Oxidative Phosphorylation Enzymes Research Articles

Mitochondrial targeted neuron focused genes in hippocampus of rats with traumatic brain injury

Context:Mild traumatic brain injury (mTBI) represents a major health problem in civilian populations as well as among the military service members due to (1) lack of effective treatments, and (2) our incomplete understanding about the progression of secondary cell injury cascades resulting in neuronal cell death due to deficient cellular energy metabolism and damaged mitochondria.Aims:The aim of this study was to identify and delineate the mitochondrial targeted genes responsible for altered brain energy metabolism in the injured brain.Settings and Design:Rats were either grouped into naïve controls or received lateral fluid percussion brain injury (2–2.5 atm) and followed up for 7 days.Materials and Methods:Rats were either grouped into naïve controls or received lateral fluid percussion brain injury (2–2.5 atm) and followed for 7 days. The severity of brain injury was evaluated by the neurological severity scale—revised (NSS-R) at 3 and 5 days post TBI and immunohistochemical analyses at 7 days post TBI. The expression profiles of mitochondrial-targeted genes across the hippocampus from TBI and naïe rats were also examined by oligo-DNA microarrays.Results:NSS-R scores of TBI rats (5.4 ± 0.5) in comparison to naïe rats (3.9 ± 0.5) and H and E staining of brain sections suggested a mild brain injury. Bioinformatics and systems biology analyses showed 31 dysregulated genes, 10 affected canonical molecular pathways including a number of genes involved in mitochondrial enzymes for oxidative phosphorylation, mitogen-activated protein Kinase (MAP), peroxisome proliferator-activated protein (PPAP), apoptosis signaling, and genes responsible for long-term potentiation of Alzheimer's and Parkinson's diseases.Conclusions:Our results suggest that dysregulated mitochondrial-focused genes in injured brains may have a clinical utility for the development of future therapeutic strategies aimed at the treatment of TBI.

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 ρ0 cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in ρ0 cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.

Effects of in utero antiretroviral exposure on mitochondrial DNA levels, mitochondrial function and oxidative stress

HIV and antiretroviral (ART) exposure in utero may have deleterious effects on the infant, but uncertainty still exists. The objective of this study was to evaluate aspects of mitochondrial DNA (mtDNA) content, mitochondrial function and oxidative stress simultaneously in placenta, umbilical cord blood and infant blood in HIV/ART-exposed infants compared with uninfected controls. HIV-1-infected pregnant women and HIV-1-uninfected healthy pregnant controls were enrolled in the study prospectively. Placenta and umbilical cord blood were obtained at delivery and infant blood was obtained within 48 h of delivery. mtDNA content was determined for each specimen. Nuclear [subunit IV of cytochrome c-oxidase (COX IV)]- and mitochondrial (COX II)-encoded polypeptides of the oxidative phosphorylation enzyme cytochrome c-oxidase were quantified in cord and infant blood. Placental mitochondria malondialdehyde (MDA) concentrations were measured as a marker of oxidative stress. Twenty HIV-positive/HIV-exposed and 26 control mother-infant pairs were enrolled in the study. All HIV-infected women and their infants received ART. Placental MDA concentration and mtDNA content in placenta and cord blood were similar between groups. The cord blood COX II:IV ratio was lower in the HIV-positive group than in the controls, whereas the infant peripheral blood mtDNA content was higher in the HIV-exposed infants, but the infant peripheral blood COX II:IV ratio was similar. No infant had clinical evidence of mitochondrial disease or acquired HIV infection. In multivariable regression analyses, the significant findings in cord and infant blood were both most associated with HIV/ART exposure. HIV-exposed infants showed reduced umbilical cord blood mitochondrial enzyme expression with increased infant peripheral blood mitochondrial DNA levels, the latter possibly reflecting a compensatory mechanism to overcome HIV/ART-associated mitochondrial toxicity.

Glucocorticoids induce mitochondrial gene transcription in HepG2 cells: Role of the mitochondrial glucocorticoid receptor

Glucocorticoids are major regulators of a plethora of cellular functions, acting on target cells through glucocorticoid receptors (GR) and modulation of gene transcription, among other mechanisms. One main site of action of glucocorticoids is the hepatocyte, which responds to the hormonal stimulus with induction of several proteins among them enzymes of oxidative phosphorylation (OXPHOS), both nuclearly and mitochondrially encoded. The induction of OXPHOS is regarded as a result of a nuclear action of the receptor on the respective nuclear genes and on genes encoding mitochondrial transcription factors. The presence of GR in mitochondria and of sequences in the mitochondrial genome similar to glucocorticoid responsive elements, suggested a direct action of GR on mitochondrial transcription. We demonstrate in HepG2 hepatocarcinoma cells specific binding of GR to the regulatory D-loop region of the mitochondrial genome and show that dexamethasone induces the mitochondrial transcription factors A, B1, and B2, the mitochondrial ribosomal RNA, and several mitochondrially encoded OXPHOS genes. Applying α-amanitin, the specific inhibitor of DNA-dependent RNA polymerase II, the dexamethasone-induced transcription of the mitochondrial genes can still proceeds, whereas the DEX effect on transcription of the mitochondrial transcription factors is suppressed. Moreover, HepG2 cells overexpressing mitochondrial targeted GR showed increased RNA synthesis, cytrochrome oxidase subunit I protein expression, and mitochondrial ATP production. We conclude that glucocorticoids can stimulate directly mitochondrial transcription by the mitochondrially localized GR, affecting OXPHOS enzyme biosynthesis. This takes place in addition to their action on mitochondrial genes by way of induction of the nuclearly encoded mitochondrial transcription factors.

Analysis of the Mitochondrial Complex I-V Enzyme Activities of Peripheral Leukocytes in Oxidative Phosphorylation Disorders

Mitochondrial oxidative phosphorylation defects are a common cause of mitochondrial diseases, which are characterized by multiorgan involvement and clinically heterogeneous manifestations. Diagnosis is difficult because of the lack of clinically feasible methods. In this study, mitochondrial complex I-V enzyme activity was measured in 64 patients with suspected mitochondrial disease and 200 healthy controls. Spectrophotometric assay was used for the analysis of mitochondrial complex I-V enzyme activity in peripheral leukocytes. Diagnosis was based on clinical presentation, magnetic resonance imaging (MRI), muscle pathology, and point mutation screening in mitochondrial DNA. The differential diagnosis of aminoacidopathies, organic acidurias, and fatty acid β-oxidation defects was made. Thirty-five patients (54.7%) were diagnosed with Leigh syndrome based on characteristic brain MRI. Complex enzyme activity in controls was found to be stable. A deficiency in the oxidative phosphorylation was found in 29 patients (45.3%). Twenty (31.2%) patients had isolated complex defects, complex I deficiency (n = 2, 3.1%), complex II deficiency (n = 3, 4.7%), complex III deficiency (n = 5, 7.8%), complex IV deficiency (n = 5, 7.8%), and complex V deficiency (n = 5, 7.8%). Nine patients were found to have combined deficiencies, 3 (4.7%) had combined deficiencies of complex I and IV, 2 (3.1%) had combined deficiencies of complex III and V, and 2 (3.1%) had a combined deficiency of complex I and V. In conclusion, the peripheral leukocyte oxidative phosphorylation enzyme activity assay was found to be a reliable method for the diagnosis of mitochondrial diseases.

Long-term Training with Electrical Stimulation Regulates Muscle Gene Expression and Bone Architecture after Spinal Cord Injury

Long-term training with electrical stimulation regulates muscle gene expression and bone architecture after SCI: Adams CM, Suneja M, McHenry CM, Littmann AL, Dudley-Javoroski S, Shields RK, Physical Therapy and Rehabilitation Science, Carver College of Medicine, University of Iowa Background: Musculoskeletal deterioration is a serious secondary complication associated with paralysis from spinal cord injury (SCI). PURPOSE: We present the long-term effects of regular electrical stimulation training on muscle gene regulation and bone architecture after SCI. METHODS: Individuals with complete paralysis from SCI trained the calf muscle (soleus) or the thigh muscle (quadricep) during novel resistive tasks. The electrical stimulation protocol induced 2-4 bouts of 60-120 isometric contractions at a 15-20 Hz stimulation. After at least 2 years of unilateral training, we obtained muscle biopsies from the calf (soleus) and thigh muscles (vastus lateralis) of (trained and untrained) subjects with SCI. We then used Affymetrix human exon 1.0 arrays to identify mRNAs that were ≥ 1.5 fold increased or decreased in the atrophied untrained muscles. We next examined the trabecular bone using mDCT to quantify the sites and quality of the trabecular lattice. RESULTS: Out of >17,000 transcripts examined, in the untrained limb 51 mRNAs were increased including mRNAs encoding fast-twitch isoforms of myosin and troponin, as well as the mRNA encoding myostatin. Fifty-four mRNAs were decreased, including mRNAs encoding slow-twitch isoforms of myosin and troponin, 11 enzymes for oxidative phosphorylation, as well as PGC1α, a transcriptional co-activator that promotes the slow twitch, oxidative phenotype and inhibits FoxO3-mediated muscle atrophy. Training increased the mRNAs encoding slow-twitch isoforms and isoforms associated with glucose metabolism. The bone trabecular lattice was denser with distinct rods and plates within the trained femur and tibia. CONCLUSIONS: These findings support that thirty minutes/day of electrical stimulation stress induced significant muscle molecular adaptations and prevented the deterioration of bone after SCI. Funded by Nielsen Foundation and National Institutes of Health (R01-NR-01028505; R01-HD-04583233; R01HD062507).

Abstract 3385: A systems view of epithelial – mesenchymal transition signaling states

Abstract Epithelial – mesenchymal transition (EMT) is an important contributor to the invasion and metastasis of epithelial-derived cancers. While considerable effort has focused in the regulators involved in the transition process, we have focused on consequences of EMT to prosurvival signaling. Changes in distinct metastable and ‘epigentically-fixed’ EMT states were measured by correlation of protein, phosphoprotein, phosphopeptide and RNA transcript abundance. The assembly of 1167 modulated components into functional systems or machines simplified biological understanding and increased prediction confidence highlighting four functional groups: cell adhesion and migration, metabolism, transcription nodes and proliferation/survival networks. A coordinate metabolic reduction in a cluster of 17 free-radical stress pathway components was observed and correlated with reduced glycolytic and increased oxidative phosphorylation enzyme capacity, consistent with reduced cell cycling and reduced need for macromolecular biosynthesis in the mesenchymal state. An attenuation of EGFR autophosphorylation and a switch from autocrine to paracrine-competent EGFR signaling was implicated in the enablement of tumor cell chemotaxis. A similar attenuation of IGF1R, MET and RON signaling with EMT was observed. In contrast, EMT increased prosurvival autocrine IL11/IL6-JAK2-STAT signaling, autocrine fibronectin-integrin α5β1 activation, autocrine Axl/Tyro3/PDGFR/FGFR RTK signaling and autocrine TGFβR signaling. A relatively uniform loss of polarity and cell-cell junction linkages to actin cytoskeleton and intermediate filaments was measured at a systems level. A more heterogeneous gain of ECM remodeling and associated with invasion and migration was observed. Correlation to stem cell, EMT, invasion and metastasis datasets revealed the greatest similarity with normal and cancerous breast stem cell populations, CD49fhi/EpCAM-/lo and CD44hi/CD24lo respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3385. doi:10.1158/1538-7445.AM2011-3385

Increase in mitochondrial DNA mutations impairs retinal function and renders the retina vulnerable to injury

Mouse models that accumulate high levels of mitochondrial DNA (mtDNA) mutations owing to impairments in mitochondrial polymerase γ (PolG) proofreading function have been shown to develop phenotypes consistent with accelerated aging. As increase in mtDNA mutations and aging are risk factors for neurodegenerative diseases, we sought to determine whether increase in mtDNA mutations renders neurons more vulnerable to injury. We therefore examined the in vivo functional activity of retinal neurons and their ability to cope with stress in transgenic mice harboring a neural-targeted mutant PolG gene with an impaired proofreading capability (Kasahara, etal. (2006) Mol Psychiatry11(6):577-93, 523). We confirmed that the retina of these transgenic mice have increased mtDNA deletions and point mutations and decreased expression of mitochondrial oxidative phosphorylation enzymes. Associated with these changes, the PolG transgenic mice demonstrated accelerated age-related loss in retinal function as measured by dark-adapted electroretinogram, particularly in the inner and middle retina. Furthermore, the retinal ganglion cell-dominant inner retinal function in PolG transgenic mice showed greater vulnerability to injury induced by raised intraocular pressure, an insult known to produce mechanical, metabolic, and oxidative stress in the retina. These findings indicate that an accumulation of mtDNA mutations is associated with impairment in neural function and reduced capacity of neurons to resist external stress in vivo, suggesting a potential mechanism whereby aging central nervous system can become more vulnerable to neurodegeneration.

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes.

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.

The effect of dietary soy isoflavones before and after ovariectomy on hippocampal protein markers of mitochondrial bioenergetics and antioxidant activity in female monkeys

Estrogen therapy can promote cognitive function if initiated within a ‘critical window’ during the menopausal transition. However, in the absence of a progestogen, estrogens increase endometrial cancer risk which has spurred research into developing estrogenic alternatives that have the beneficial effects of estrogen but which are clinically safer. Soy protein is rich in isoflavones, which are a class of potential estrogenic alternatives. We sought to determine the effects of two diets, one with casein-lactalbumin as the main protein source and the other with soy protein containing isoflavones, on protein markers of hippocampal bioenergetic capacity in adult female cynomolgus macaques ( Macaca fascicularis). Further, we assessed the effects of dietary soy isoflavones before or after ovariectomy. Animals receiving soy diet premenopausally then casein/lactalbumin post-ovariectomy had higher relative hippocampal content of glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase subunit e1α. Post-ovariectomy consumption of soy was associated with higher succinate dehydrogenase α levels and lower levels of isocitrate dehydrogenase, both proteins involved in the tricarboxylic acid cycle, significantly decreased expression of the antioxidant enzyme peroxiredoxin-V, and a non-significant trend towards decreased manganese superoxide dismutase expression. None of the diet paradigms significantly affected expression levels of oxidative phosphorylation enzyme complexes, or of mitochondrial fission and fusion proteins. Together, these data suggest that long-term soy diet produces minimal effects on hippocampal expression of proteins involved in bioenergetics, but that switching between a diet containing primarily animal protein and one containing soy isoflavones before and after menopause may result in complex effects on brain chemistry.

Loss of mitochondria in ganglioneuromas

A shift in cellular energy production from oxidative phosphorylation (OXPHOS) to glycolysis, even under aerobic conditions, is called the Warburg effect. To elucidate changes of the mitochondrial energy metabolism in ganglioneuroma (GN) individual OXPHOS enzymes were analyzed by activity assays and by immunohistochemical staining methods. GN (n=7) showed a significant reduction in the activity and content of OXPHOS enzymes. Citrate synthase activity was also severely diminished in GN compared to normal cortical kidney (p=0.0002) and adrenal (p=0.0024) tissues. Furthermore, the mitochondrial membrane protein porin was undetectable or significantly reduced. Accordingly, a reduction of the copy number of mitochondrial DNA was observed in GN compared to cortical kidney tissue. The striking decline of mitochondrial mass is specific for GN but not for neuroblastoma, in which a reduction of the OXPHOS complexes without reduction of mitochondrial mass was reported. Knowledge of the mechanism by which tumor cells achieve the Warburg effect will provide a starting point for functional studies aimed at restoring aerobic energy metabolism as a potential new therapeutic strategy to treat malignancies.

Heterogeneity of mitochondrial energy metabolism in classical triphasic Wilms tumor

Metabolic changes are observed in a variety of tumors. The nature of the changes in aerobic energy metabolism differs between tumor types. Therefore, immunohistochemical staining, enzymatic measurements and immunoblot analysis were used to determine alterations of oxidative phosphorylation (OXPHOS) in classic triphasic Wilms' Tumor (WT). Our studies revealed that the epithelial, stromal and blastemal elements of this tumor differ in their energy metabolism. Compared to unaffected kidney tissue, normal mitochondrial mass was observed in the epithelial and blastemal regions of WT, whereas the stroma showed a massive down-regulation of mitochondria, as indicated by low porin content, low citrate synthase activity, and reduced mtDNA copy number. All OXPHOS enzyme activities were reduced in all WT samples, with the exception of two epithelial-dominant cases, which showed up-regulation of complex III activity compared to control kidney tissues. Interestingly, our studies show that, even within a specific tumor entity, cell-type-specific alterations of aerobic energy metabolism can occur, although all cell types showed a clear tendency toward a reduced aerobic energy metabolism.

A systems view of epithelial–mesenchymal transition signaling states

AbtractEpithelial–mesenchymal transition (EMT) is an important contributor to the invasion and metastasis of epithelial-derived cancers. While considerable effort has focused in the regulators involved in the transition process, we have focused on consequences of EMT to prosurvival signaling. Changes in distinct metastable and ‘epigentically-fixed’ EMT states were measured by correlation of protein, phosphoprotein, phosphopeptide and RNA transcript abundance. The assembly of 1167 modulated components into functional systems or machines simplified biological understanding and increased prediction confidence highlighting four functional groups: cell adhesion and migration, metabolism, transcription nodes and proliferation/survival networks. A coordinate metabolic reduction in a cluster of 17 free-radical stress pathway components was observed and correlated with reduced glycolytic and increased oxidative phosphorylation enzyme capacity, consistent with reduced cell cycling and reduced need for macromolecular biosynthesis in the mesenchymal state. An attenuation of EGFR autophosphorylation and a switch from autocrine to paracrine-competent EGFR signaling was implicated in the enablement of tumor cell chemotaxis. A similar attenuation of IGF1R, MET and RON signaling with EMT was observed. In contrast, EMT increased prosurvival autocrine IL11/IL6-JAK2-STAT signaling, autocrine fibronectin-integrin α5β1 activation, autocrine Axl/Tyro3/PDGFR/FGFR RTK signaling and autocrine TGFβR signaling. A relatively uniform loss of polarity and cell–cell junction linkages to actin cytoskeleton and intermediate filaments was measured at a systems level. A more heterogeneous gain of ECM remodeling and associated with invasion and migration was observed. Correlation to stem cell, EMT, invasion and metastasis datasets revealed the greatest similarity with normal and cancerous breast stem cell populations, CD49fhi/EpCAM-/lo and CD44hi/CD24lo, respectively.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-010-9367-3) contains supplementary material, which is available to authorized users.

Development of Impaired Cytochrome C Oxidase Activity In Apheresis Platelets During Storage

AbstractAbstract 3347 Background:With current storage requirements, the shelf life of platelet (PLT) products is largely limited by the development of deleterious in vitro changes associated with overall reduction in therapeutic efficacy collectively known as the “Platelet Storage Lesion” (PSL). PSL is characterized by a number of biochemical changes including lactate accumulation, bicarbonate depletion and a fall in pH. We hypothesize that these changes reflect a state of impaired oxidative phosphorylation associated with increased reliance on anaerobic glycolysis that evolves during PLT storage. In this study we evaluated the function and expression of “Cytochrome C Oxidase” (COX), a key mitochondrial enzyme in oxidative phosphorylation, with relation to several in vitro markers of PSL. The studies were performed in apheresis PLT stored for up to 7 days under standard blood bank conditions. Methods:Apheresis PLT concentrates were collected in 100% plasma using a Trima Cell Separator (CaridianBCT, Lakewood CO) to provide products with at least 3 × 1011 PLT. All products were collected in Trima® storage bags and were stored in a flatbed reciprocal agitator at 22 ± 2°C for 7 days. Multiple standard in vitro assays were performed on Days 0 (baseline measurement), 2, 4 and 7 of storage including PLT count, mean PLT volume, pH, pO2, pCO2, bicarbonate, lactate & glucose levels, aggregation and ATP release studies (ADP/Collagen), soluble CD40 ligand levels (supernatant) and total intracellular PLT ATP content. In addition, steady state COX kinetics and protein immunoblotting for COX subunits I and IV, were performed using isolated PLT mitochondria from simultaneously collected samples. Data are reported as mean ± 2 SD (n =10 experiments). One-way Analysis of Variance (ANOVA) and post-hoc Tukey's range test were used to compare data obtained at different time points. Differences were considered statistically significant only if the p value was <0.001 taking into the account that performing such large number of tests in the study increased the probability that a significant p value was incorrectly obtained by random chance. Results:PLT COX function declined significantly throughout storage (Table). Steady-state levels of COX I and IV remained essentially unchanged. This decrease in COX function paralleled progressive ATP depletion and time-dependent changes that were consistent with the development of the PSL. Conclusion:During storage of apheresis PLT for 7 days, COX function decreased progressively in association with ATP depletion indicating acquired impairment in oxidative phosphorylation. These findings suggest that bioenergetic failure is associated with PSL. Further studies are required to determine if mitochondrial dysfunction is a cause of PSL, and if it can be prevented or is amenable to therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Differential expression, phosphorylation of COX subunit 1 and COX activity during diapause phase in the cotton bollworm, Helicoverpa armigera

Helicoverpa armigera (Lepidoptera, Noctuidae) is an important agricultural pest with a pupal diapause. Cytochrome c oxidase (COX) is a key speed-limited enzyme of oxidative phosphorylation in mitochondria for ATP production. A differentially expressed cDNA fragment encoding COX subunit 1 (cox1) was cloned by differential display-PCR from the pupal brain at diapause termination with an injection of ecdysone. We then obtained the full length of H. armigera cox1 (Hea-cox1) cDNA which has an open reading frame of 1530 nucleotides encoding a putative protein of 510 amino acid residues, with CGA as a start codon. To evaluate the response to different energy demands during pupal development and at diapause termination, we assessed the expression of Hea-cox1 mRNA and protein, COX activity and its phosphorylation. The results show that Hea-cox1 expression at the mRNA and protein levels is associated with COX activity, and high levels of Hea-cox1 expression and COX activity are present in nondiapause pupae, suggesting that low energy metabolism provided by oxidative phosphorylation in mitochondria in diapause individuals is necessary. After diapause is broken by injection of 20-hydroxyecdysone, expression of Hea-cox1 mRNA and protein increases gradually and COX activity increases significantly. Furthermore, Hea-cox1 phosphorylation is closely correlated with COX activity, suggesting that reversible protein phosphorylation may play a key role in insect diapause by suppressing the rate of energy production.

Does adiponectin mimic exercise?

Does adiponectin mimic exercise?

Progressive mitochondrial compromise in brains and livers of primates exposed in utero to nucleoside reverse transcriptase inhibitors (NRTIs).

Mitochondrial compromise has been documented in infants born to women infected with the human immunodeficiency virus (HIV-1) who received nucleoside reverse transcriptase inhibitor (NRTI) therapy during pregnancy. To model these human exposures, we examined mitochondrial integrity at birth and 1 year in brain cortex and liver from offspring of retroviral-free Erythrocebus patas dams-administered human-equivalent NRTI doses for the last half (10 weeks) of gestation. Additional infants, followed for 1 year, were given the same drugs as their mothers for the first 6 weeks of life. Exposures included: no drug, Zidovudine (AZT), Lamivudine (3TC), AZT/3TC, AZT/Didanosine (ddI), and Stavudine (d4T)/3TC. In brain and liver, oxidative phosphorylation (OXPHOS) enzyme activities (complexes I, II, and IV) showed minimal differences between unexposed and NRTI-exposed offspring at both times. Brain and liver mitochondria from most NRTI-exposed patas, both at birth and 1 year of age, contained significant (p < 0.05) morphological damage observed by electron microscopy (EM), based on scoring of coded photomicrographs. Brain and liver mitochondrial DNA (mtDNA) levels in NRTI-exposed patas were depleted significantly in the 3TC and d4T/3TC groups at birth and were depleted significantly (p < 0.05) at 1 year in all NRTI-exposed groups. In 1-year-old infants exposed in utero to NRTIs, mtDNA depletion was 28.8-51.8% in brain and 37.4-56.5% in liver. These investigations suggest that some NRTI-exposed human infants may sustain similar mitochondrial compromise in brain and liver and should be followed long term for cognitive integrity and liver function.

Annual Symposium of the Society for the Study of Inborn Errors of Metabolism

Background: Recently, TMEM70 was identified as a novel ancillary factor essential in biogenesis of mammalian complex V. We report clinical, biochemical and genetic data of two unrelated patients with pathogenic mutations in TMEM70. The two patients presented with remarkable difference in age of onset and severity of clinical symptoms.Material and Methods: Oxidative phosphorylation (OXPHOS) enzyme activities were measured using spectrophotometrical analysis. Functional integrity of the five complexes was evaluated using BN-PAGE followed by in-gel activity staining. Western blotting was performed to document amounts of residual protein in the OXPHOS complexes. All coding exons and part of flanking introns of TMEM70 were PCR amplified and sequenced.Results: In one patient, who presented with severe neonatal phenotype, sequencing of TMEM70 showed a homozygous splice site mutation (c.317-2A>G). The adolescent patient with milder symptoms was found to be compound heterozygote for the c.317-2A>G mutation and c.251delC deletion. In the adolescent patient, 3-methylglutaconic acid was detected in urine. Almost complete absence of functional holocomplex V was demonstrated in several tissues from both patients.Conclusions: Complex V deficiency caused by mutations in TMEM70 is apparently not extremely rare. Patients can present with a severe neonatal form, as well as with milder non progressive encephalopathy.

Mitochondrial Dysfunction in NnaD Mutant Flies and Purkinje Cell Degeneration Mice Reveals a Role for Nna Proteins in Neuronal Bioenergetics

The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in 3 weeks. Loss of function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 ortholog (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD/Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss of function results in altered bioenergetics and mitochondrial dysfunction.

Enhancing T3 and cAMP responsive gene participation in the thermogenic regulation of fuel oxidation pathways

We sought to identify glycolysis, glycogenolysis, lipolysis, Krebs cycle, respiratory chain, and oxidative phosphorylation enzymes simultaneously regulated by T3 and cAMP. We performed in silico analysis of 56 promoters to search for cis-cAMP (CREB) and cis-thyroid (TRE) response elements, considering UCP1, SERCA2 and glyceraldehyde 3-phosphate dehydrogenase as reference. Only regulatory regions with prior in vitro validation were selected. 29/56 enzymes presented potential TREs in their regulatory sequence, and some scored over 0.80 (better predictive value 1): citrate synthase, phosphoglucose isomerase, succinate dehydrogenases A/C, UCP3, UCP2, UCP4, UCP5, phosphoglycerate mutase, glyceraldehyde 3-P dehydrogenase, glucokinase, malate dehydrogenase, acyl-CoA transferase (thiolase), cytochrome a3, and lactate dehydrogenase. Moreover, some enzymes have not yet been described in the literature as genomically regulated by T3. Our results point to other enzymes which may possibly be regulated by T3 and CREB, and speculate their joint roles in contributing to the optimal thermogenic acclimation.