Mitochondrial Proton Research Articles

Transient heat release during induced mitochondrial proton uncoupling

Non-shivering thermogenesis through mitochondrial proton uncoupling is one of the dominant thermoregulatory mechanisms crucial for normal cellular functions. The metabolic pathway for intracellular temperature rise has widely been considered as steady-state substrate oxidation. Here, we show that a transient proton motive force (pmf) dissipation is more dominant than steady-state substrate oxidation in stimulated thermogenesis. Using transient intracellular thermometry during stimulated proton uncoupling in neurons of Aplysia californica, we observe temperature spikes of ~7.5 K that decay over two time scales: a rapid decay of ~4.8 K over ~1 s followed by a slower decay over ~17 s. The rapid decay correlates well in time with transient electrical heating from proton transport across the mitochondrial inner membrane. Beyond ~33 s, we do not observe any heating from intracellular sources, including substrate oxidation and pmf dissipation. Our measurements demonstrate the utility of transient thermometry in better understanding the thermochemistry of mitochondrial metabolism.

Brown seaweed (AquaArom) supplementation increases food intake and improves growth, antioxidant status and resistance to temperature stress in Atlantic salmon, Salmo salar.

Seaweeds represent a vast resource that remains underutilized as an ingredient in aquafeeds. Here we probed the effect of addition of AquaArom, a seaweed meal derived from brown seaweeds (Laminaria sp., kelp), to fish feed on growth performance, antioxidant capacity and temperature responsiveness of mitochondrial respiration. A commercial salmonid feed was mixed with 0 (control), 3, 6 and 10% seaweed and fed to Atlantic salmon (Salmo salar) smolts for 30 days. The smolts consumed more of the seaweed-supplemented food relative to the control and there were no mortalities. Compared with the control, the final fish weight, standard length, weight gain and SGR were higher in fish fed diets supplemented with the 3 and 10% seaweed, while growth performance for fish maintained on 6% seaweed remained neutral. Importantly, seaweed supplementation increased protein efficiency ratio (PER) and tended to improve food conversion ratio (FCR). Although the hepatosomatic and visceral indices did not change, whole gut and intestinal weights and lengths were higher in fish maintained on seaweed-supplemented diets suggesting increased retention time and a larger surface area for food digestion and nutrient absorption. Measurement of antioxidant status revealed that seaweed supplementation dose-dependently increased plasma total antioxidant capacity as well as the level of glutathione, and activities of catalase and superoxide dismutase in liver mitochondria. Moreover, seaweed supplementation reduced the effect of acute temperature rise on mitochondrial respiration and proton leak. Overall, these data suggest that AquaArom can be mixed with fish food up to 10% to increase food consumption and enhance growth performance, as well as to improve antioxidant capacity and alleviate adverse effects of stressors such as temperature in fish.

Abstract 800: ESI-09 and HJC0197, known EPAC inhibitors, sensitize lung cancer cells to glucose starvation by uncoupling mitochondrial electron transport, leading to bioenergetic crisis

Abstract Cancer cells utilize aerobic glycolysis and proliferate continuously beyond the capacity of their blood supply, leading to microenvironmental stresses such as hypoxia, nutrient (glucose) deprivation and extracellular acidosis. We have previously showed that extracellular acidosis prevents glucose starvation-induced death of lung cancer cells by reducing glycolytic energy production and de novo protein/RNA synthesis that consume ATP (AACR 2018). This acidosis-dependent, glucose starvation-resistant, and ATP-saving phenotype is thought to serve as an adaptive response to glucose deprivation in cancer cells to survive an energy-restricted tumor microenvironment. In the present study, we screened small-molecule inhibitors to explore therapeutic reagents that can exert cytotoxicity against the glucose starvation-resistant phenotype in the lung cancer cell lines, A549 and H1299 cells. Among the various inhibitors tested, we found that ESI-09 and HJC0197, known as inhibitors of the exchanger protein directly activated by cAMP (EPAC), reduced the cellular ATP levels and survival under glucose deprivation stress under both acidic (pH 6.8) and neutral (pH 7.4) conditions. Interestingly, the ATP-reducing effect of ESI-09 and HJC0197 was not due to inhibition of the EPAC function, because the effect cannot be reproduced with other EPAC inhibitors, such as CE3F4, EPAC5376753 and ESI-05. Rather, the effect of ESI-09 and HJC0197 was attributed to their previously unknown action of uncoupling the mitochondrial electron transport chain (ETC). We found that ESI-09 and HJC0197 induced mitochondrial proton leak, causing ATP deficiency and cell death via the following three mechanisms: 1) decreased mitochondrial ATP production due to ETC uncoupling, 2) increased ATP consumption resulting from reversal of F0/F1-ATPsynthase/ATPase to maintain the mitochondrial membrane potential, and 3) glucose deficiency as a result of increased glucose consumption due to a compensatory stimulation of glycolytic ATP production. The energy-saving phenotype of cancer cells is thought to serve as an adaptive strategy that allows cell survival in an energy-restricted, acidic tumor microenvironment. Our results suggest that ESI-09 and HJC0197 disrupt bioenergetic tumor metabolism for energy homeostasis in both acidic and neutral microenvironment, and may exert a therapeutic effect against the starvation-resistant phenotype of cancer cells. The complete chemical structures of ESI-09 and HJC0197 will be divulged at the time of the presentation at the meeting. Citation Format: Yuki Iwai, Ryota Kikuchi, Hiroyuki Nakamura, Nobuyuki Koyama, Koichi Hagiwara, Kazutetsu Aoshiba. ESI-09 and HJC0197, known EPAC inhibitors, sensitize lung cancer cells to glucose starvation by uncoupling mitochondrial electron transport, leading to bioenergetic crisis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 800.

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.

2020: murburn concept heralds a new era in cellular bioenergetics

Cellular bioenergetics has been interpreted for several decades using the Keilin-Mitchell-Boyer (KMB) model of oxidative phosphorylation (OxPhos), and for understanding/managing of the pertinent mitochondrial pathophysiological states. Although decades of research had revealed many faulty chemico-physical aspects of KMB perspective, recent critical insights from our group’s writings have sufficiently brought out the errors in the KMB model, rendering it obsolete/redundant. The murburn model proposed in lieu is a compelling alternative for explaining OxPhos because it reasons several facets of mitochondrial structure-function correlations, reaction chemistry and thermodynamics. However, the mitochondrial research community appears to be recalcitrant, and continues to follow the erstwhile erroneous ideas and not take cognizance of the new insights. Hence, we deemed it opportune to make a clarion call for a jettisoning of the superseded terminologies (or keywords) and concepts routinely used by researchers in this field. First, we present a statistical perspective of the usage of these terms in the past and recent times, to support our claims and call. Then, we articulate simplified arguments why the key elements/terms of the KMB model like “electron-transfer/electron-transport/respiratory chain”, “mitochondrial proton pumps”, “mitochondrial membrane po-tential”, “chemiosmosis”, “proton motive force” and “rotary ATP synthase/synthesis” violate scientific/semantic logic. Finally, we conclude with summative statements projecting the importance of our claims and call.

SUN-216 High Fat Diet Regulate Energy Metabolism of Sertoli Cells

The harmfulness of high fat diet (HFD) to male fertility remains conflict. Sertoli cells (SCs) are essential for spermatogenesis as they provide developing germ cells (GCs) with physical support, nutrients and energy (1). Lactate produced by SCs represents as the preferential energy substrate to GCs (2). The aim of this study was to investigate the effects of HFD on energy supply of SCs to GCs and to themselves. In vivo, in order to assess the effects of HFD on male fertility and SCs, male Wistar rats were fed with HFD for 8 weeks. The results showed that the sperm concentration (P<0.01) and pups per litter (7.33 ± 1.20 vs 12.33 ± 0.33,P<0.05) significantly decreased in HFD rats compared to those normal diet (ND) fed rats. Furthermore, the seminiferous epithelia were slightly thinned and disorganized in HFD rats, accompanied by an increased number of vacuoles and lipid droplets in SCs. In vitro, primary SCs obtained from 19-20 days of Wistar male rats were cultured with 0.25 and 0.50 mM palmitic acid for 24 hours for further studies. We firstly assessed the energy supply of SCs to GCs, and found that palmitic acid promoted glycolysis and improved lactate production (P<0.01) and transport by increasing the protein expression or activity associated with lactate biosynthesis and transport. To investigate the effect of palmitic acid on SCs energy metabolism, we assessed mitochondrial function via extracellular flux analyzer. The mitochondrial ATP synthesis (P<0.01) and maximum respiratory capacity (P<0.01) were decreased, while mitochondrial proton leakage was increased in SCs cultured with 0.5mM palmitic acid. Consistent with these, palmitic acid increased mitochondrial ROS and decreased mitochondrial membrane potential. For SCs, fatty acid β-oxidation is suggested to be used to satisfy the vast majority of their own energy needs (3). In this study, compared to control cells, the protein level of β-oxidative Key enzyme, carnitine palmitoyl-transferase 1α in SCs exposed to palmitic acid was decreased (P<0.01), along with the increase of intracellular lipid droplets. In conclusion, palmitic acid increased lactate production. At the same time, palmitic acid also impaired mitochondrial respiratory and fatty acid β-oxidation. We speculate that SCs yields more lactate could not only ensure the energy supply of SCs to GCs under harsh conditions, but also compensate for its own energy metabolism disorder. (1) Mruk DD et al., Endocrine reviews 2004; 25: 747-806. (2) Smith LB et al., Seminars in cell & developmental biology 2014; 30: 2-13. (3) Xionget al., Reproduction 2009 137,469-479

Mitochondrial Rich Proton Pumping Cells in the Kidney and Epididymis are Highly Glycolytic

The kidney collecting duct (CD) has a crucial role in regulating acid‐base balance. Alpha intercalated cells (ICs) of the CD maintain acid‐base homeostasis by acidifying the urine, via apical V‐ATPase H+ pumps. ICs are characterized in appearance by a very high density of mitochondria, but remarkably little is known about metabolism in these cells, due to a lack of suitable techniques to study this and the inability to generate a stable IC cell line. By using different innovative imaging techniques, we aimed to better understand how metabolism might be adapted in ICs to their specialized H+ pumping function.Using multiphoton microscopy of intact murine kidney slices, we have found that mitochondria in ICs have some highly unusual features compared with other tubular cells. IC mitochondria are considerably more mobile and dynamic, and display a high NADH signal but low uptake of voltage dependent dyes. Moreover, they are energized by a pH gradient that is functionally coupled to the proton pumping activity of the V‐ATPase. However, high resolution confocal microscopy and 3D electron microscopy (FIB‐SEM) in fixed mouse kidney tissue revealed that the mitochondria and V‐ATPase expressing vesicles were distant from each other, reducing the likelihood that the mitochondria are the only energy source for the V‐ATPase H+ pumps in ICs. Furthermore, we observed that the expression of respiratory chain complex V (ATP synthase) is lower in ICs than in surrounding cells, implying reduced oxidative capacity. Conversely, expression of enzymes in the glycolytic pathway, including lactate dehydrogenase, was markedly higher in ICs, consistent with high anaerobic ATP‐generating capacity. Interestingly, expression of glycolytic enzymes was also high in clear cells in the epididymis, suggesting that this is a common feature of proton pumping cells.As a surrogate of cellular ATP levels, we imaged cytosolic Ca2+ in kidney slices using a fluorescent reporter (GcaMP6s), and found that inhibition of mitochondrial ATP production with cyanide produced striking rises in Ca2+ in all tubular cell types except ICs. However, Ca2+ also increased in ICs when glycolysis was simultaneously inhibited. Finally, direct functional measurement of V‐ATPase function in microperfused isolated outermedullary collecting ducts (OMCDs) revealed that inhibition of glycolysis leads to a significant decrease in H+ pumping in ICs.In summary, we have provided several lines of evidence that mitochondrial rich cells in the kidney and epididymis are surprisingly glycolytic, and that glycolysis is functionally important for V‐ATPase activity. These findings provide new insights into coupling of transport and metabolism in proton pumping cells.Support or Funding InformationThis research is supported by The Swiss National Centre for Competence in Research Kidney Control of Homeostasis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoxia Inducible Factor Activation Prevents Renal Mitochondria Dysfunction and Improves Cortical Oxygenation in Type 1 Diabetic Mice

Hypoxia‐inducible factors (HIFs) are transcription factors that are activated during hypoxia in order to regulate a hypoxic gene response. During normoxia, prolyl hydroxylase domain‐containing protein 2 (PHD2) is one of three isoforms that acts on HIF as a first step towards its degradation and its knockdown consequently results in HIF activation. Previous studies have demonstrated that diabetes induces mitochondria dysfunction resulting in renal cortical hypoxia, yet studies indicate that hyperglycemia impairs HIF stabilization resulting in suboptimal activation and an inability to correct renal hypoxia. In this study, we hypothesize that increased HIF‐activation resulting from PHD2 knockdown, prevents mitochondria dysfunction and improves renal oxygenation in type 1 diabetic mice.Balb/c mice from four groups (Wild type normoglycemic (n=16), wild type diabetic (n=10), PHD2+/− normoglycemic (n=16) and PHD2+/− diabetic (n=11)) were placed in metabolic cages for 24 h urine collection followed by conscious determination glomerular filtration rate (GFR), using a single bolus injection of FITC‐inulin. Diabetes was induced by a low dose injection of strepzotocin (i.p.) for 5 consecutive days and diabetes duration was 5–6 weeks. Renal cortical oxygenation was measured with Clark electrodes and kidney mitochondria function, including mitochondria proton leak, was assessed by high resolution respirometry. Urinary excretion of albumin and thiobarbituric acid reactive species were measured and corrected for 24 h urine volume.Diabetes resulted in increased GFR in both groups compared to normoglycemic mice. Wild type diabetic mice presented with increased total mitochondrial leak respiration compared to normoglycemic mice, which was prevented in the PHD2+/− mice. Interestingly, regulated leak, defined as mitochondrial proton leak regulated by uncoupling proteins and adenosine nucleotide translocase, was increased in diabetic PHD2+/− only, while unregulated leak, defined as total leak minus regulated leak, was increased in wild type diabetic mice. Both diabetic groups displayed increased albuminuria and thiobarbituric acid reactive species compared to normoglycemic control mice.In conclusion, activation of HIF by reducing PHD2 expression prevents mitochondria dysfunction and renal cortical hypoxia in type 1 diabetic mice. This could imply a novel target for the PHD inhibitors currently in clinical trials.Support or Funding InformationThis study was supported financially by the Swedish Research Council, the Swedish Diabetes Foundation and the Family Ernfors Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of AnnexinA1 as an Endogenous Regulator of RhoA, and Its Role in the Pathophysiology and Experimental Therapy of Type-2 Diabetes.

Annexin A1 (ANXA1) is an endogenously produced anti-inflammatory protein, which plays an important role in the pathophysiology of diseases associated with chronic inflammation. We demonstrate that patients with type-2 diabetes have increased plasma levels of ANXA1 when compared to normoglycemic subjects. Plasma ANXA1 positively correlated with fatty liver index and elevated plasma cholesterol in patients with type-2 diabetes, suggesting a link between aberrant lipid handling, and ANXA1. Using a murine model of high fat diet (HFD)-induced insulin resistance, we then investigated (a) the role of endogenous ANXA1 in the pathophysiology of HFD-induced insulin resistance using ANXA1−/− mice, and (b) the potential use of hrANXA1 as a new therapeutic approach for experimental diabetes and its microvascular complications. We demonstrate that: (1) ANXA1−/− mice fed a HFD have a more severe diabetic phenotype (e.g., more severe dyslipidemia, insulin resistance, hepatosteatosis, and proteinuria) compared to WT mice fed a HFD; (2) treatment of WT-mice fed a HFD with hrANXA1 attenuated the development of insulin resistance, hepatosteatosis and proteinuria. We demonstrate here for the first time that ANXA1−/− mice have constitutively activated RhoA. Interestingly, diabetic mice, which have reduced tissue expression of ANXA1, also have activated RhoA. Treatment of HFD-mice with hrANXA1 restored tissue levels of ANXA1 and inhibited RhoA activity, which, in turn, resulted in restoration of the activities of Akt, GSK-3β and endothelial nitric oxide synthase (eNOS) secondary to re-sensitization of IRS-1 signaling. We further demonstrate in human hepatocytes that ANXA1 protects against excessive mitochondrial proton leak by activating FPR2 under hyperglycaemic conditions. In summary, our data suggest that (a) ANXA1 is a key regulator of RhoA activity, which restores IRS-1 signal transduction and (b) recombinant human ANXA1 may represent a novel candidate for the treatment of T2D and/or its complications.

Mitoflash biogenesis and its role in the autoregulation of mitochondrial proton electrochemical potential.

Respiring mitochondria undergo an intermittent electrical and chemical excitation called mitochondrial flash (mitoflash), which transiently uncouples mitochondrial respiration from ATP production. How a mitoflash is generated and what specific role it plays in bioenergetics remain incompletely understood. Here, we investigate mitoflash biogenesis in isolated cardiac mitochondria by varying the respiratory states and substrate supply and by dissecting the involvement of different electron transfer chain (ETC) complexes. We find that robust mitoflash activity occurs once mitochondria are electrochemically charged by state II/IV respiration (i.e., no ATP synthesis at Complex V), regardless of the substrate entry site (Complex I, Complex II, or Complex IV). Inhibiting forward electron transfer abolishes, while blocking reverse electron transfer generally augments, mitoflash production. Switching from state II/IV to state III respiration, to allow for ATP synthesis at Complex V, markedly diminishes mitoflash activity. Intriguingly, when mitochondria are electrochemically charged by the ATPase activity of Complex V, mitoflashes are generated independently of ETC activity. These findings suggest that mitoflash biogenesis is mechanistically linked to the build up of mitochondrial electrochemical potential rather than ETC activity alone, and may functionally counteract overcharging of the mitochondria and hence serve as an autoregulator of mitochondrial proton electrochemical potential.

Mitochondrial matrix pH acidifies during anoxia and is maintained by the F1Fo-ATPase in anoxia-tolerant painted turtle cortical neurons.

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine‐123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF‐1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo‐ATPase was inhibited with oligomycin‐A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.

The yeast mitochondrial proteins Rcf1 and Rcf2 support the enzymology of the cytochrome c oxidase complex and generation of the proton motive force

The yeast mitochondrial proteins Rcf1 and Rcf2 are associated with a subpopulation of the cytochrome bc1-cytochrome c oxidase supercomplex and have been proposed to play a role in the assembly and/or modulation of the activity of the cytochrome c oxidase (complex IV, CIV). Yeast mutants deficient in either Rcf1 or Rcf2 proteins can use aerobic respiration-based metabolism for growth, but the absence of both proteins results in a strong growth defect. In this study, using assorted biochemical and biophysical analyses of Rcf1/Rcf2 single and double null-mutant yeast cells and mitochondria, we further explored how Rcf1 and Rcf2 support aerobic respiration and growth. We show that the absence of Rcf1 physically reduces the levels of CIV and diminishes the ability of the CIV that is present to maintain a normal mitochondrial proton motive force (PMF). Although the absence of Rcf2 did not noticeably affect the physical content of CIV, the PMF generated by CIV was also lower than normal. Our results indicate that the detrimental effects of the absence of Rcf1 and Rcf2 proteins on the CIV complex are distinct in terms of CIV assembly/accumulation and additive in terms of the ability of CIV to generate PMF. Thus, the combined absence of Rcf1 and Rcf2 alters both CIV physiology and assembly. We conclude that the slow aerobic growth of the Rcf1/Rcf2 double null mutant results from diminished generation of mitochondrial PMF by CIV and limits the level of CIV activity required for maintenance of the PMF and growth under aerobic conditions.

Constitutive Activation of NAD-Dependent Sirtuin 3 Plays an Important Role in Tumorigenesis of Chromium(VI)-Transformed Cells.

Chronic exposure of human bronchial epithelial BEAS-2B cells to hexavalent chromium [Cr(VI)] causes malignant cell transformation. Sirtuin-3 (SIRT3) regulates mitochondrial adaptive response to stress, such as metabolic reprogramming and antioxidant defense mechanisms. In Cr(VI)-transformed cells, SIRT3 was upregulated and mitochondrial adenosine triphosphate (ATP) production and proton leak were reduced. Knockdown of SIRT3 by its shRNA further decreased mitochondrial ATP production, proton leak, mitochondrial mass, and mitochondrial membrane potential, indicating that SIRT3 positively regulates mitochondrial oxidative phosphorylation and maintenance of mitochondrial integrity. Mitophagy is critical to maintain proper cellular functions. In Cr(VI)-transformed cells expressions of Pink 1 and Parkin, twomitophagy proteins, were elevated, and mitophagy remained similar as that in passage-matched normal BEAS-2B cells, indicating that in -Cr(VI)-transformed cells mitophagy is suppressed. Knockdown of SIRT3 induced mitophagy, suggesting that SIRT3 plays an important role in mitophagy suppression of Cr(VI)-transformed cells. In Cr(VI)-transformed cells, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was constitutively activated, and protein levels of p62 and p-p62Ser349 were elevated. Knockdown of SIRT3 or treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) decreased the binding of p-p62Ser349 to Keap1, resulting in increased binding of Keap1 to Nrf2 and consequently reduced Nrf2 activation. The results from CHIP assay showed that in Cr(VI)-transformed cells binding of Nrf2 to antioxidant response element (ARE) of SIRT3 gene promoter was dramatically increased. Knockdown of SIRT3 suppressed cell proliferation and tumorigenesis of Cr(VI)-transformed cells. Overexpression of SIRT3 in normal BEAS-2B cells exhibited mitophagy suppression phenotype and increased cell proliferation and tumorigenesis. The present study demonstrated that upregulation of SIRT3 causes mitophagy suppression and plays an important role in cell survival and tumorigenesis of Cr(VI)-transformed cells.

ATP synthase is required for male fertility and germ cell maturation in Drosophila testes.

Germ cell maturation is essential for spermatogenesis and testis homeostasis. ATP synthase serves significant roles in energy storage in germ cell survival and is catalyzed by alterations in the mitochondrial membrane proton concentration. The intrinsic cellular mechanisms governing stem cell maturation remain largely unknown. In the present study, in vivo RNA interference (RNAi) screening of major ATP synthase subunits was performed, and the function of ATP synthase for male fertility and spermatogenesis in Drosophila was explored. A Upstream Activation Sequence/Gal4 transcription factor system was used to knock down gene expression in specific cell types, and immunofluorescence staining was conducted to assess the roles of ATP synthase subunits in Drosophila testes. It was identified that knockdown of ATP synthase resulted in male infertility and abnormal spermatogenesis in Drosophila testes. In addition, knockdown of the ATP synthase β subunit in germ cells resulted in defects in male infertility and germ cell maturation, while the hub and cyst cell populations were maintained. Other major ATP synthase subunits were also examined and similar phenotypes in Drosophila testes were identified. Taken together, the data from the present study revealed that ATP synthase serves important roles for male fertility during spermatogenesis by regulating germ cell maturation in Drosophila testes.

Reduced nuclear DNA methylation and mitochondrial transcript changes in adenomas do not associate with mtDNA methylation

BackgroundAltered mitochondrial function and large-scale changes to DNA methylation patterns in the nuclear genome are both hallmarks of colorectal cancer (CRC). Mitochondria have multiple copies of a 16 kb circular genome that contains genes that are vital for their function. While DNA methylation is known to alter the nuclear genome in CRC, it is not clear whether it could have a similar influence in mtDNA; indeed, currently, the issue of whether mitochondrial genome (mtDNA) methylation occurs is controversial. Thus our goal here was to determine whether the methylation state of mtDNA is linked to mitochondrial gene transcription in colorectal adenomas, and to assess its suitability as a biomarker in CRC.MethodsTo investigate the relationship between DNA methylation and mitochondrial transcripts in adenomas, we performed RNA-sequencing and Whole Genome Bisulphite Sequencing (WGBS) of mtDNA-enriched DNA from normal mucosa and paired adenoma patient samples.ResultsTranscriptional profiling indicated that adenomas had reduced mitochondrial proton transport versus normal mucosa, consistent with altered mitochondrial function. The expression of 3 tRNAs that are transcribed from mtDNA were also decreased in adenoma. Overall methylation of CG dinucleotides in the nuclear genome was reduced in adenomas (68%) compared to normal mucosa (75%, P < 0.01). Methylation in mtDNA was low (1%) in both normal and adenoma tissue but we observed clusters of higher methylation at the ribosomal RNA genes. Levels of methylation within these regions did not differ between normal and adenoma tissue.ConclusionsWe provide evidence that low-level methylation of specific sites does exist in the mitochondrial genome but that it is not associated with mitochondrial gene transcription changes in adenomas. Furthermore, as no large scale changes to mtDNA methylation were observed it is unlikely to be a suitable biomarker for early-stage CRC.

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Omega-3 Monoacylglyceride Effects on Longevity, Mitochondrial Metabolism and Oxidative Stress: Insights from Drosophila melanogaster.

During the last decade, essential polyunsaturated fatty acids (PUFAs) such as eicosatetraenoic acid (EPA) and docosahexaenoic acid (DHA) derived from marine sources have been investigated as nonpharmacological dietary supplements to improve different pathological conditions, as well as aging. The aim of this study was to determine the effects of dietary n-3 PUFA monoacylglycerides (MAG, both EPA and DHA) on the mitochondrial metabolism and oxidative stress of a short-lifespan model, Drosophila melanogaster, sampled at five different ages. Our results showed that diets supplemented with MAG-EPA and MAG-DHA increased median lifespan by 14.6% and decreased mitochondrial proton leak resulting in an increase of mitochondrial coupling. The flies fed on MAG-EPA also had higher electron transport system capacity and mitochondrial oxidative capacities. Moreover, both n-3 PUFAs delayed the occurrence of lipid peroxidation but only flies fed the MAG-EPA diet showed maintenance of superoxide dismutase activity during aging. Our study therefore highlights the potential of n-3 PUFA monoacylglycerides as nutraceutical compounds to delay the onset of senescence by acting directly or indirectly on the mitochondrial metabolism and suggests that Drosophila could be a relevant model for the study of the fundamental mechanisms linking the effects of n-3 PUFAs to aging.

Metformin attenuates renal medullary hypoxia in diabetic nephropathy through inhibition uncoupling protein‐2

The purpose of the study is to examine the effect of metformin on oxygen metabolism and mitochondrial function in the kidney of an animal model of insulinopenic diabetes in order to isolate any renoprotective effect from any concomitant effect on blood glucose homeostasis. Sprague-Dawley rats were injected with streptozotocin (STZ) (50mgkg-1 ) and when stable started on metformin treatment (250mgkg-1 ) in the drinking water. Rats were prepared for in vivo measurements 25 to 30days after STZ injection, where renal function, including glomerular filtration rate and sodium transport, was estimated in anesthetized rats. Intrarenal oxygen tension was measured using oxygen sensors. Furthermore, mitochondrial function was assessed in mitochondria isolated from kidney cortex and medulla analysed by high-resolution respirometry, and superoxide production was evaluated using electron paramagnetic resonance. Insulinopenic rats chronically treated with metformin for 4weeks displayed improved medullary tissue oxygen tension despite of no effect of metformin on blood glucose homeostasis. Metformin reduced UCP2-dependent LEAK and differentially affected medullary mitochondrial superoxide radical production in control and diabetic rats. Metformin attenuates diabetes-induced renal medullary tissue hypoxia in an animal model of insulinopenic type 1 diabetes. The results suggest that the mechanistic pathway to attenuate the diabetes-induced medullary hypoxia is independent of blood glucose homeostasis and includes reduced UCP2-mediated mitochondrial proton LEAK.

Quercetin preserves redox status and stimulates mitochondrial function in metabolically-stressed HepG2 cells

Hyperglycemia augments formation of intracellular reactive oxygen species (ROS) with associated mitochondrial damage and increased risk of insulin resistance in type 2 diabetes. We examined whether quercetin could reverse chronic high glucose-induced oxidative stress and mitochondrial dysfunction. Following long-term high glucose treatment, complex I activity was significantly decreased in isolated mitochondria from HepG2 cells. Quercetin dose-dependently recovered complex I activity and lowered cellular ROS generation under both high and normal glucose conditions. Respirometry studies showed that quercetin could counteract the detrimental increase in inner mitochondrial membrane proton leakage resulting from high glucose while it increased oxidative respiration, despite a decrease in electron transfer system (ETS) capacity, and lower non-ETS oxygen consumption. A quercetin-stimulated increase in cellular NAD+/NADH was evident within 2 h and a two-fold increase in PGC-1α mRNA within 6 h, in both normal and high glucose conditions. A similar pattern was also found for the mRNA expression of the repulsive guidance molecule b (RGMB) and its long non-coding RNA (lncRNA) RGMB-AS1 with quercetin, indicating a potential change of the glycolytic phenotype and suppression of aberrant cellular growth which is characteristic of the HepG2 cells. Direct effects of quercetin on PGC-1α activity were minimal, as quercetin only weakly enhanced PGC-1α binding to PPARα in vitro at higher concentrations. Our results suggest that quercetin may protect mitochondrial function from high glucose-induced stress by increasing cellular NAD+/NADH and activation of PGC-1α-mediated pathways. Lower ROS in combination with improved complex I activity and ETS coupling efficiency under conditions of amplified oxidative stress could reinforce mitochondrial integrity and improve redox status, beneficial in certain metabolic diseases.

Heptanoate is neuroprotective in vitro but triheptanoin post-treatment did not protect against middle cerebral artery occlusion in rats

Triheptanoin, the medium-chain triglyceride of heptanoate, has been shown to be anticonvulsant and neuroprotective in several neurological disorders. In the gastrointestinal tract, triheptanoin is cleaved to heptanoate, which is then taken up by the blood and most tissues, including liver, heart and brain. Here we evaluated the neuroprotective effects of heptanoate and its effects on mitochondrial oxygen consumption in vitro. We also investigated the neuroprotective effects of triheptanoin compared to long-chain triglycerides when administered after stroke onset in rats. Heptanoate pre-treatment protected cultured neurons against cell death induced by oxygen glucose deprivation and N-methyl-D-aspartate. Incubation of cultured astrocytes with heptanoate for 2 h increased mitochondrial proton leak and also enhanced basal respiration and ATP turnover, suggesting that heptanoate protects against oxidative stress and is used as fuel. However, continuous 72 h infusion of triheptanoin initiated 1 h after middle cerebral artery occlusion in rats did not alter stroke volume at 3 days or neurological deficit at 1 and 3 days relative to long-chain triglyceride control treatment.