Proton Leak Rate Research Articles

How does mitochondria function contribute to aerobic performance enhancement in lizards?

Aims: Aerobic exercise typically enhances endurance across vertebrates so that chronically high energy demands can be met. Some known mechanisms of doing this include increases in red blood cell numbers, angiogenesis, muscle fiber adaptions, mitochondria biogenesis, and changes to cellular metabolism and oxidative phosphorylation. We used green anole lizards (Anolis carolinensis) to test for an effect of aerobic exercise on metabolism, mitochondria densities, and mitochondrial function. Methods: We first tested the response of green anoles to endurance training and pyrroloquinoline quinone (PQQ) supplementation, which has been shown to increase mitochondria biogenesis. We also conducted a mitochondrial stress test to determine how training affected mitochondrial function in skeletal muscle fibers. Results: Aerobic exercise led to increased endurance and decreased standard metabolic rate (SMR), while PQQ did not affect endurance and increased SMR. In a second experiment, aerobic exercise increased endurance and decreased resting metabolic rate (RMR) in both male and female green anoles. Higher counts of mitochondrial gene copies in trained lizards suggested additional mitochondria adaptations to achieve increased endurance and decreased metabolism. A mitochondrial stress test revealed no effect on baseline oxygen consumption rates of muscle fibers, but untrained lizards had higher maximal oxygen consumption rates with the addition of metabolic fuel. Conclusion: It is likely that trained lizards exhibited lower maximal oxygen consumption rates by developing higher mitochondria efficiency. This adaptation allows for high ATP demand to be met by making more ATP per oxygen molecule consumed. On the other hand, it is possible that untrained lizards prioritized limiting reactive oxygen species (ROS) production at rest, while sacrificing higher levels of proton leak and higher oxygen consumption rates when working to meet high ATP demand.

Violacein switches off low molecular weight tyrosine phosphatase and rewires mitochondria in colorectal cancer cells

In the last decade, emerging evidence has shown that low molecular weight protein tyrosine phosphatase (LMWPTP) not only contributes to the progression of cancer but is associated with prostate low survival rate and colorectal cancer metastasis. We report that LMWPTP favors the glycolytic profile in some tumors. Therefore, the focus of the present study was to identify metabolic enzymes that correlate with LMWPTP expression in patient samples. Exploratory data analysis from RNA-seq, proteomics, and histology staining, confirmed the higher expression of LMWPTP in CRC. Our descriptive statistical analyses indicate a positive expression correlation between LMWPTP and energy metabolism enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In addition, we examine the potential of violacein to reprogram energetic metabolism and LMWPTP activity. Violacein treatment induced a shift of glycolytic to oxidative metabolism associated with alteration in mitochondrial efficiency, as indicated by higher oxygen consumption rate. Particularly, violacein treated cells displayed higher proton leak and ATP-linked oxygen consumption rate (OCR) as an indicator of the OXPHOS preference. Notably, violacein is able to bind and inhibit LMWPTP. Since the LMWPTP acts as a hub of signaling pathways that offer tumor cells invasive advantages, such as survival and the ability to migrate, our findings highlight an unexplored potential of violacein in circumventing the metabolic plasticity of tumor cells.

Pharmacological Inhibition of NOX4 Improves Mitochondrial Function and Survival in Human Beta-Cells.

Previous studies have reported beneficial effects of NADPH oxidase 4 (NOX4) inhibition on beta-cell survival in vitro and in vivo. The mechanisms by which NOX4 inhibition protects insulin producing cells are, however, not known. The aim of the present study was to investigate the effects of a pharmacological NOX4 inhibitor (GLX7013114) on human islet and EndoC-βH1 cell mitochondrial function, and to correlate such effects with survival in islets of different size, activity, and glucose-stimulated insulin release responsiveness. We found that maximal oxygen consumption rates, but not the rates of acidification and proton leak, were increased in islets after acute NOX4 inhibition. In EndoC-βH1 cells, NOX4 inhibition increased the mitochondrial membrane potential, as estimated by JC-1 fluorescence; mitochondrial reactive oxygen species (ROS) production, as estimated by MitoSOX fluorescence; and the ATP/ADP ratio, as assessed by a bioluminescent assay. Moreover, the insulin release from EndoC-βH1 cells at a high glucose concentration increased with NOX4 inhibition. These findings were paralleled by NOX4 inhibition-induced protection against human islet cell death when challenged with high glucose and sodium palmitate. The NOX4 inhibitor protected equally well islets of different size, activity, and glucose responsiveness. We conclude that pharmacological alleviation of NOX4-induced inhibition of beta-cell mitochondria leads to increased, and not decreased, mitochondrial ROS, and this was associated with protection against cell death occurring in different types of heterogeneous islets. Thus, NOX4 inhibition or modulation may be a therapeutic strategy in type 2 diabetes that targets all types of islets.

Cadmium inhibits neural stem/progenitor cells proliferation via MitoROS-dependent AKT/GSK-3β/β-catenin signaling pathway.

Cadmium (Cd) is a toxic heavy metal widely found in the environment. Cd is also a potential neurotoxicant, and its exposure is associated with impairment of cognitive function. However, the underlying mechanisms by which Cd induces neurotoxicity are unclear. In this study, we investigated the in vitro effect of Cd on primary murine neural stem/progenitor cells (mNS/PCs) isolated from the subventricular zone. Our results show that Cd exposure leads to mNS/PCs G1/S arrest, promotes cell apoptosis, and inhibits cell proliferation. In addition, Cd increases intracellular and mitochondrial reactive oxygen species (ROS) that activates mitochondrial oxidative stress, decreases ATP production, and increases mitochondrial proton leak and glycolysis rate in a dose-dependent manner. Furthermore, Cd exposure decreases phosphorylation of protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK3β) in mNS/PCs. In addition, pretreatment mNS/PCs with MitoTEMPO, a mitochondrial-targeted antioxidant, improves mitochondrial morphology and functions and attenuates Cd-induced inhibition of mNS/PCs proliferation. It also effectively reverses Cd-induced changes of phosphorylation of AKT and the expression of β-catenin and its downstream genes. Taken together, our data suggested that AKT/GSK3β/β-catenin signaling pathway is involved in Cd-induced mNS/PCs proliferation inhibition via MitoROS-dependent pattern.

Ellagic Acid and Its Microbial Metabolite Urolithin A Alleviate Diet-Induced Insulin Resistance in Mice.

This work aims at evaluating the effect of dietary ellagic acid (EA) and its microbial metabolite urolithin A (UA) on glucose metabolism and insulin resistance (IR) in mice with diet-induced IR. DBA2J mice are fed a high fat/high sucrose diet (HF/HS) for 8 weeks to induce IR and then 0.1% EA, UA, or EA and UA (EA+UA) are added to the HF/HS-diet for another 8 weeks. UA significantly decreases fasting glucose and increases adiponectin compared with HF/HS-controls. During intraperitoneal insulin tolerance test, EA+UA significantly improve insulin-mediated glucose lowering effects at 15 and 120min and reduce blood triglycerides compared with HF/HS-controls. Serum free fatty acids are significantly decreased by EA, UA, and EA+UA. Differential expression of genes related to mitochondrial function by EA, UA, and EA+UA in liver and skeletal muscle is observed. Primary hepatocytes from IR-mice have higher proton leak, basal and ATP-linked oxygen consumption rates compared with healthy controls. EA and EA+UA but not UA reduce the proton leak in hepatocytes from IR-mice. EA and UA induce different metabolic benefits in IR mice. The effects of EA and UA on mitochondrial function suggest a potentially novel mechanism modulating metabolism.

Protective Therapy with Mitochondria Isolated from Mesenchymal Stromal Cells in a Sepsis Model

Background & Aim In vivo mitochondrial transfer of mesenchymal stromal cells (MSCs) to immune cells, alveolar and bronchial epithelial cells resulted in metabolic and functional improvements. Thus, we hypothesized that naked mitochondria isolated from MSCs can be internalized by pulmonary structural cells and could be used as systemic therapy in a sepsis model. Methods, Results & Conclusion MSCs-derived mitochondria were incubated with septic murine lung endothelial and epithelial cells. Mitochondrial internalization was assessed by fluorescence microscopy, and its impact on oxidative stress and cellular respiration were evaluated. C57Bl6 mice were divided into control group, sepsis group (CLP, cecal ligation and puncture) and sepsis group with mito-treatment (CLP-mito, intravenous administration of mitochondria derived from 3 × 106 MSCs, 24 hours after CLP). The effects on mortality, pulmonary function and morphometry, liver and kidney histopathological and molecular changes were evaluated after 24 hours of mito-treatment. Mitochondria internalization by septic endothelial and epithelial cells led to reduce oxidative stress which was increased by 250% in endothelial cells and 150% in epithelial cells in CLP mice compared to control. Likewise, by oxygraphy, we analyzed the rate of oxygen consumption (OCR) of these cells. We have seen that, in epithelial cells, only maximal (MR) repolarization was reduced by about 30% after sepsis. In endothelial, both basal (BR) and maximal respiration (MR) were reduced by 50 and 48% respectivelly after sepsis, as well as a significant increase in the proton-leak (PL) rate in relation to the control group (33%). In both cell types, the altered parameters were re-established at control levels after mitocondrial treatment. Mito-treatment induced higher survival rates (60%) and reduced lung elastance (24%). Lung morphometry analysis demonstrated that mito-treatment reversed increases found in septic animals in alveolar collapse (230%), lung neutrophils (90%), lung collagen collagen (300%), elastogenesis (38%). Histological analysis of the liver demonstrates the increase of vacuolization (210%) and inflammatory infiltrate (105%) in septic animals, which were completely normalized after mito-treatment. Treatment also improved sepsis-induced kidney brush border damage (350%) and interstitial edema (205%). This work demonstrates the ability to internalize mitochondria isolated from MSCs by structural cells and the therapeutic potential of mito-treatment in a sepsis model.

Queuine Micronutrient Deficiency Promotes Warburg Metabolism and Reversal of the Mitochondrial ATP Synthase in Hela Cells.

Queuine is a eukaryotic micronutrient, derived exclusively from eubacteria. It is incorporated into both cytosolic and mitochondrial transfer RNA to generate a queuosine nucleotide at position 34 of the anticodon loop. The transfer RNA of primary tumors has been shown to be hypomodified with respect to queuosine, with decreased levels correlating with disease progression and poor patient survival. Here, we assess the impact of queuine deficiency on mitochondrial bioenergetics and substrate metabolism in HeLa cells. Queuine depletion is shown to promote a Warburg type metabolism, characterized by increased aerobic glycolysis and glutaminolysis, concomitant with increased ammonia and lactate production and elevated levels of lactate dehydrogenase activity but in the absence of significant changes to proliferation. In intact cells, queuine deficiency caused an increased rate of mitochondrial proton leak and a decreased rate of ATP synthesis, correlating with an observed reduction in cellular ATP levels. Data from permeabilized cells demonstrated that the activity of individual complexes of the mitochondrial electron transport chain were not affected by the micronutrient. Notably, in queuine free cells that had been adapted to grow in galactose medium, the re-introduction of glucose permitted the mitochondrial F1FO-ATP synthase to operate in the reverse direction, acting to hyperpolarize the mitochondrial membrane potential; a commonly observed but poorly understood cancer trait. Together, our data suggest that queuosine hypomodification is a deliberate and advantageous adaptation of cancer cells to facilitate the metabolic switch between oxidative phosphorylation and aerobic glycolysis.

Abstract 282: Mitochondrial Complex I Induced Myocardial Stunning Following Cardiopulmonary Resuscitation

Background: Cardiogenic shock following cardiopulmonary resuscitation (CPR) for sudden cardiac arrest is common, occurring even in the absence of acute coronary artery occlusion, and contributes to high rates of post-CPR mortality. The pathophysiology of this shock is unclear and effective therapies for improving clinical outcomes are lacking. Methods and Results: Using a murine model of asystolic cardiac arrest, we investigated the pathophysiology of post-CPR cardiogenic shock and discovered that duration of cardiac arrest (4, 8, 12 or 16-minute) prior to CPR determined post-resuscitation success rates, degree of neurological injury, and severity of myocardial dysfunction. Post-CPR cardiac dysfunction was not associated with myocardial necrosis, apoptosis, inflammation, or mitochondrial permeability transition pore opening and recovered within several days, indicative of myocardial stunning. Post-CPR myocardial stunning was associated with increases in ventricular and mitochondrial reactive oxygen species (ROS, P <0.001 vs Sham, respectively). Seahorse micropolarimetry of isolated post-CPR cardiac mitochondria revealed decreased rates of maximal oxygen consumption rates (OCR) for both Complex I and Complex II vs controls ( P <0.01 vs Sham, respectively), indicating inhibition of mitochondrial oxidative phosphorylation. Paradoxically, in the presence of ADP stimulated coupled respiration, post-CRP mitochondria demonstrated increased OCR ( P <0.05 vs Sham) and increased rates of proton leak ( P <0.05 vs Sham), suggesting Complex I as the site of ROS generation. These findings were not observed at complex II. S1QEL, a complex I-specific superoxide inhibitor, administered during CPR, decreased myocardial ROS generation while improving post-CPR myocardial function ( P <0.01 vs CPR control), neurological injury ( P <0.01 vs CPR control), and survival ( P <0.01 vs CPR control). Conclusions: Our results demonstrate that cardiogenic shock following resuscitation from cardiac arrest is consistent with myocardial stunning mediated by mitochondrial complex I injury and ROS generation. Targeting this mechanism represents a novel and practical therapy for improving sudden cardiac arrest resuscitation outcomes.

Mitochondrial oxidative stress-induced transcript variants of ATF3 mediate lipotoxic brain microvascular injury

Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for cardiovascular disease and vascular dementia (VaD). Accumulating evidence indicates that both atherosclerosis and VaD are linked to vascular inflammation. However, the role of TGRL in vascular inflammation, which increases risk for VaD, remains largely unknown and its underlying mechanisms are still unclear. We strived to determine the effects of postprandial TGRL exposure on brain microvascular endothelial cells, the potential risk factor of vascular inflammation, resulting in VaD. We showed in Aung et al., J Lipid Res., 2016 that postprandial TGRL lipolysis products (TL) activate mitochondrial reactive oxygen species (ROS) and increase the expression of the stress-responsive protein, activating transcription factor 3 (ATF3), which injures human brain microvascular endothelial cells (HBMECs) in vitro. In this study, we deployed high-throughput sequencing (HTS)-based RNA sequencing methods and mito stress and glycolytic rate assays with an Agilent Seahorse XF analyzer and profiled the differential expression of transcripts, constructed signaling pathways, and measured mitochondrial respiration, ATP production, proton leak, and glycolysis of HBMECs treated with TL. Conclusions: TL potentiate ROS by mitochondria which activate mitochondrial oxidative stress, decrease ATP production, increase mitochondrial proton leak and glycolysis rate, and mitochondria DNA damage. Additionally, CPT1A1 siRNA knockdown suppresses oxidative stress and prevents mitochondrial dysfunction and vascular inflammation in TL treated HBMECs. TL activates ATF3-MAPKinase, TNF, and NRF2 signaling pathways. Furthermore, the NRF2 signaling pathway which is upstream of the ATF3-MAPKinase signaling pathway, is also regulated by the mitochondrial oxidative stress. We are the first to report differential inflammatory characteristics of transcript variants 4 (ATF3-T4) and 5 (ATF3-T5) of the stress responsive gene ATF3 in HBMECs induced by postprandial TL. Specifically, our data indicates that ATF3-T4 predominantly regulates the TL-induced brain microvascular inflammation and TNF signaling. Both siRNAs of ATF3-T4 and ATF3-T5 suppress cells apoptosis and lipotoxic brain microvascular endothelial cells. These novel signaling pathways triggered by oxidative stress-responsive transcript variants, ATF3-T4 and ATF3-T5, in the brain microvascular inflammation induced by TGRL lipolysis products may contribute to pathophysiological processes of vascular dementia.

Ellagic Acid and Its Microbial Metabolite Urolithin a Alleviate Diet-induced Insulin Resistance in Mice (OR24-03-19)

ObjectivesPomegranates have high levels of ellagitannins (ETs) and ellagic acid (EA). ETs and EA are converted to urolithins (U) by gut microbiota in the GI tract. Both are absorbed systemically. We aimed at evaluating the metabolic effects of EA and its microbial metabolite UA in mice with diet-induced insulin resistance (IR). MethodsMale DBA2J mice were fed a Western diet (42% energy from fat, 32% from sucrose, 0.2% cholesterol) for 8 weeks to induce IR. After IR was confirmed the WD was supplemented with 0.1% EA, UA, or EA and UA combined (EAUA) for another 8 weeks. At the end of the intervention body composition, intraperitoneal insulin and glucose tolerance test (IPITT and IPGTT), serum and tissue (liver, adipose and skeletal muscle (SM)) lipids, inflammatory, metabolic and mitochondrial markers were determined. ResultsCompared to WD control, blood glucose and free fatty acids were significantly decreased with EA, UA and EAUA, and IR and triglycerides were improved by EAUA only. Hepatic lipid content, histology markers of hepatic steatosis, as well as inflammatory markers (TNFα, MCP1 and IL6) in blood and all three tissues were not changed by the intervention. We observed differential regulation of genes related to mitochondrial function but not mitochondrial DNA content by EA, UA and EAUA in liver and SM. Markers of mitochondrial fusion (Mfn2) and mitophagy (Parkin and Pink) were increased by UA and EAUA compared with IR mice. In addition, primary hepatocytes from IR mice had significant higher proton leak, basal and ATP-linked oxygen consumption rates than healthy control mice. EA and EAUA but not UA reduced the proton leak in hepatocytes from IR mice. ConclusionsDietary EA and UA differentially improved the glucose control and modified markers of mitochondrial function in liver and SM of mice with diet induced IR. Future studies are needed to understand how EA/UA mediated changes of mitochondrial function contribute to the improvement of IR. Funding SourcesCenter for Human Nutrition, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles.

Brain cytochrome-c-oxidase as a marker of mitochondrial function: A pilot study in major depression using NIRS.

Brain mitochondrial dysfunction is implicated in the pathophysiology of mood disorders. Brain cytochrome-c-oxidase (COX) activity is associated with the mitochondrial function. Near-infrared spectroscopy (NIRS) noninvasively measures oxidized COX (oxCOX) and tissue oxygenation index (TOI) reflecting cerebral blood flow and oxygenation. oxCOX and TOI were assessed in prefrontal cortex (Fp1/2, Brodmann area 10) in patients in a major depressive episode (N = 13) with major depressive disorder (MDD; N = 7) and bipolar disorder (BD; N = 6) compared with the controls (N = 10). One patient with MDD and all the patients with BD were taking medications. Computational modeling estimated oxCOX and TOI related indices of mitochondrial function and cerebral blood flow, respectively. oxCOX was lower in patients than controls (p = .014) correlating inversely with depression severity (r = -.72; p = .006), driven primarily by lower oxCOX in BD compared with the controls. Computationally modeled mitochondrial parameters of the electron transport chain, such as the nicotinamide adenine dinucleotide ratio (NAD+ /NADH; p = .001) and the proton leak rate across the inner mitochondrial membrane (klk2 ; p = .008), were also lower in patients and correlated inversely with depression severity. No such effects were found for TOI. In this pilot study, oxCOX and related mitochondrial parameters assessed by NIRS indicate an abnormal cerebral metabolic state in mood disorders proportional to depression severity, potentially providing a biomarker of antidepressant effect. Because the effect was driven by the medicated BD group, findings need to be evaluated in a larger, medication-free population.

Evaluation of free testosterone measurement on the ids-isys automated platform as compared to RIA

Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for cardiovascular disease and vascular dementia (VaD). Accumulating evidence indicates that both atherosclerosis and VaD are linked to vascular inflammation. However, the role of TGRL in vascular inflammation, which increases risk for VaD, remains largely unknown and its underlying mechanisms are still unclear. We strived to determine the effects of postprandial TGRL exposure on brain microvascular endothelial cells, the potential risk factor of vascular inflammation, resulting in VaD. We showed in Aung et al., J Lipid Res., 2016 that postprandial TGRL lipolysis products (TL) activate mitochondrial reactive oxygen species (ROS) and increase the expression of the stress-responsive protein, activating transcription factor 3 (ATF3), which injures human brain microvascular endothelial cells (HBMECs) in vitro. In this study, we deployed high-throughput sequencing (HTS)-based RNA sequencing methods and mito stress and glycolytic rate assays with an Agilent Seahorse XF analyzer and profiled the differential expression of transcripts, constructed signaling pathways, and measured mitochondrial respiration, ATP production, proton leak, and glycolysis of HBMECs treated with TL. Conclusions: TL potentiate ROS by mitochondria which activate mitochondrial oxidative stress, decrease ATP production, increase mitochondrial proton leak and glycolysis rate, and mitochondria DNA damage. Additionally, CPT1A1 siRNA knockdown suppresses oxidative stress and prevents mitochondrial dysfunction and vascular inflammation in TL treated HBMECs. TL activates ATF3-MAPKinase, TNF, and NRF2 signaling pathways. Furthermore, the NRF2 signaling pathway which is upstream of the ATF3-MAPKinase signaling pathway, is also regulated by the mitochondrial oxidative stress. We are the first to report differential inflammatory characteristics of transcript variants 4 (ATF3-T4) and 5 (ATF3-T5) of the stress responsive gene ATF3 in HBMECs induced by postprandial TL. Specifically, our data indicates that ATF3-T4 predominantly regulates the TL-induced brain microvascular inflammation and TNF signaling. Both siRNAs of ATF3-T4 and ATF3-T5 suppress cells apoptosis and lipotoxic brain microvascular endothelial cells. These novel signaling pathways triggered by oxidative stress-responsive transcript variants, ATF3-T4 and ATF3-T5, in the brain microvascular inflammation induced by TGRL lipolysis products may contribute to pathophysiological processes of vascular dementia.

Compatible osmolytes modulate mitochondrial function in a marine osmoconformer Crassostrea gigas (Thunberg, 1793)

Salinity is an important environmental factor affecting physiology of marine organisms. Osmoconformers such as marine mollusks maintain metabolic function despite changes of the osmolarity and composition of the cytosol during salinity shifts. Currently, metabolic responses to the salinity-induced changes of the intracellular milieu are not well understood. We studied the effects of osmolarity (450 vs. 900 mOsm) and compatible osmolytes (70–590 mM of taurine or betaine) on isolated gill mitochondria of a marine osmoconformer, the Pacific oyster Crassostrea gigas. Physiological concentrations of taurine enhanced mitochondrial ATP synthesis and electron transport system (ETS) capacity, increased mitochondrial coupling and stimulated the forward flux through the Complex I. Notably, the stimulatory effects of taurine were more pronounced at 900 mOsm compared to 450 mOsm. In contrast, betaine proportionally increased the rates of the mitochondrial proton leak, oxidative phosphorylation and ETS flux (with no net effect on the mitochondrial coupling) and suppressed the activity of cytochrome c oxidase in oyster mitochondria. However, the effective concentration of betaine (590 mM) was higher than typically found in bivalves, and thus betaine is not likely to affect oyster mitochondria under the physiological conditions in vivo. Our findings indicate that taurine may support the mitochondrial bioenergetics during hyperosmotic stress in oysters. Compatibility of taurine with the metabolic functions and its beneficial effects on mitochondria may have contributed to its broad distribution as an osmolyte in marine osmoconformers and might explain the earlier reports of the positive effects of taurine supplementation on energy metabolism of other organisms, including mammals.

The single IGF-1 partial deficiency is responsible for mitochondrial dysfunction and is restored by IGF-1 replacement therapy

Background & aimsWe previously described in cirrhosis and aging, both conditions of IGF-1 deficiency, a clear hepatic mitochondrial dysfunction with increased oxidative damage. In both conditions, the hepatic mitochondrial function was improved with low doses of IGF-1. The aim of this work was to explore if the only mere IGF-1 partial deficiency, without any exogenous insult, is responsible for hepatic mitochondrial dysfunction. MethodsHeterozygous (igf1+/−) mice were divided into two groups: untreated and treated mice with low doses of IGF-1. WT group was used as controls. Parameters of hepatic mitochondrial function were determined by flow cytometry, antioxidant enzyme activities were determined by spectrophotometry, and electron chain transport enzyme levels were determined by immunohistochemistry and immunofluorescence analyses. Liver expression of genes coding for proteins involved in mitochondrial protection and apoptosis was studied by microarray analysis and RT-qPCR. ResultsHz mice showed a significant reduction in hepatic mitochondrial membrane potential (MMP) and ATPase activity, and an increase in intramitochondrial free radical production and proton leak rates, compared to controls. These parameters were normalized by IGF-1 replacement therapy. No significant differences were found between groups in oxygen consumption and antioxidant enzyme activities, except for catalase, whose activity was increased in both Hz groups. Relevant genes coding for proteins involved in mitochondrial protection and survival were altered in Hz group and were reverted to normal in Hz+IGF-1 group. ConclusionsThe mere IGF-1 partial deficiency is per se associated with hepatic mitochondrial dysfunction sensitive to IGF-1 replacement therapy. Results in this work prove that IGF-1 is involved in hepatic mitochondrial protection, because it is able to reduce free radical production, oxidative damage and apoptosis. All these IGF-1 actions are mediated by the modulation of the expression of genes encoding citoprotective and antiapoptotic proteins.

Lower mitochondrial proton leak and decreased glutathione redox in primary muscle cells of obese diet-resistant versus diet-sensitive humans.

Weight loss success in response to energy restriction is highly variable. This may be due in part to differences in mitochondrial function and oxidative stress. The objective of the study was to determine whether mitochondrial function, content, and oxidative stress differ in well-matched obese individuals in the upper [obese diet sensitive (ODS)] vs lower quintiles [obese diet resistant (ODR)] for rate of weight loss. Primary myotubes derived from muscle biopsies of individuals identified as ODS or ODR were studied. Compliant ODS and ODR females who completed in the Ottawa Hospital Weight Management Program and identified as ODS and ODR participated in this study. Eleven ODS and nine ODR weight-stable females matched for age, body mass, and body mass index participated in this study. Vastus lateralis muscle biopsies were obtained and processed for muscle satellite cell isolation. Mitochondrial respiration, content, reactive oxygen species, and glutathione redox ratios were measured in the myotubes of ODS and ODR individuals. Mitochondrial proton leak was increased in myotubes of ODS compared with ODR (P < .05). Reduced and oxidized glutathione was decreased in the myotubes of ODR vs ODS (P < .05), indicating a more oxidized glutathione redox state. There were no differences in myotube mitochondrial content, uncoupling protein 3, or adenine nucleotide translocase levels. Lower rate of mitochondrial proton leak in muscle is a cell autonomous phenomenon in ODR vs ODS individuals, and this is associated with a more oxidized glutathione redox state in ODR vs ODS myotubes. The muscle of ODR subjects may thus have a lower capacity to adapt to oxidative stress as compared with ODS.

Alterations in proton leak, oxidative status and uncoupling protein 3 content in skeletal muscle subsarcolemmal and intermyofibrillar mitochondria in old rats.

BackgroundWe considered of interest to evaluate how aging affects mitochondrial function in skeletal muscle.MethodsWe measured mitochondrial oxidative capacity and proton leak, together with lipid oxidative damage, superoxide dismutase specific activity and uncoupling protein 3 content, in subsarcolemmal and intermyofibrillar mitochondria from adult (six months) and old (two years) rats. Body composition, resting metabolic rate and plasma non esterified fatty acid levels were also assessed.ResultsOld rats displayed significantly higher body energy and lipids, while body proteins were significantly lower, compared to adult rats. In addition, plasma non esterified fatty acid levels were significantly higher, while resting metabolic rates were found to be significantly lower, in old rats compared to adult ones. Significantly lower oxidative capacities in whole tissue homogenates and in intermyofibrillar and subsarcolemmal mitochondria were found in old rats compared to adult ones. Subsarcolemmal and intermyofibrillar mitochondria from old rats exhibited a significantly lower proton leak rate, while oxidative damage was found to be significantly higher only in subsarcolemmal mitochondria. Mitochondrial superoxide dismutase specific activity was not significantly affected in old rats, while significantly higher content of uncoupling protein 3 was found in both mitochondrial populations from old rats compared to adult ones, although the magnitude of the increase was lower in subsarcolemmal than in intermyofibrillar mitochondria.ConclusionsThe decrease in oxidative capacity and proton leak in intermyofibrillar and subsarcolemmal mitochondria could induce a decline in energy expenditure and thus contribute to the reduced resting metabolic rate found in old rats, while oxidative damage is present only in subsarcolemmal mitochondria.

Immunomodulation by the interactive effects of cadmium and hypercapnia in marine bivalves Crassostrea virginica and Mercenaria mercenaria

Estuarine organisms are exposed to multiple stressors including large fluctuations in partial pressure of carbon dioxide (PCO2) and concentrations of trace metals such as cadmium (Cd) that can affect their survival and fitness. Ocean acidification due to the increasing atmospheric PCO2 leads to a decrease in pH and shifts in the carbonate chemistry of seawater which can change bioavailability and toxicity of metals. We studied the interactive effects of PCO2 and Cd exposure on metal levels, metabolism and immune-related functions in hemocytes of two ecologically and economically important bivalve species, Mercenaria mercenaria (hard shell clam) and Crassostrea virginica (Eastern oyster). Clams and oysters were exposed to combinations of three PCO2 levels (∼400, 800 and 2000 μatm PCO2, corresponding to the present day conditions and the projections for the years 2100 and 2250, respectively) and two Cd concentrations (0 and 50 μg l−1) in seawater. Following four weeks of exposure to Cd, hemolymph of both species contained similar Cd levels (50–70 μg l−1), whereas hemocytes accumulated intracellular Cd burdens up to 15–42 mg l−1, regardless of the exposure PCO2. Clam hemocytes had considerably lower Cd burdens than those of oysters (0.7–1 ng 10−6 cells vs. 4–6 ng 10−6 cells, respectively). Cd exposure suppressed hemocyte metabolism and increased the rates of mitochondrial proton leak in normocapnia indicating partial mitochondrial uncoupling. This Cd-induced mitochondrial uncoupling was alleviated in hypercapnia. Cd exposure suppressed immune-related functions in hemocytes of clams and oysters, and these effects were exacerbated at elevated PCO2. Thus, elevated PCO2 combined with Cd exposure resulted in decrease in phagocytic activity and adhesion capacity as well as lower expression of mRNA for lectin and heat shock protein (HSP70) in clam and oyster hemocytes. In oysters, combined exposure to elevated PCO2 and Cd also led to reduced activity of lysozyme in hemocytes and hemolymph. Overall, our study shows that moderately elevated PCO2 (∼800–2000 μatm PCO2) potentiates the negative effects of Cd on immunity and thus may sensitize clams and oysters to pathogens and diseases during seasonal hypercapnia and/or ocean acidification in polluted estuaries.

Mitochondrial dysfunction in rabies virus infection of neurons

Infection with the challenge virus standard-11 (CVS) strain of fixed rabies virus induces neuronal process degeneration in adult mice after hindlimb footpad inoculation. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal protein adduct staining, indicating a critical role of oxidative stress. Mitochondrial dysfunction is the major cause of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection significantly increased maximal uncoupled respiration and complex IV respiration and complex I and complex IV activities, but did not affect complex II-III or citrate synthase activities. Increases in complex I activity, but not complex IV activity, correlated with susceptibility of the cells to CVS infection. CVS infection maintained coupled respiration and rate of proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by a high NADH/NAD+ ratio. The basal production of reactive oxygen species (ROS) was not affected in CVS-infected neurons. However, a higher rate of ROS generation occurred in CVS-infected neurons in the presence of mitochondrial substrates and inhibitors. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration and oxidative stress.

Interactive effects of pH and metals on mitochondrial functions of intertidal bivalves Crassostrea virginica and Mercenaria mercenaria

Intertidal bivalves experience broad fluctuations of environmental temperature, pH and oxygen content which could change their intracellular pH. They are also exposed to trace metals such as cadmium (Cd) and copper (Cu) that accumulate in their tissues and may negatively affect mitochondrial functions and bioenergetics. We determined the interactive effects of pH and trace metals (25μM Cd or Cu) on mitochondrial functions (including respiration and membrane potentials in both ADP-stimulated (state 3) and resting (state 4) states) of two common marine bivalves, the hard clams (Mercenaria mercenaria) and eastern oysters (Crassostrea virginica). In the absence of the trace metals, mitochondrial functions of C. virginica and M. mercenaria were insensitive to pH in a broad physiologically relevant range (6.6–7.8). Mitochondrial respiration was generally suppressed by 25μM Cd or Cu (with the stronger effects observed for ADP-stimulated compared to the resting respiration) while the mitochondrial membrane potential was unaffected. pH modulated the effects of Cu and Cd on mitochondrial respiration of the bivalves. In oysters, Cu suppressed ADP-stimulated mitochondrial respiration at high and low pH values (6.6 and 7.8, respectively), but had no effect in the intermediate pH range (7.0–7.4). In clams, the negative effect of Cu on ADP-stimulated respiration was only observed at extremely high pH (7.8). A decrease in pH was also protective against Cd in mitochondria of clams and oysters. In clams, 25μM Cd suppressed ADP-stimulated respiration at all pH; however, at low pH (6.6–7.0) this suppression was paralleled by a decrease in the rates of proton leak thereby effectively restoring mitochondrial coupling. In oysters, the inhibitory effects of Cd on ADP-stimulated respiration were fully abolished at low pH (6.6–7.0). This indicates that moderate acidosis (such as occurs during exposure to air, extreme salinities or elevated CO2 levels in the intertidal zone) may have a beneficial side-effect of protecting mitochondria of clams and oysters against the toxic effects of trace metals in polluted estuaries.

Investigating The Metabolic Basis for Weight loss Variability in Obese Humans

Response to weight loss treatments is highly variable and this may be due to biological factors such as altered skeletal muscle oxidative capacity and mitochondrial function. Diet adherent patients who completed the Ottawa Hospital Weight Management Program (OHWMP) and demonstrated the highest (Obese Diet Sensitive; ODS) and the lowest (Obese Diet Resistant; ODR) rates of weight loss have been recruited for studies of whole body and skeletal muscle energy metabolism. Our previous research demonstrated that ODS, compared to ODR have a higher proportion of oxidative fibres and mitochondrial proton leak in vastus lateralis muscle. We and others also demonstrated that proton leak through uncoupling protein 3 mitigates oxidative stress. It is proposed that a higher rate of mitochondrial proton leak contributes to higher rates of weight loss and may protect against oxidative stress. Purpose: To investigate potential metabolic mechanisms in muscle that contribute to weight loss variability in humans. Highly diet adherent, obese, weight stable women who have previously completed the OHWP and classified as ODS or ODR have been recruited to participate in this study. Participants arrive at the study location early in the morning for determinants of resting metabolic rate. Prior to and 6 hrs following a high fat meal (60% dietary fat, 35% of total daily caloric requirements), vastus lateralis muscle biopsies are obtained for assessment of mitochondrial respiration and determinants of mitochondrial function and oxidative stress. During the 6 hrs, blood samples are obtained every half hour and metabolic rate is measured every hour. Hypothesis: Skeletal muscle from ODS individuals will demonstrate higher rates of mitochondrial respiration and proton leak under basal conditions and in response to a high fat meal which in turn will minimize oxidative stress in muscle. This research will provide valuable insight into the biological basis for weight loss variability in humans.