Electron Transport Chain Enzymes Research Articles

Correction of Mitochondrial Enzyme Activities in the Skeletal Muscles of Old Rats in Response to Addition of Olive Oil to the Ration.

Activities of mitochondrial electron transport chain enzymes NADH-CoQ oxidoreductase (complex I), cytochrome C-oxidase (complex IV), and citrate synthase were measured by spectrophotometry in m. quadriceps femoris homogenate from old rats receiving olive oil with the ration. Reduced activities of complexes I and IV in old animals were restored to the level of young animals after 6-week consumption of olive oil. Activity of citrate synthase did not change with age. Positive effect of olive oil on fatty-acid composition of the muscle tissue in old animals was demonstrated. The content of summary monounsaturated fatty acids, reduced with aging, and of summary polyunsaturated ones, increasing with age, were restored in old rats to the levels virtually not differing from the levels of young animals.

Protective effects of exercise against sepsis-induced energy metabolism dysfunction in skeletal muscle of rats

We evaluated effects of aerobic physical preconditioning on general performance and energy metabolism in skeletal muscle of septic rats. Forty-eight 10-wk-old male Wistar rats were randomly assigned to either Untrained or Trained groups. Aerobic exercise training protocol (AETP) consisted of an 8-week treadmill program. After AETP, performance was evaluated by graded treadmill and functional ambulation testing. Afterwards animals from both groups were randomly assigned to Sham or CLP surgery (cecal ligation and perforation), resulting in the following groups: Sham untrained (ShamU), CLP untrained (CLPU), Sham trained (ShamT), and CLP trained (CLPT). Two days after surgery, animals repeated the ambulation test, and were euthanized after this. Diaphragm, soleus and plantaris muscles were harvested. Mitochondrial electron transport chain enzyme (METC) and creatine kinase (CK) activity were measured. AETP led to significant improvement in performance of distance run and in skeletal muscle function of the Trained group. Forty-eight hours after surgery the CLPT group was able to maintain similar muscle performance as Sham groups. Dysfunction was shown in the diaphragm in METC complexes I and II-III and in locomotive soleus muscles in complex I; CK enzyme activity was significantly increased in sedentary CLPU group in soleus and plantaris muscle, but in the diaphragm there was only a tendency (p=0.07). CLPT animals that were submitted to AETP avoided all these negative results. Taken together our results provide evidence of the positive effects obtained with an aerobic physical preconditioning program on METC and CK enzyme activity related to the diaphragm and locomotive muscles mitigating sepsis-induced energy metabolism dysfunction.

Contribution of mitochondrial function to exercise-induced attenuation of renal dysfunction in spontaneously hypertensive rats.

It is well known that exercise training exhibits renal protective effects in animal models of hypertension and chronic renal failure. However, the mechanisms regulating these effects of exercise training remain unclear. This study aimed to investigate the role of mitochondrial function in exercise-induced attenuation of renal injury in spontaneously hypertensive rats (SHR). The adult male SHR and age-matched normotensive Wistar-Kyoto rats (WKY) were given moderate-intensity exercise for 12 weeks or treated with MitoQ10 for 8 weeks. In this work, exercise training in SHR reduced blood pressure, and effectively attenuated renal dysfunction, marked by reduced creatinine excretion, albuminuria, blood urea nitrogen, and glomerular sclerosis. Exercise training in SHR reduced MDA levels in plasma and kidneys and suppressed formation of 3-nitrotyrosine in kidneys. Exercise training suppressed mitochondrial ROS and [Formula: see text] formation, enhanced ATP formation, reduced mitochondrial swelling, and restored electron transport chain enzyme activity in kidneys of SHR. Furthermore, exercise training upregulated protein expression of uncoupling protein 2 and manganese superoxide dismutase in kidneys of SHR. In addition, treatment with mitochondria-targeted antioxidant MitoQ10 exhibited similar renal protective effects in SHR. In conclusion, chronic aerobic exercise training preserved mitochondrial function and abated oxidative stress in the kidneys of SHR, which may in part explain the protective effect of exercise on renal function and structure in hypertensive individuals.

Mitochondrial modulation by Epigallocatechin 3-Gallate ameliorates cisplatin induced renal injury through decreasing oxidative/nitrative stress, inflammation and NF-kB in mice.

Cancer chemotherapy drug cisplatin is known for its nephrotoxicity. The aim of this study is to investigate whether Epigallocatechin 3-Gallate (EGCG) can reduce cisplatin mediated side effect in kidney and to understand its mechanism of protection against tissue injury. We used a well-established 3-day cisplatin induced nephrotoxicity mice model where EGCG were administered. EGCG is a major active compound in Green Tea and have strong anti-oxidant and anti-inflammatory properties. EGCG protected against cisplatin induced renal dysfunction as measured by serum creatinine and blood urea nitrogen (BUN). EGCG improved cisplatin induced kidney structural damages such as tubular dilatation, cast formation, granulovaculoar degeneration and tubular cell necrosis as evident by PAS staining. Cisplatin induced kidney specific mitochondrial oxidative stress, impaired activities of mitochondrial electron transport chain enzyme complexes, impaired anti-oxidant defense enzyme activities such as glutathione peroxidase (GPX) and manganese superoxide dismutase (MnSOD) in mitochondria, inflammation (tumor necrosis factor α and interleukin 1β), increased accumulation of NF-κB in nuclear fraction, p53 induction, and apoptotic cell death (caspase 3 activity and DNA fragmentation). Treatment of mice with EGCG markedly attenuated cisplatin induced mitochondrial oxidative/nitrative stress, mitochondrial damages to electron transport chain activities and antioxidant defense enzyme activities in mitochondria. These mitochondrial modulations by EGCG led to protection mechanism against cisplatin induced inflammation and apoptotic cell death in mice kidney. As a result, EGCG improved renal function in cisplatin mediated kidney damage. In addition to that, EGCG attenuated cisplatin induced apoptotic cell death and mitochondrial reactive oxygen species (ROS) generation in human kidney tubular cell line HK-2. Thus, our data suggest that EGCG may represent new promising adjunct candidate for cisplatin.

Alterations in mitochondrial number and function in Alzheimer's disease fibroblasts.

Mitochondrial dysfunction is observed in brains of Alzheimer's Disease patients as well as many rodent model systems including those modeling mutations in preseinilin 1 (PSEN1). The aim of our study was to characterize mitochondrial function and number in fibroblasts from AD patients with PSEN1 mutations. We used biochemical assays, metabolic profiling and fluorescent labeling to assess mitochondrial number and function in fibroblasts from three AD patients compared to fibroblasts from three controls. The mutant AD fibroblasts had increased Aβ42 relative to controls along with reduction in ATP, basal and maximal mitochondrial respiration as well as impaired spare mitochondrial respiratory capacity. Fluorescent staining and expression of genes encoding electron transport chain enzymes showed diminished mitochondrial content in the AD fibroblasts. This study demonstrates that mitochondrial dysfunction is observable in AD fibroblasts and provides evidence that this model system could be useful as a tool to screen disease-modifying compounds.

Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency: FUNCTIONAL ROLE OF NEURONAL NITRIC OXIDE SYNTHASE DURING MITOPHAGY

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser(1412)) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease.

4-hydroxy tempo improves mitochondrial and neurobehavioral deficits in experimental model of Huntington's disease.

Mitochondrial dysfunctions have been implicated in the progression of Huntington's disease (HD). To date, several free radical scavengers have been tested in experimental HD, but only a few have shown promise. Although most antioxidants rapidly reduce ROS but in the process they are oxidized, which limits their ability to protect. Therefore, in the present study we employed a potent recycling antioxidant, 4-hydroxy tempo (4-HT), because it can reinstate its reduced state even after its oxidation during scavenging of ROS. Female Wistar rats were administered 3-nitropropionic acid (3-NP) and/or 4-HT for 21 days, after which animals were subjected to biochemical and behavioral assessments. Our results showed that 4-HT treatment significantly attenuated the 3-NP induced decrease in the activities of mitochondrial electron transport chain enzymes. In addition, 4-HT administration restored the increased nitrite and lipid peroxidation levels. Apart from this, 4-HT also attenuated the 3-NP induced decrease in superoxide dismutase and catalase activities. Further, 4-HT administration resulted in significant improvement in 3-NP induced cognitive and motor impairments. Taken together, the results of the study demonstrate that 4-HT is beneficial in 3-NP induced model of HD and thus could be a potential therapeutic agent in management of this disease.

Activity assay for plant mitochondrial enzymes.

Mitochondria are sites for respiration to produce chemical energy via oxidative phosphorylation. Their primary role has been viewed as the oxidation of organic acids via the tricarboxylic acid (TCA) cycle and the synthesis of ATP coupled to the transfer of electrons to O2. TCA cycle enzymes are essential for plant carbon metabolism and provide the reductant for the electron transport chain (ETC) enzymes that in turn drives ATP synthesis. The activity of individual enzymes will determine the flux of metabolism and thus the downstream consequences for respiration rate. Measurements of activities of mitochondrial enzymes, such as components of TCA cycle and the ETC, can provide insight into regulation of mitochondrial function. The activities of these enzymes vary between developmental stages, in different tissues, and in response to environmental conditions. In this chapter, methods for enzymatic assay of TCA cycle enzymes and a number of the ETC complex enzymes are described in detail.

Mitochondrial Dihydrolipoamide Dehydrogenase is Upregulated in Response to Intermittent Hypoxic Preconditioning.

Intermittent hypoxia preconditioning (IHP) has been shown to protect neurons against ischemic stroke injury. Studying how proteins respond to IHP may identify targets that can help fight stroke. The objective of the present study was to investigate whether mitochondrial dihydrolipoamide dehydrogenase (DLDH) would respond to IHP and if so, whether such a response could be linked to neuroprotection in ischemic stroke injury. To do this, we subjected male rats to IHP for 20 days and measured the content and activity of DLDH as well as the three α-keto acid dehydrogenase complexes that contain DLDH. We also measured mitochondrial electron transport chain enzyme activities. Results show that DLDH content was indeed upregulated by IHP and this upregulation did not alter the activities of the three α-keto acid dehydrogenase complexes. Results also show that the activities of the five mitochondrial complexes (I-V) were not altered either by IHP. To investigate whether IHP-induced DLDH upregulation is linked to neuroprotection against ischemic stroke injury, we subjected both DLDH deficient mouse and DLDH transgenic mouse to stroke surgery followed by measurement of brain infarction volume. Results indicate that while mouse deficient in DLDH had exacerbated brain injury after stroke, mouse overexpressing human DLDH also showed increased brain injury after stroke. Therefore, the physiological significance of IHP-induced DLDH upregulation remains to be further investigated.

An active lifestyle induces positive antioxidant enzyme modulation in peripheral blood mononuclear cells of overweight/obese postmenopausal women

AimsThe aim of this study was to investigate the effects of an active lifestyle on mitochondrial functioning, viability, bioenergetics, and redox status markers in peripheral blood mononuclear cells (PBMC) of overweight/obese postmenopausal women. Materials and methodsWe performed a cross-sectional study with postmenopausal women aged 45–64years and body mass index>25kg/m2, divided into physically active (n=23) and sedentary (n=12) groups. Mitochondria functioning and viability, bioenergetics and redox status parameters were assessed in PBMC with spectrophotometric and fluorometric assays. Key findingsNo differences were found in the enzyme activity of complexes I and II of the electron transport chain (ETC), mitochondrial superoxide dismutase (MnSOD) activity, methyl-tetrazolium reduction levels and reduced glutathione and oxidized glutathione levels between the groups. However, the physically active group presented higher levels of reactive oxygen species (ROS) (P=0.04) and increased catalase (CAT) (P=0.029), total (P=0.011) and cytosolic SOD (CuZnSOD) (P=0.009) activities. SignificanceAn active lifestyle that includes aerobic exercise for at least 30min, three times per week may improve antioxidant enzyme activities in PBMC in overweight/obese postmenopausal women, without changes in the activity of the ETC enzymes. However, this low intensity physical activity is not able to induce relevant mitochondrial adaptations.

Betaine is a positive regulator of mitochondrial respiration

Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.

Basic regulatory principles of Escherichia coli's electron transport chain for varying oxygen conditions.

For adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system. In the following a mathematical model of the E. coli ETC linked to basic modules for substrate uptake, fermentation product excretion and biomass formation is introduced. The kinetic modelling focusses on regulatory principles of the ETC for varying oxygen conditions in glucose-limited continuous cultures. The model is based on the balance of electron donation (glucose) and acceptance (oxygen or other acceptors). Also, it is able to account for different chemostat conditions due to changed substrate concentrations and dilution rates. The parameter identification process is divided into an estimation and a validation step based on previously published and new experimental data. The model shows that experimentally observed, qualitatively different behaviour of the ubiquinone redox state and the ArcA activity profile in the micro-aerobic range for different experimental conditions can emerge from a single network structure. The network structure features a strong feed-forward effect from the FNR regulatory system to the ArcBA regulatory system via a common control of the dehydrogenases of the ETC. The model supports the hypothesis that ubiquinone but not ubiquinol plays a key role in determining the activity of ArcBA in a glucose-limited chemostat at micro-aerobic conditions.

Axial mitochondrial myopathy in a patient with rapidly progressive adult-onset scoliosis

Axial myopathy can be the underlying cause of rapidly progressive adult-onset scoliosis; however, the pathogenesis of this disorder remains poorly understood. Here we present a case of a 69-year old woman with a family history of scoliosis affecting both her mother and her son, who over 4 years developed rapidly progressive scoliosis. The patient had a history of stable scoliosis since adolescence that worsened significantly at age 65, leading to low back pain and radiculopathy. Paraspinal muscle biopsy showed morphologic evidence of a mitochondrial myopathy. Diagnostic deficiencies of electron transport chain enzymes were not detected using standard bioassays, but mitochondrial immunofluorescence demonstrated many muscle fibers totally or partially deficient for complexes I, III, IV-I, and IV-IV. Massively parallel sequencing of paraspinal muscle mtDNA detected multiple deletions as well as a 40.9% heteroplasmic novel m.12293G > A (MT-TL2) variant, which changes a G:C pairing to an A:C mispairing in the anticodon stem of tRNA LeuCUN. Interestingly, these mitochondrial abnormalities were not detected in the blood of either the patient or her son, suggesting that the patient’s rapidly progressive late onset scoliosis was due to the acquired paraspinal mitochondrial myopathy; the cause of non-progressive scoliosis in the other two family members currently remains unexplained. Notably, this case illustrates that isolated mitochondrial myopathy can underlie rapidly-progressive adult-onset scoliosis and should be considered in the differential diagnosis of the primary axial myopathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0137-3) contains supplementary material, which is available to authorized users.

An experimental study to investigate the impact of p-coumaric acid, a common dietary polyphenol, on cadmium chloride-induced renal toxicity.

Cadmium, a well-known environmental pollutant and a toxic transitional metal causes severe damage to many organs, such as liver, kidney, lungs, heart, etc. The current study has been designed to assess the impact of p-coumaric acid, a common dietary polyphenol on cadmium chloride-induced renal toxicity in rats. Therefore, the activities of membrane bound ATPases, mitochondrial TCA cycle and electron transport chain enzymes, gluconeogenic and glycolytic enzymes, and glycogen content were estimated in kidney tissue homogenates of control and experimental rats. In addition, the serum levels of glucose and pro-inflammatory cytokines, such as TNF-α and IL-1β were also estimated. The cadmium chloride administered rats (3 mg per kg per b. wt per s.c.) showed significant decrease in the levels of membrane bound ATPases, mitochondrial TCA cycle and electron transport chain enzymes, glycolytic enzymes, and glycogen content as compared with controls. Conversely, the levels of glucose, gluconeogenic enzymes and pro-inflammatory cytokines (TNF-α, and IL-1β) were found to be increased. However, the administration of p-coumaric acid (100 mg per kg per b. wt per s.c.) along with the cadmium chloride significantly modulated these biochemical and immunological changes to near normal, as compared to cadmium chloride treated rats. Thus, the results provide strong evidence that p-coumaric acid has a protective action against cadmium-induced renal toxicity in rats.

Fluoxetine-induced pancreatic beta cell dysfunction: New insight into the benefits of folic acid in the treatment of depression

BackgroundMajor depressive disorder is a common psychiatric illness with reported prevalence rates of 12–16% in persons aged 12 and over. Depression is also associated with a high risk of new onset of type 2 diabetes (T2D). This relationship between depression and diabetes may be related to depression itself and/or drugs prescribed. Importantly, the use of selective serotonin reuptake inhibitors (SSRIs), the most commonly prescribed class of antidepressants, increases the risk of developing T2D. However, the mechanism(s) underlying this association remains elusive. MethodsHere we examine the effects of the SSRI fluoxetine (Prozac®) on beta cell function utilizing INS-1E cells, a rat beta cell line, to elucidate the underlying molecular mechanisms. ResultsFluoxetine treatment significantly reduced glucose stimulated insulin secretion (GSIS). This decreased beta cell function was concomitant with an increased production of reactive oxygen species and oxidative damage which may contribute to decreased mitochondrial electron transport chain enzyme (ETC) activity. Importantly the fluoxetine-induced deficits in beta cell function were prevented by the addition of the antioxidant folic acid. LimitationsThese studies were conducted in vitro; the in vivo relevance remains to be determined. ConclusionsThese findings suggest that use of SSRI antidepressants may increase the risk of new-onset T2D by causing oxidative stress in pancreatic beta cells. However, folic acid supplementation in patients taking SSRIs may reduce the risk of new onset diabetes via protection of normal beta cell function.

Essential Roles of Neutral Ceramidase and Sphingosine in Mitochondrial Dysfunction Due to Traumatic Brain Injury

In addition to immediate brain damage, traumatic brain injury (TBI) initiates a cascade of pathophysiological events producing secondary injury. The biochemical and cellular mechanisms that comprise secondary injury are not entirely understood. Herein, we report a substantial deregulation of cerebral sphingolipid metabolism in a mouse model of TBI. Sphingolipid profile analysis demonstrated increases in sphingomyelin species and sphingosine concurrently with up-regulation of intermediates of de novo sphingolipid biosynthesis in the brain. Investigation of intracellular sites of sphingosine accumulation revealed an elevation of sphingosine in mitochondria due to the activation of neutral ceramidase (NCDase) and the reduced activity of sphingosine kinase 2 (SphK2). The lack of change in gene expression suggested that post-translational mechanisms are responsible for the shift in the activities of both enzymes. Immunoprecipitation studies revealed that SphK2 is complexed with NCDase and cytochrome oxidase (COX) subunit 1 in mitochondria and that brain injury hindered SphK2 association with the complex. Functional studies showed that sphingosine accumulation resulted in a decreased activity of COX, a rate-limiting enzyme of the mitochondrial electron transport chain. Knocking down NCDase reduced sphingosine accumulation in mitochondria and preserved COX activity after the brain injury. Also, NCDase knockdown improved brain function recovery and lessened brain contusion volume after trauma. These studies highlight a novel mechanism of secondary TBI involving a disturbance of sphingolipid-metabolizing enzymes in mitochondria and suggest a critical role for mitochondrial sphingosine in promoting brain injury after trauma.

Influence of the oxidative stress induced by the organophosphate pesticide bromopropylate on the mitochondrial respiratory chain in Trichoderma harzianum

The present study was designed to investigate the effect of bromopropylate on its own transport rate, glycolysis and tricarboxylic acid cycle metabolite levels, adenine nucleotides, and membrane lipid peroxidation (LPO) as well as the activities of mitochondrial electron transport chain (ETC) enzymes in eukaryotic Trichoderma harzianum. The transport rate of bromopropylate reached a maximum level within the first 24h of incubation for all studied concentrations. The succinate dehydrogenase (SDH) and cytochrome c oxidase (CCO) activities reached their maxima at 72h for 2.5 and 10mg/L of bromopropylate, respectively. In addition, the intracellular pyruvate levels increased for bromopropylate concentrations up to 2.5mg/L. The maximum intracellular α-ketoglutarate level was determined at 5mg/L, while the intracellular fumarate and citrate levels reached their maximums at 7.5mg/L of bromopropylate. The variations in the adenine nucleotide levels showed a positive correlation with both α-ketoglutarate and fumarate levels. Nevertheless, the LPO levels increased with increasing bromopropylate concentrations. These results may indicate that the membrane becomes more damaged from an impaired respiratory chain, which may then cause an increase in electron leakage.

Investigating mitochondrial metabolism in contracting HL-1 cardiomyocytes following hypoxia and pharmacological HIF activation identifies HIF-dependent and independent mechanisms of regulation.

Hypoxia is a consequence of cardiac disease and downregulates mitochondrial metabolism, yet the molecular mechanisms through which this occurs in the heart are incompletely characterized. Therefore, we aimed to use a contracting HL-1 cardiomyocyte model to investigate the effects of hypoxia on mitochondrial metabolism. Cells were exposed to hypoxia (2% O2) for 6, 12, 24, and 48 hours to characterize the metabolic response. Cells were subsequently treated with the hypoxia inducible factor (HIF)-activating compound, dimethyloxalylglycine (DMOG), to determine whether hypoxia-induced mitochondrial changes were HIF dependent or independent, and to assess the suitability of this cultured cardiac cell line for cardiovascular pharmacological studies. Hypoxic cells had increased glycolysis after 24 hours, with glucose transporter 1 and lactate levels increased 5-fold and 15-fold, respectively. After 24 hours of hypoxia, mitochondrial networks were more fragmented but there was no change in citrate synthase activity, indicating that mitochondrial content was unchanged. Cellular oxygen consumption was 30% lower, accompanied by decreases in the enzymatic activities of electron transport chain (ETC) complexes I and IV, and aconitase by 81%, 96%, and 72%, relative to controls. Pharmacological HIF activation with DMOG decreased cellular oxygen consumption by 43%, coincident with decreases in the activities of aconitase and complex I by 26% and 30%, indicating that these adaptations were HIF mediated. In contrast, the hypoxia-mediated decrease in complex IV activity was not replicated by DMOG treatment, suggesting HIF-independent regulation of this complex. In conclusion, 24 hours of hypoxia increased anaerobic glycolysis and decreased mitochondrial respiration, which was associated with changes in ETC and tricarboxylic acid cycle enzyme activities in contracting HL-1 cells. Pharmacological HIF activation in this cardiac cell line allowed both HIF-dependent and independent mitochondrial metabolic changes to be identified.

Effect of Coenzyme Q10 supplementation on mitochondrial electron transport chain activity and mitochondrial oxidative stress in Coenzyme Q10 deficient human neuronal cells

Primary Coenzyme Q10 (CoQ10) deficiency is an autosomal recessive disorder with a heterogeneous clinical presentation. Common presenting features include both muscle and neurological dysfunction. Muscle abnormalities can improve, both clinically and biochemically following CoQ10 supplementation, however neurological symptoms are only partially ameliorated. At present, the reasons for the refractory nature of the neurological dysfunction remain unknown. In order to investigate this at the biochemical level we evaluated the effect of CoQ10 treatment upon a previously established neuronal cell model of CoQ10 deficiency. This model was established by treatment of human SH-SY5Y neuronal cells with 1mM para-aminobenzoic acid (PABA) which induced a 54% decrease in cellular CoQ10 status. CoQ10 treatment (2.5μM) for 5 days significantly (p<0.0005) decreased the level of mitochondrial superoxide in the CoQ10 deficient neurons. In addition, CoQ10 treatment (5μM) restored mitochondrial membrane potential to 90% of the control level. However, CoQ10 treatment (10μM) was only partially effective at restoring mitochondrial electron transport chain (ETC) enzyme activities. ETC complexes II/III activity was significantly (p<0.05) increased to 82.5% of control levels. ETC complexes I and IV activities were restored to 71.1% and 77.7%, respectively of control levels.In conclusion, the results of this study have indicated that although mitochondrial oxidative stress can be attenuated in CoQ10 deficient neurons following CoQ10 supplementation, ETC enzyme activities appear partially refractory to treatment. Accordingly, treatment with >10μM CoQ10 may be required to restore ETC enzyme activities to control level. Accordingly, these results have important implication for the treatment of the neurological presentations of CoQ10 deficiency and indicate that high doses of CoQ10 may be required to elicit therapeutic efficacy.

Signalling pathways in succinate dehydrogenase B‐associated renal carcinoma

Renal tumours have recently been described in association with mutations in the gene encoding the B subunit of succinate dehydrogenase, a mitochondrial Krebs cycle and electron transport chain enzyme (SDHB-associated renal cell carcinomas). The aim of this study was to investigate the roles of different signalling pathways in the pathogenesis of these tumours. We used immunohistochemistry and antibodies against phospho-specific epitopes to examine the activity of three potential signalling pathways in tumour cells of three genetically confirmed cases of SDHB-associated renal cell carcinomas. We found no evidence supporting a role for either the mTOR [p-mTOR (Ser2448), p-S6 riboprotein (Ser235/236)] or hypoxia-inducible (carbonic anhydrase 9 and EGFR) pathways. However, there was immunohistochemical reactivity for phosphorylated AMP-dependent kinase (p-AMPK Thr172) and glycogen synthase kinase 3 (GSK3) phosphorylation (p-GSK3 Ser12), and nuclear expression of cyclin D1. We suggest that these tumours may arise through a mechanism involving ATP depletion, activation of AMPK, and induction of cyclin D1, and that this may be a unique pathway of tumour development that has the potential for therapeutic intervention in these rare tumours.