p-p53 (ser15)-Phb1-Bak, and chemoresponsiveness in paired chemosensitive and chemoresistant gynecologic cancer cells (ovarian and cervical cancer cell lines) using western blot, immunoprecipitation, sea horse, and immunofluorescence. Translational strategy with proximity ligation assessment in phb1-p-p53 (ser15) in human ovarian tumor sections further confirmed in vitro finding, associated with clinical outcome.Results: We report that: (1) Knock-down of Phb1 prevents Cisplatin (cis‐diamine-dichloroplatinum; CDDP) -induced changes in mitochondrial fragmentation and Oma1 mediated cleavage, and Opa1 processing; (2) In response to CDDP, Phb1 facilitates the p-p53 (ser15)-Phb1-Bak interaction in mitochondria in chemosensitive gynecologic cancer cells but not in chemoresistant cells; (3) Akt overexpression results in suppressed p-p53(Ser15)-Phb1 interaction and dysregulated mitochondrial dynamics, and (4) Consistent with in vitro findings, proximity ligation assessment (PLA) in human ovarian tumor sections demonstrated that p-p53(ser15)-Phb1-Bak interaction in mitochondria is associated with better chemoresponsiveness and clinical outcome of patients. Determining the molecular mechanisms by which Phb1 facilitates mitochondrial fragmentation and interacts with p53 may advance the current understanding of chemoresistance and pathogenesis of gynecologic cancer.Conclusion: Determining the key molecular mechanisms by which Phb1 facilitates the formation of p-p53 (ser15)-Bak-Phb1 and its involvement in the regulation of mitochondrial dynamics and apoptosis may ultimately contribute to the current understanding of molecular and cellular basis of chemoresistance in this gynecologic cancer.

Chemotherapy resistance is a critical barrier in cancer treatment. Metabolic adaptations have been shown to fuel therapy resistance; however, little is known regarding the generality of these changes and whether specific therapies elicit unique metabolic alterations. Using a combination of metabolomics, transcriptomics, and functional genomics, we show that two anthracyclines, doxorubicin and epirubicin, elicit distinct primary metabolic vulnerabilities in human breast cancer cells. Doxorubicin-resistant cells rely on glutamine to drive oxidative phosphorylation andde novoglutathione synthesis, while epirubicin-resistant cells display markedly increased bioenergetic capacity and mitochondrial ATP production. The dependence on these distinct metabolic adaptations is revealed by the increased sensitivity of doxorubicin-resistant cells and tumor xenografts to buthionine sulfoximine (BSO), a drug that interferes with glutathione synthesis, compared with epirubicin-resistant counterparts that are more sensitive to the biguanide phenformin. Overall, our work reveals that metabolic adaptations can vary with therapeutics and that these metabolic dependencies can be exploited as a targeted approach to treat chemotherapy-resistant breast cancer.

Background IL-15 is believed to play a role in the beneficial impact of exercise on muscle energy metabolism. However, previous studies have generally used supraphysiological levels of IL-15 that do not represent contraction-induced IL-15 secretion. Methods L6 myotubes were treated acutely (3 h) and chronically (48 h) with concentrations of IL-15 mimicking circulating (1–10 pg/ml) and muscle interstitial (100 pg/ml −20 ng/ml) IL-15 levels with the aim to better understand its autocrine/paracrine role on muscle glucose uptake and mitochondrial function. Results Acute exposure to IL-15 levels representing muscle interstitial IL-15 increased basal glucose uptake without affecting insulin sensitivity. This was accompanied by increased mitochondrial oxidative functions in association with increased AMPK pathway and formation of complex III-containing supercomplexes. Conversely, chronic IL-15 exposure resulted in a biphasic effect on mitochondrial oxidative functions and ETC supercomplex formation was increased with low IL-15 levels but decreased with higher IL-15 concentrations. The AMPK pathway was activated only by high levels of chronic IL-15 treatment. Similar results were obtained in skeletal muscle from muscle-specific IL-15 overexpressing mice that show very high circulating IL-15 levels. Conclusions Acute IL-15 treatment that mimics local IL-15 concentrations enhances muscle glucose uptake and mitochondrial oxidative functions. That mitochondria respond differently to different levels of IL-15 during chronic treatments indicates that IL-15 might activate two different pathways in muscle depending on IL-15 concentrations. General significance Our results suggest that IL-15 may act in an autocrine/paracrine fashion and be, at least in part, involved in the positive effect of exercise on muscle energy metabolism.

Elevated metabolism is a key hallmark of multiple cancers, serving to fulfill high anabolic demands. Ovarian cancer (OVCA) is the fifth leading cause of cancer deaths in women with a high mortality rate (45%). Chemoresistance is a major hurdle for OVCA treatment. Although substantial evidence suggests that metabolic reprogramming contributes to anti-apoptosis and the metastasis of multiple cancers, the link between tumor metabolism and chemoresistance in OVCA remains unknown. While clinical trials targeting metabolic reprogramming alone have been met with limited success, the synergistic effect of inhibiting tumor-specific metabolism with traditional chemotherapy warrants further examination, particularly in OVCA. This review summarizes the role of key glycolytic enzymes and other metabolic synthesis pathways in the progression of cancer and chemoresistance in OVCA. Within this context, mitochondrial dynamics (fission, fusion and cristae structure) are addressed regarding their roles in controlling metabolism and apoptosis, closely associated with chemosensitivity. The roles of multiple key oncogenes (Akt, HIF-1α) and tumor suppressors (p53, PTEN) in metabolic regulation are also described. Next, this review summarizes recent research of metabolism and future direction. Finally, we examine clinical drugs and inhibitors to target glycolytic metabolism, as well as the rationale for such strategies as potential therapeutics to overcome chemoresistant OVCA.

Glutaredoxin 2 (GRX2), a mitochondrial glutathione-dependent oxidoreductase, is central to glutathione homeostasis and mitochondrial redox, which is crucial in highly metabolic tissues like the heart. Previous research showed that absence of Grx2, leads to impaired mitochondrial complex I function, hypertension and cardiac hypertrophy in mice but the impact on mitochondrial structure and function in intact cardiomyocytes and in humans has not been explored. We hypothesized that Grx2 controls cardiac mitochondrial dynamics and function in cellular and mouse models, and that low expression is associated with human cardiac dysfunction. Here we show that Grx2 absence impairs mitochondrial fusion, ultrastructure and energetics in primary cardiomyocytes and cardiac tissue. Moreover, provision of the glutathione precursor, N-acetylcysteine (NAC) to Grx2-/- mice did not restore glutathione redox or prevent impairments. Using genetic and histopathological data from the human Genotype-Tissue Expression consortium we demonstrate that low GRX2 is associated with fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, GRX2 is important in the control of cardiac mitochondrial structure and function, and protects against human cardiac pathologies., Graphical abstract fx1

Simple Summary Hexokinase II (HKII) is a key glycolysis enzyme associated with tumorigenesis, but its molecular mechanism and pathophysiological role in chemoresistant ovarian cancer remain elusive. In this study, we delineate the novel mechanism showing that activated phosphorylated-p53 (P-p53 Ser15) is required for the regulation of HKII intracellular trafficking and metabolic regulation in chemosensitive ovarian cancer, but not in chemoresistant ovarian cancer harboring p53 mutation. We have observed that increased nuclear HKII-P-p53 (Ser15) interaction is likely associated with chemosensitivity and better survival outcomes in epithelial ovarian cell lines, human primary epithelial ovarian cancer cells, and tumor sections. Nuclear HKII-P-p53 (Ser15) interaction may function as a promising prognostic biomarker, enabling prediction of patients with poor prognosis for deciding better clinical strategies. Abstract In epithelial ovarian cancer (EOC), carboplatin/cisplatin-induced chemoresistance is a major hurdle to successful treatment. Aerobic glycolysis is a common characteristic of cancer. However, the role of glycolytic metabolism in chemoresistance and its impact on clinical outcomes in EOC are not clear. Here, we show a functional interaction between the key glycolytic enzyme hexokinase II (HKII) and activated P-p53 (Ser15) in the regulation of bioenergetics and chemosensitivity. Using translational approaches with proximity ligation assessment in cancer cells and human EOC tumor sections, we showed that nuclear HKII-P-p53 (Ser15) interaction is increased after chemotherapy, and functions as a determinant of chemoresponsiveness as a prognostic biomarker. We also demonstrated that p53 is required for the intracellular nuclear HKII trafficking in the control of glycolysis in EOC, associated with chemosensitivity. Mechanistically, cisplatin-induced P-p53 (Ser15) recruits HKII and apoptosis-inducing factor (AIF) in chemosensitive EOC cells, enabling their translocation from the mitochondria to the nucleus, eliciting AIF-induced apoptosis. Conversely, in p53-defective chemoresistant EOC cells, HKII and AIF are strongly bound in the mitochondria and, therefore, apoptosis is suppressed. Collectively, our findings implicate nuclear HKII-P-p53(Ser15) interaction in chemosensitivity and could provide an effective clinical strategy as a promising biomarker during platinum-based therapy.

Mitochondria play a crucial role in neuronal survival through efficient energy metabolism. In pathological conditions, mitochondrial stress leads to neuronal death, which is regulated by the anti-apoptotic BCL-2 family of proteins. MCL-1 is an anti-apoptotic BCL-2 protein localized to mitochondria either in the outer membrane (OM) or inner membrane (Matrix), which have distinct roles in inhibiting apoptosis and promoting bioenergetics, respectively. While the anti-apoptotic role for Mcl1 is well characterized, the protective function of MCL-1 Matrix remains poorly understood. Here, we show MCL-1OM and MCL-1Matrix prevent neuronal death through distinct mechanisms. We report that MCL-1Matrix functions to preserve mitochondrial energy transduction and improves respiratory chain capacity by modulating mitochondrial oxygen consumption in response to mitochondrial stress. We show that MCL-1Matrix protects neurons from stress by enhancing respiratory function, and by inhibiting mitochondrial permeability transition pore opening. Taken together, our results provide novel insight into how MCL-1Matrix may confer neuroprotection under stress conditions involving loss of mitochondrial function.

Metabolic reprogramming (including the Warburg effect) is a hallmark of cancer, yet the association between the altered metabolism and chemoresistance remains elusive. Hexokinase II (HKII) is a key metabolic enzyme and is upregulated in multiple cancers. In this study, we examined the impact of targeting metabolism via silencing of HKII on chemoresistance in ovarian cancer (OVCA). In addition, the regulatory molecular mechanism of tumor metabolism was examined using gain- and loss-of-function approaches in epithelial OVCA cell lines of various histological subtypes. We demonstrated that cisplatin (CDDP)-induced p53-mediated HKII downregulation is a determinant of chemosensitivity in OVCA. Silencing of HKII sensitized chemoresistant OVCA cells to apoptosis in a p53-dependent manner. As a negative regulator, p53 suppressed HKII transcription by promoter binding and decreased glycolysis. Pyruvate dehydrogenase kinase-1 (PDK1) is a key regulator of cell proliferation involved in Akt signaling axis. Our Gene Expression Profiling Interactive Analysis (GEPIA) and molecular studies also revealed that PDK1, an upstream activator strongly correlates with HKII expression and regulates its metabolic activity. Finally, we demonstrated that the clinically approved drug metformin sensitizes chemoresistant OVCA cells to CDDP via PDK1-HKII pathway. Collectively, our data implicate that p53--PDK1-HKII axis is a central regulatory component of metabolism conferring chemoresistance in OVCA.

SUMMARY TSC2 inactivating mutations elicit mTORC1 hyperactivation and underlie neural and mesenchymal tumorigenesis in tuberous sclerosis complex (TSC). Given the paucity of humanized and lineage-relevant experimental systems relevant to TSC we have developed the first multi-system TSC model, based on CRISPR/Cas9-mediated knockout of TSC2 in human pluripotent stem cells (hPSCs) and their differentiation into putative cell types driving TSC tumors and neurological dysfunction. Mesenchymal neural crest cell (NCC) differentiation recapitulates critical features of lung and kidney tumors, while neural lineage induction of TSC2−/− hPSCs progressively models aberrant neuronal, glial and neural precursor cells (NPCs) that constitute TSC brain tumors. We additionally provide an RNA-sequencing (RNA-seq) resource in which multiple time-points during induction of WT and TSC2−/− cells into neural and neural crest lineages were profiled. Temporal RNA-seq analyses exposed a massive proteostatic stress response activated acutely during neuroepithelial induction in TSC2-deficient cells. Remarkably, this is resolved upon NCC specification but leads to endosomal and metabolic reprogramming in NPCs. Furthermore, TSC2−/− NPCs, but not NCCs, are uniquely vulnerable to death via proteasome inhibition independent of mTORC1 regulation, highlighting cell type-specific proteostatic states. Thus, our data reveal that mechanisms regulating proteostasis stress are strikingly lineage-specific in TSC2−/−cells, and these differences are acquired developmentally. This drives long-term catabolic adaptations specifically in neural cells that counter typical mTORC1 paradigms, resulting in previously unrecognized tumor/lineage-specific therapeutic sensitivities. Overall, our work exemplifies the complexity of elucidating underlying biological mechanisms and therapeutic approaches for multisystem diseases, illustrating the power of utilizing hPSC disease models with tissue-specific relevance.

Mitochondria are highly dynamic organelles that respond rapidly to a number of stressors to regulate energy transduction, cell death signaling, and reactive oxygen species generation. We hypothesized that mitochondrial remodeling, comprising both structural and functional alterations, following ionizing radiation (IR) may underlie some of the tenets of radiobiology. Mesenchymal stem cells (MSCs) are precursors of bone marrow stroma and are altered in acute myeloid leukemia and by radiation and chemotherapy. Here, we report on changes in mitochondrial remodeling in human MSCs following X-ray IR. Mitochondrial function was significantly increased in MSCs 4 h after IR as measured by mitochondrial oxygen consumption. Consistent with this elevated functional effect, electron transport chain supercomplexes were also increased in irradiated samples. In addition, mitochondria were significantly, albeit modestly, elongated, as measured by high-throughput automated confocal imaging coupled with automated mitochondrial morphometric analyses. We also demonstrate in fibroblasts that mitochondrial remodeling is required for the adaptation of cells to IR. To determine novel mechanisms involved in mitochondrial remodeling, we performed quantitative proteomics on isolated mitochondria from cells following IR. Label-free quantitative mitochondrial proteomics revealed notable changes in proteins in irradiated samples and identified prosaposin, and potentially its daughter protein saposin-B, as a potential candidate for regulating mitochondrial function following IR. Whereas research into the biologic effects of cellular irradiation has long focused on nuclear DNA effects, our experimental work, along with that of others, is finding that mitochondrial effects may have broader implications in the field of stress adaptation and cell death in cancer (including leukemia) and other disease states.—Patten, D. A., Ouellet, M., Allan, D. S., Germain, M., Baird, S. D., Harper, M.-E., Richardson, R. B. Mitochondrial adaptation in human mesenchymal stem cells following ionizing radiation.

Mitochondrial reactive oxygen species (ROS) are important cellular signaling molecules, but can cause oxidative damage if not kept within tolerable limits. An important proximal form of ROS in mitochondria is superoxide. Its production is thought to occur in regulated stochastic bursts, but current methods using mitochondrial targeted cpYFP to assess superoxide flashes are confounded by changes in pH. Accordingly, these flashes are generally referred to as ‘mitoflashes’. Here we provide regulatory insights into mitoflashes and pH fluctuations in skeletal muscle, and the role of uncoupling protein-3 (UCP3). Using quantitative confocal microscopy of mitoflashes in intact muscle fibers, we show that the mitoflash magnitude significantly correlates with the degree of mitochondrial inner membrane depolarization and ablation of UCP3 did not affect this correlation. We assessed the effects of the absence of UCP3 on mitoflash activity in intact skeletal muscle fibers, and found no effects on mitoflash frequency, amplitude or duration, with a slight reduction in the average size of mitoflashes. We further investigated the regulation of pH flashes (pHlashes, presumably a component of mitoflash) by UCP3 using mitochondrial targeted SypHer (mt-SypHer) in skeletal muscle fibers. The frequency of pHlashes was significantly reduced in the absence of UCP3, without changes in other flash properties. ROS scavenger, tiron, did not alter pHlash frequency in either WT or UCP3KO mice. High resolution respirometry revealed that in the absence of UCP3 there is impaired proton leak and Complex I-driven respiration and maximal coupled respiration. Total cellular production of hydrogen peroxide (H(2)O(2)) as detected by Amplex-UltraRed was unaffected. Altogether, we demonstrate a correlation between mitochondrial membrane potential and mitoflash magnitude in skeletal muscle fibers that is independent of UCP3 and a role for UCP3 in the control of pHlash frequency and of proton leak- and Complex I coupled- respiration in skeletal muscle fibers. The differential regulation of mitoflashes and pHlashes by UCP3 and tiron also indicate that the two events, though may be related, are not identical events.

Mitochondria are highly dynamic organelles. Alterations in mitochondrial dynamics are causal or are linked to numerous neurodegenerative, neuromuscular, and metabolic diseases. It is generally thought that cells with altered mitochondrial structure are prone to mitochondrial dysfunction, increased reactive oxygen species generation and widespread oxidative damage. The objective of the current study was to investigate the relationship between mitochondrial dynamics and the master cellular antioxidant, glutathione (GSH). We reveal that mouse embryonic fibroblasts (MEFs) lacking the mitochondrial fusion machinery display elevated levels of GSH, which limits oxidative damage. Moreover, targeted metabolomics and 13C isotopic labeling experiments demonstrate that cells lacking the inner membrane fusion GTPase OPA1 undergo widespread metabolic remodeling altering the balance of citric acid cycle intermediates and ultimately favoring GSH synthesis. Interestingly, the GSH precursor and antioxidant n-acetylcysteine did not increase GSH levels in OPA1 KO cells, suggesting that cysteine is not limiting for GSH production in this context. Post-mitotic neurons were unable to increase GSH production in the absence of OPA1. Finally, the ability to use glycolysis for ATP production was a requirement for GSH accumulation following OPA1 deletion. Thus, our results demonstrate a novel role for mitochondrial fusion in the regulation of GSH synthesis, and suggest that cysteine availability is not limiting for GSH synthesis in conditions of mitochondrial fragmentation. These findings provide a possible explanation for the heightened sensitivity of certain cell types to alterations in mitochondrial dynamics.

Objective High levels of reactive oxygen species (ROS) are intricately linked to obesity and associated pathologies, notably insulin resistance and type 2 diabetes. However, ROS are also thought to be important in intracellular signaling, which may paradoxically be required for insulin sensitivity. Many theories have been developed to explain this apparent paradox, which have broadened our understanding of these important small molecules. While many sites for intracellular ROS production have been described, mitochondrial generated ROS remain a major contributor in most cell types. Mitochondrial ROS generation is controlled by a number of factors described in this review. Moreover, these studies have established both a demand for novel sensitive approaches to measure ROS, as well as a need to standardize and review their suitability for different applications. Methods To properly assess levels of ROS and mitochondrial ROS in the development of obesity and its complications, a growing number of tools have been developed. This paper reviews many of the common methods for the investigation of ROS in mitochondria, cell, animal, and human models. Results Available approaches can be generally divided into those that measure ROS-induced damage (e.g., DNA, lipid, and protein damage); those that measure antioxidant levels and redox ratios; and those that use novel biosensors and probes for a more direct measure of different forms of ROS (e.g., 2′,7′-di-chlorofluorescein (DCF), dihydroethidium (DHE) and its mitochondrial targeted form (MitoSOX), Amplex Red, roGFP, HyPer, mt-cpYFP, ratiometric H2O2 probes, and their derivatives). Moreover, this review provides caveats and strengths for the use of these techniques in different models. Conclusions Advances in these techniques will undoubtedly advance the understanding of ROS in obesity and may help resolve unanswered questions in the field.

Objectives Type 2 diabetes mellitus is a chronic progressive disease that is associated with increased risk for cardiovascular diseases and with impaired mitochondrial metabolism in cardiac and skeletal muscles. Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with significant morbidity and mortality. Type 2 diabetes is also one of the prevalent concomitant diseases in patients with AF. During AF, myocardial energy demand is high due to electrical activity. To date, however, very little is known about the effects of AF on atrial muscle mitochondrial energetics. We hypothesized that preexisting fibrillation or type 2 diabetes impacts atrial mitochondrial energetics and electron transport chain supercomplexes. Methods Atrial appendages were collected from patients who had consented and who had and did not have preexisting AF and were undergoing coronary artery bypass graft surgery. Mitochondrial functional analyses were conducted in permeabilized myofibers using high-resolution respirometry. Results Results show impaired complex I and II function in addition to impaired electron transport chain supercomplex assembly in patients with diabetes and AF compared to patients with diabetes but without AF. There were no differences in mitochondrial content in atrial muscle between the groups. There was a strong trend for increased oxidative damage (protein carbonyls) in patients with diabetes and AF compared to patients with diabetes but without AF. Conclusions Overall, findings suggest impaired mitochondrial function in AF and type 2 diabetes.

Background/Objectives: Inter-individual variability in weight loss during obesity treatment is complex and poorly understood. Here we use whole body and tissue approaches to investigate fuel oxidation characteristics in skeletal muscle fibers, cells and distinct circulating protein biomarkers before and after a high fat meal (HFM) challenge in those who lost the most (obese diet-sensitive; ODS) vs the least (obese diet-resistant; ODR) amount of weight in a highly controlled weight management program. Subjects/Methods: In 20 weight stable-matched ODS and ODR women who previously completed a standardized clinical weight loss program, we analyzed whole-body energetics and metabolic parameters in vastus lateralis biopsies and plasma samples that were obtained in the fasting state and 6 h after a defined HFM, equivalent to 35% of total daily energy requirements. Results: At baseline (fasting) and post-HFM, muscle fatty acid oxidation and maximal oxidative phosphorylation were significantly greater in ODS vs ODR, as was reactive oxygen species emission. Plasma proteomics of 1130 proteins pre and 1, 2, 5 and 6 h after the HFM demonstrated distinct group and interaction differences. Group differences identified S-formyl glutathione hydratase, heat shock 70 kDA protein 1A/B (HSP72), and eukaryotic translation initiation factor 5 (eIF5) to be higher in ODS vs ODR. Group-time differences included aryl hydrocarbon interacting protein (AIP), peptidylpropyl isomerase D (PPID) and tyrosine protein-kinase Fgr, which increased in ODR vs ODS over time. HSP72 levels correlated with muscle oxidation and citrate synthase activity. These proteins circulate in exosomes; exosomes isolated from ODS plasma increased resting, leak and maximal respiration rates in C2C12 myotubes by 58%, 21% and 51%, respectively, vs those isolated from ODR plasma. Conclusions: Findings demonstrate distinct muscle metabolism and plasma proteomics in fasting and post-HFM states corresponding in diet-sensitive vs diet-resistant obese women.

Taking insurgency sponsorship as an instrument states have available for achieving foreign policy objectives, I consider how state-sponsors could best manipulate their support to maximize control o...

High levels of reactive oxygen species (ROS) are intricately linked to obesity and associated pathologies, notably insulin resistance and type 2 diabetes. However, ROS are also thought to be important in intracellular signaling, which may paradoxically be required for insulin sensitivity. Many theories have been developed to explain this apparent paradox, which have broadened our understanding of these important small molecules. While many sites for intracellular ROS production have been described, mitochondrial generated ROS remain a major contributor in most cell types. Mitochondrial ROS generation is controlled by a number of factors described in this review. Moreover, these studies have established both a demand for novel sensitive approaches to measure ROS, as well as a need to standardize and review their suitability for different applications.To properly assess levels of ROS and mitochondrial ROS in the development of obesity and its complications, a growing number of tools have been developed. This paper reviews many of the common methods for the investigation of ROS in mitochondria, cell, animal, and human models.Available approaches can be generally divided into those that measure ROS-induced damage (e.g., DNA, lipid, and protein damage); those that measure antioxidant levels and redox ratios; and those that use novel biosensors and probes for a more direct measure of different forms of ROS (e.g., 2',7'-di-chlorofluorescein (DCF), dihydroethidium (DHE) and its mitochondrial targeted form (MitoSOX), Amplex Red, roGFP, HyPer, mt-cpYFP, ratiometric HAdvances in these techniques will undoubtedly advance the understanding of ROS in obesity and may help resolve unanswered questions in the field.

Mitochondrial dysfunction plays an important role in the etiology of neurodegenerative diseases. However, the progressive nature of neuronal loss in genetic models of mitochondrial dysfunction suggests the presence of compensatory mechanisms promoting neuronal survival under these conditions. Here, we identified the energy metabolism kinase LKB1 as a key regulator of the compensatory mechanisms activated in neurons, following mitochondrial dysfunction. To accomplish this, we have created an in vivo neurodegenerative model based on the deletion of the mitochondrial protein apoptosis-inducing factor (AIF) in postmitotic neurons. Loss of mitochondrial function caused by AIF deletion induced several adaptive mechanisms, including increased glycolysis and mitochondrial biogenesis. Importantly, the activation of these adaptive mechanisms was abrogated by the deletion of one allele of LKB1, resulting in impaired neuronal survival. Because loss of mitochondrial function is a central mechanism implicated in neurodegenerative diseases, modulation of LKB1-dependent pathways may represent an important strategy to preserve neuronal survival and function.

Werner syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of genes essential for adaptation to low oxygen conditions. HIF-1 is also implicated in the molecular mechanisms of ageing. Here, we show that the cellular depletion of WRN protein (by siRNA targeting) leads to increased HIF-1 complex stabilization and activation. HIF-1 activation in the absence of WRN involves the generation of mitochondrial reactive oxygen species (mtROS) since SkQ1, a mitochondrial-targeted antioxidant, and stigmatellin, an inhibitor of mitochondrial complex III, blocked increased HIF-1 levels. Ascorbate, an essential co-factor involved in HIF-1 stability, was decreased in WRN-depleted cells. Interestingly, expression levels of GLUT1, a known dehydroascorbic acid transporter, were also decreased in WRN-depleted cells. Ascorbate supplementation of WRN-depleted cells led to a dose-dependent inhibition of HIF-1 activation. These results indicate that WRN protein regulates HIF-1 activation by affecting mitochondrial ROS production and intracellular ascorbate levels. This work provides a novel mechanistic link between HIF-1 activity and different age-associated pathologies.

Neuronal cell loss associated with neurodegeneration has recently been linked to mitochondrial dysfunction. Electron transport chain defects and reactive oxygen species (ROS) production are emerging as important players in the etiology of neurodegenerative diseases. Proper management of ROS and disposal of damaged cellular components are vital to the survival and function of neurons. Proteins involved in these pathways are often mutated in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. In this review, we will discuss the roles of ROS in normal physiology, how changes in ROS production affect neuronal survival in neurodegenerative diseases, and the recent advances in mitochondrial antioxidants as potential therapeutics.

Skeletal muscle mitochondrial dysfunction has been documented in patients with type 2 diabetes mellitus; however, specific respiratory defects and their mechanisms are poorly understood. The aim of the current study was to examine oxidative phosphorylation and electron transport chain (ETC) supercomplex assembly in rectus abdominis muscles of 10 obese diabetic and 10 obese non-diabetic individuals.Twenty obese women undergoing Roux-en-Y gastric bypass surgery were recruited for this study. Muscle samples were obtained intraoperatively and subdivided for multiple analyses, including high-resolution respirometry and assessment of supercomplex assembly. Clinical data obtained from referring physicians were correlated with laboratory findings.Participants in both groups were of a similar age, weight and BMI. Mitochondrial respiration rates were markedly reduced in diabetic vs non-diabetic patients. This defect was observed during maximal ADP-stimulated respiration in the presence of complex I-linked substrates and complex I- and II-linked substrates, and during maximal uncoupled respiration. There were no differences in fatty acid (octanoyl carnitine) supported respiration, leak respiration or isolated activity of cytochrome c oxidase. Intriguingly, significant correlations were found between glycated haemoglobin (HbA1c) levels and maximal respiration or respiration supported by complex I, complex I and II or fatty acid. In the muscle of diabetic patients, blue native gel electrophoresis revealed a striking decrease in complex I, III and IV containing ETC supercomplexes.These findings support the hypothesis that ETC supercomplex assembly may be an important underlying mechanism of muscle mitochondrial dysfunction in type 2 diabetes mellitus.

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.

Sustained cellular function and viability of high-energy demanding post-mitotic cells rely on the continuous supply of ATP. The utilization of mitochondrial oxidative phosphorylation for efficient ATP generation is a function of oxygen levels. As such, oxygen deprivation, in physiological or pathological settings, has profound effects on cell metabolism and survival. Here we show that mild extracellular acidosis, a physiological consequence of anaerobic metabolism, can reprogramme the mitochondrial metabolic pathway to preserve efficient ATP production regardless of oxygen levels. Acidosis initiates a rapid and reversible homeostatic programme that restructures mitochondria, by regulating mitochondrial dynamics and cristae architecture, to reconfigure mitochondrial efficiency, maintain mitochondrial function and cell survival. Preventing mitochondrial remodelling results in mitochondrial dysfunction, fragmentation and cell death. Our findings challenge the notion that oxygen availability is a key limiting factor in oxidative metabolism and brings forth the concept that mitochondrial morphology can dictate the bioenergetic status of post-mitotic cells., In hypoxic conditions, cells depend on anaerobic respiration, which results in extracellular acidosis. Khacho et al. find that acidosis serves a protective function, enhancing mitochondrial respiratory capacity and sustaining ATP synthesis despite limited oxygen availability, by both promoting mitochondrial fusion and inhibiting fission.

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Here we report that the generation of mitochondrial reactive oxygen species are essential for regulating HIF-1 in normal oxygen conditions in the vasculature., Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1α transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22phox siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1α protein stability, HIF-1–dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase–generated ROS in Ang II–mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions.

Overexpression of the EGF receptor (EGFR) is a recurrent theme in human cancer and is thought to cause aggressive phenotypes and resistance to standard therapy. There has, thus, been a concerted effort in identifying EGFR gene mutations to explain misregulation of EGFR expression as well as differential sensitivity to anti-EGFR drugs. However, such genetic alterations have proven to be rare occurrences in most types of cancer, suggesting the existence of a more general physiological trigger for aberrant EGFR expression. Here, we provide evidence that overexpression of wild-type EGFR can be induced by the hypoxic microenvironment and activation of hypoxia-inducible factor 2-α (HIF2α) in the core of solid tumors. Our data suggest that hypoxia/HIF2α activation represents a common mechanism for EGFR overexpression by increasing EGFR mRNA translation, thereby diminishing the necessity for gene mutations. This allows for the accumulation of elevated EGFR levels, increasing its availability for the autocrine signaling required for tumor cell growth autonomy. Taken together, our findings provide a nonmutational explanation for EGFR overexpression in human tumors and highlight a role for HIF2α activation in the regulation of EGFR protein synthesis.

Aim: To determine the effect of saposin C (a known trophic domain of prosaposin) on proliferation, migration and invasion, as well as its effect on the expression of urokinase plasmonogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMP)-2 and -9 in normal and malignant prostate cells. In addition, we tested whether saposin C can activate p42/44 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signal transduction pathways of the mitogen-activated protein kinase (MAPK) superfamily. Methods: We employed Western blot analysis, phospho-specific antibodies, cell proliferation assay, reverse transcriptase-polymerase chain reaction, in vitro kinase assays and migration and invasion to determine the effect of saposin C on various biological behaviors of prostate stromal and cancer cells. Results: Saposin C, in a cell type-specific manner, upregulates uPA/uPAR and immediate early gene c-Jun expression, stimulates cell proliferation, migration and invasion and activates p42/44 and SAPK/JNK MAPK pathways in prostate stromal and cancer cells. Normal prostate epithelial cells were not responsive to saposin C treatment in the above studies. Conclusion: Saposin C functions as a multipotential modulator of diverse biological activities in prostate cancer and stromal cells. These results strongly suggest that saposin C functions as a potent growth factor for prostatic cells and may contribute to prostate carcinogenesis and/or the development of hormone-refractory prostate cancer. (Asian J Androl 2005 Jun; 7: 147–158)

Lymph node imaging has been helpful in managing patients with lymphoma, melanoma, and breast cancer. To evaluate 38 patients with head and neck cancers, 99mTc minicolloid was injected adjacent to the tumor and into a similar area on the uninvolved side. Lymphoscintigraphy of the neck was performed at 3- and 5-hour intervals after injection and bilateral cervical lymphatic drainage was observed. Each patient then underwent a neck dissection. The pathologic node findings were then correlated with the neck scans. Results confirmed that cervical lymphatic drainage is unpredictable in 50% of the patients once the channels are involved with metastatic disease. Lymphoscintigraphy is not a reliable method of detecting early metastatic cervical carcinoma.

Technetium 99m isotope venography was performed in 116 extremities. By x-ray contrast venographic confirmation in forty-eight extremities, it was accurate in 92 per cent of the patients with a “definitive” diagnosis of thrombosis and in 78 per cent with normal results of the study. Radioactive transit time from foot to groin was not a statistically reliable parameter for detecting thrombosis. The excellent visualization of the venous system, the ease of performance, and the acceptance by the patient of this relatively simple technic make isotope venography an attractive method for the diagnosis of major venous thrombosis. The indications for isotope venography are: (1) as a diagnostic alternative to x-ray venography; (2) as an improved means of detecting pulmonary embolism when combined with a lung scan; (3) as a simple means of following thrombolytic phenomena in the veins of the lower extremities.

In a prospective study of twenty-six patients with ischemic ulcerations of the lower extremity, the predictive reliability with regard to spontaneous wound healing of diabetes, pedal pulses, ankle blood pressure (ABP) as measured by doppler ultrasound, and "leg ulcer scan" as performed by the intra-arterial injection of radioactive albumin was evaluated. The results suggest that only the leg ulcer scan is significantly reliable in predicting the likelihood of spontaneous healing. The following format for the evaluation of the ischemic leg ulcer is therefore suggested: (1) If pedal pulses are present, a three week trial of conservative therapy is indicated before further evaluation. (2) If the doppler ABP is 50 mm Hg or less, the ulcer will not heal spontaneously. (3) Leg ulcer scan is indicated: (a) in the absence of pedal pulses if ABP is less than 50 mm Hg; (b) in the presence of pedal pulses if there is no evidence of spontaneous healing after three weeks of conservative therapy.