Background Endurance exercise training promotes the catabolism of branched-chain amino acids (BCAAs) in skeletal muscles. We have previously shown that mitochondrial DNA (mtDNA) haplogroups J and K are markers of low responders in endurance training. In this paper, we hypothesize that BCAA catabolism is a surrogate marker of lower respiratory chain activity attributed to these haplogroups. We evaluated whether exercise-induced changes in amino acid concentrations differ between subjects harbouring mtDNA haplogroups J or K and those with non-JK haplogroups. Methods Finnish male conscripts (N = 633) undertook the 12-min Cooper running test at the beginning and end of their military service. The intervention during the service mainly included endurance aerobic exercise and sports-related muscle training. Concentrations of seven amino acids were analysed in the serum using a high-throughput 1 H NMR metabolomics platform. Total DNA was extracted from whole blood, and restriction fragment analysis was used to determine mtDNA haplogroups J and K. Results The concentrations of the seven amino acids were higher following the intervention, with the exception of phenylalanine; interestingly, the increase in the concentrations of three BCAAs was larger in subjects with haplogroup J or K than in subjects with non-JK haplogroups ( p = 0.029). MtDNA haplogroups J and K share two common nonsynonymous variants. Structural analysis based on crystallographic data on bovine complexes I and III revealed that the Leu18 variant in cytochrome b encoded by m.14798T > C may interfere with ubiquinone binding at the Qi site in complex III. Conclusions The increase in the concentrations of serum BCAAs following exercise intervention differs between subjects harbouring mtDNA haplogroup J or K and those harbouring non-JK haplogroups. Lower response in endurance training and difference in exercise-induced increase in the concentrations of serum BCAAs suggest decreased respiratory chain activity. Haplogroups J and K share m.14798T > C in MT-CYB , which may hamper the function of complex III.

Purpose: Next-generation sequencing (NGS) has made genetic testing of patients with epileptic encephalopathies easier novel variants are discovered and new phenotypes described. Variants in the same gene even the same variant can cause different types of epilepsy and neurodevelopmental disorders. Our aim was to identify the genetic causes of epileptic encephalopathies in paediatric patients with complex phenotypes. Methods: NGS was carried out for three patients with epileptic encephalopathies. Detailed clinical features, brain magnetic resonance imaging and electroencephalography were analysed. We searched the Human Gene Mutation Database for the published GABRG2 variants with clinical description of patients and composed a summary of the variants and their phenotypic features. Results: We identified two novel de novo GABRG2 variants, p.P282T and p.S306F, with new phenotypes including neuroradiological evidence of neurodegeneration and epilepsy of infancy with migrating focal seizures (EIMFS). One patient carried previously reported p.P83S variant with autism spectrum disorder (ASD) phenotype that has not yet been described related to GABRG2 disorders and a more severe epilepsy phenotype than reported earlier. In all, the literature search yielded twenty-two articles describing 27 different variants that were divided into two categories: those with self-limiting epilepsies and febrile seizures and those with more severe drug-resistant epileptic encephalopathies. Conclusion: This study further expands the genotypic and phenotypic spectrum of epilepsies associated with GABRG2 variants. More knowledge is still needed about the influence of the environment, genetic background and other epilepsy susceptibility genes on the phenotype of the specific GABRG2 variants.

Significance: NAD+ and NADP+ are important cosubstrates in redox reactions and participate in regulatory networks operating in adjustment of metabolic pathways. Moreover, NAD+ is a cosubstrate in post-translational modification of proteins and is involved in DNA repair. NADPH is indispensable for reductive syntheses and the redox chemistry involved in attaining and maintaining correct protein conformation. Recent Advances: Within a couple of decades, a wealth of information has been gathered on NAD(H)+/NADP(H) redox imaging, regulatory role of redox potential in assembly of spatial protein structures, and the role of ADP-ribosylation of regulatory proteins affecting both gene expression and metabolism. All these have a bearing also on disease, healthy aging, and longevity. Critical Issues: Knowledge of the signal propagation pathways of NAD+-dependent post-translational modifications is still fragmentary for explaining the mechanism of cellular stress effects and nutritional state on these actions. Evaluation of the cosubstrate and regulator roles of NAD(H) and NADP(H) still suffers from some controversies in experimental data. Future Directions: Activating or inhibiting interventions in NAD+-dependent protein modifications for medical purposes has shown promise, but restraining tumor growth by inhibiting DNA repair in tumors by means of interference in sirtuins is still in the early stage. The same is true for the use of this technology in improving health and healthy aging. New genetically encoded specific NAD and NADP probes are expected to modernize the research on redox biology.

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD+ couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD+ couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled.

Mitochondrial mutations impair glucose oxidation and increase glucose uptake in cell cultures and lead to cardiomyopathy in patients. Here we characterize cardiac glucose uptake in 14 patients with the m.3243A > G mutation in mitochondrial DNA. The 14 patients with m.3243A > G and 13 controls were similar in age, physical activity and body mass index. Ten patients had diabetes. Left ventricular glucose uptake per tissue mass (LVGU) was measured with 2-[ 18 F]fluoro-2-deoxyglucose positron emission tomography during euglycemic hyperinsulinemia. Cardiac morphology and function were assessed with magnetic resonance imaging. We found that the LVGU was 25 % lower in the patients than that in the controls ( P = 0.029). LVGU was inversely correlated with mutation heteroplasmy, glycated haemoglobin and fasting lactate in patients. The seven patients with mutation heteroplasmy ≥ 49 % had 44 % lower LVGU than the seven patients with heteroplasmy < 49 %. This difference remained significant after adjustment for concurrent free fatty acid concentration or glycated haemoglobin or glucose uptake in skeletal muscle or all ( p < 0.048 [All]). Patients with m.3243A > G had a lower stroke volume and a higher heart rate than the controls, whereas cardiac output and work were similar. Myocardial glucose uptake is not increased but decreased with a threshold effect pattern in patients with the m.3243A > G mutation. The glucose hypometabolism adds to the impaired cardiac energetics and likely contributes to the progression of the mitochondrial cardiomyopathy.

Due to the relative rarity of mitochondrial diseases, generating reference ranges is problematic in evaluation of respiratory chain activities particularly in pediatric cases. We determined the sample distribution of respiratory chain enzyme activities in skeletal muscle biopsies collected from pediatric patients suspected of neuromuscular disorders. Activities of NADH-ubiquinone reductase, NADH-cytochrome c reductase, succinate-cytochrome c reductase; ubiquinol-cytochrome c reductase and cytochrome c oxidase activities have log-normal distributions even when confirmed mitochondrial diseases were ruled out. Impact of the log-normal distribution of the respiratory chain enzyme activities on clinical diagnostics is discussed.

Effects of Complex I mutations were studied by modeling in NuoH, NuoJ or NuoK subunits of Escherichia coli NDH-1 by simultaneous optical monitoring of deamino-NADH oxidation and proton translocation and fitting to the data a model equation of transmembrane proton transport. A homolog of the ND1-E24 LHON/MELAS mutation caused 95% inhibition of d-NADH oxidation and proton translocation. The NuoJ-Y59F replacement decreased proton translocation. The NuoK-E72Q mutation lowered the enzyme activity, but proton pumping could be rescued by the double mutation NuoK-E72Q/I39D. Moving the NuoK-E72/E36 pair one helix turn towards the periplasm did not affect redox activity but decreased proton pumping.

Development of the use of flavin and nicotinamide-adenine nucleotide fluorescence in monitoring the redox state of the free mitochondrial NADH/NAD(+) couple in cells, tissues and organs is reviewed. A break-through was the identification of dihydrolipoamide dehydrogenase (FpL) as the major NAD-linked fluorescent flavoprotein of mitochondria. This mitochondrial matrix flavoprotein is in equilibrium with the free NADH/NAD(+) pool and its mid-potential is sufficiently near to that of NADH/NAD(+) so that its percentage reduction follows that of the latter. Possibilities of monitoring mitochondrial and cytosolic NADH depend on the population density of mitochondria and thus are tissue-dependent. Upon a shift toward reduction, fluorescence intensities of NADH and flavins swing to reciprocal directions, so that the NADH/flavin fluorescence ratio can be used to increase the sensitivity of redox monitoring. This method is attaining widening use in studies on metabolic regulation under normal and pathological conditions.

Life creates and maintains order, which dees the general tendency of entropy increase and therefore demands energy supplies from the surrounding. In living organisms, endergonic processes are running at the expense of a universal cellular energy currency, which must be continuously regenerated. e enzymatic machinery for this provision can be dened as energy metabolism. In aerobic cells, this is accomplished by means of oxidation-reduction reactions, which are linked to conservation and conversion of the combustion energy to be saved in "energy-rich" molecules, such as adenosine triphosphate (ATP). is molecule functions as a universal energy currency whose breakdown can be coupled to the endergonic reactions of biosynthesis, transport, and motion. e majority (~95%) of the biological energy demand in aerobic cells is supplied by the machinery of oxidative phosphorylation residing in mitochondria. Cells, tissues, and organs with continuously high energy consumption are particularly dependent on mitochondria and also vulnerable from their disorders. is chapter highlights the different aspects of mitochondrial role in energy metabolism needed for cellular function and survival and outlines the optical methods of energy metabolism network studies.

Aim: The ultrastructural changes in the intestine were studied during experimental acute edematous and necrotizing porcine pancreatitis. The immunohistochemical expression of E-cadherin and beta-catenin in the jejunum and colon was assessed to characterize changes in the adherens junctions.Methods: Twenty-four pigs were randomized to controls (n = 8) or to develop mild edematous (n = 8, saline infusion to pancreatic duct) or severe necrotizing pancreatitis (n = 8, taurocholic acid infusion). The ultrastructure of the mesenteric artery and the vein and epithelium of the jejunum and colon was analyzed at baseline and after 540 min with electron microscopy. The expression of E-cadherin and beta-catenin was assessed with immunohistochemistry.Results: In the colon the microvilli and their glycocalyx shortened and reduced in density the most in necrotizing pancreatitis. In necrotizing pancreatitis adherens and tight junctions were occasionally open in the colon but rarely in the jejunum. Mitochondria in the colon epithelial cells were degenerated in necrotizing pancreatitis, swollen in edematous pancreatitis, and remained intact in the control case. In necrotizing pancreatitis, capillaries of the colon showed a broken endothelial lining with narrow lumens. The expression of E-cadherin immunoreactivity showed a trend toward a decrease in the colon in both edematous and necrotizing pancreatitis.Conclusion: Ultrastructural abnormalities in acute pancreatitis appear early in the colon, where they seem to be more damaging than in jejunum. Epithelial cell damage seems to include mitochondrial injury and an opening of tight and adherens junctions, which are more pronounced in necrotizing pancreatitis. Damage is seen in the mucosal and mesenteric endothelial cells.

Reactive oxygen species (ROS) have been implicated in many aspects of tissue/cellular metabolic signaling and pathology, including cardioprotection against ischemia–reperfusion damage. Recent reports of enhanced ROS production under global or simulated ischemia in intact heart or isolated cardiomyocytes, respectively, and its decrease again upon reperfusion are paradoxical. Mechanisms for increasing ROS production with decreasing reactant (oxygen) concentration remain elusive, making it important to critically evaluate the experimental methods used to measure ROS production. In the present paper superoxide production in isolated perfused rat hearts was monitored by lucigenin chemiluminescence or dihydroethidine (DHE) oxidation product fluorescence in parallel with redox state of flavin and cytochrome oxidase. Lucigenin luminescence decreased in ischemia and increased again upon reperfusion, transiently reaching values eightfold the control value coincidently with an overshoot of mitochondrial oxygen concentration. Hypoxic perfusion decreased lucigenin chemiluminescence in spite of coronary flow increase, whereas change in lucigenin concentration in the perfusate had negligible effect. In contrast to lucigenin luminescence, the fluorescence of the DHE oxidation product increased continuously during a 30-min global ischemia and decreased precipitously upon reperfusion, this change is coincident with absorption changes of the oxygen-binding protein myoglobin. The time course of DHE oxidation product fluorescence during ischemia and reperfusion was similar to that of the mitochondrial membrane potential probe safranin as shown in perfused heart previously [Ylitalo KV, Ala-Rämi A, Liimatta EV, Peuhkurinen KJ, Hassinen IE. J Mol Cell Cardiol 2000;32:1223–38]. In solution under high oxygen partial pressure DHE was mainly oxidized to a product, whose fluorescence, absorbance and mass spectra were similar to ethidium, and this product behaved like a mitochondrial membrane potential probe in isolated mitochondria. As a membrane permeable cation it accumulates into the mitochondria when the membrane potential is high (high intramitochondrial concentration quenches fluorescence) and then is released (increased fluorescence) during hypoxia/ischemia. Upon reperfusion it is re-accumulated in the mitochondria as the membrane potential recovers. The non-specific oxidation of DHE makes this dye less suitable for superoxide detection in experiments on isolated perfused hearts that necessitate high oxygen partial pressure in the perfusate. The time course of lucigenin luminescence during ischemia/reperfusion is consistent with decreased ROS production during ischemia/hypoxia, while the oxygen concentration is decreased, followed by an overshoot when the heart tissue is reperfused and the oxygen pressures return to normal or above normal.

Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G > A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent Km for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A > G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G > A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G > A affecting the same extramembrane loop as the m.3376G > A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G > A transition in MTND1 is the pathogenic mutation and m.3865A > G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49 kD/PSST subunits. In all, m.3376G > A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.

Advanced therapies medicinal products (ATMPs) have introduced innovative cell-based products. However, the regulatory demands for characterization of ATMPs are currently unable to adequately address the safety of such products. As recent studies have emphasized the role of mitochondria in the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we have studied in detail the viability and osteogenic differentiation potency of the hMSCs intended for use as ATMPs based on analyses of the mitochondrial inner membrane potential (Delta psi(m)). Flow cytometric measurement of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), propidium iodide fluorescence, and AnnexinV was employed to determine Delta psi(m), plasmamembrane integrity, and organization of phosphatidylserine in plasma membrane, respectively, in cultured hMSCs. Apoptosis was induced by incubating cells at critical concentration (20 mu M) of menadione. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used as an indicator for cell proliferation and alkaline phosphatase activity and calcium deposition as indicators of osteogenic differentiation. Based on JC-1 fluorescence, cell morphology, organization of phosphatidylserine, and plasma membrane integrity, we could sort cells into four categories that represented different cell quality. A strong correlation between JC-1 and osteogenic differentiation was demonstrated for the first time and thus this analytical tool is suitable not only to determine cell viability but also to predict osteogenic differentiation of hMSC.

Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2gt/gt) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34–47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hif-p4h-2gt/gt hearts. When isolated Hif-p4h-2gt/gt hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2gt/gt hearts, indicating enhanced glycolysis and an improved cellular energy state. Our data suggest that chronic stabilization of Hif-1α and Hif-2α by genetic knockdown of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function, and blood pressure.

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

MtDNA sequence variation is presumed to be neutral in effect, but associations with diseases and mtDNA haplogroups have been reported. The aim here was to evaluate the functional consequences of m.4216T>C present in haplogroup J. Furthermore, we evaluated m.3866T>C in MT-ND1, a variant detected in a child belonging to haplogroup J and with an isolated complex I deficiency. Homologous substitutions were introduced into Escherichia coli. NADH dehydrogenase domain activity of NDH-1 with either one or both mutations was markedly decreased suggesting that m.4216T>C and m.3866T>C may have an effect on the structural integrity of complex I.

Background The c.2447G>A (p.R722H) mutation in the gene POLG1 of the catalytic subunit of human mitochondrial polymerase gamma has been previously found in a few occasions but its pathogenicity has remained uncertain. We set out to ascertain its contribution to neuromuscular disease. Methods Probands from two families with probable mitochondrial disease were examined clinically, muscle and buccal epithelial DNA were analyzed for mtDNA deletions, and the POLG1, POLG2, ANT1 and Twinkle genes were sequenced. Results An adult proband presented with progressive external ophthalmoplegia, sensorineural hearing impairment, diabetes mellitus, dysphagia, a limb myopathy and dementia. Brain MRI showed central and cortical atrophy, and 18 F-deoxyglucose PET revealed reduced glucose uptake. Histochemical analysis of muscle disclosed ragged red fibers and cytochrome c oxidase-negative fibers. Electron microscopy showed subsarcolemmal aggregates of morphologically normal mitochondria. Multiple mtDNA deletions were found in the muscle, and sequencing of the POLG1 gene revealed a homozygous c.2447G>A (p.R722H) mutation. His two siblings were also homozygous with respect to the p.R722H mutation and presented with dementia and sensorineural hearing impairment. In another family the p.R722H mutation was found as compound heterozygosity with the common p.W748S mutation in two siblings with mental retardation, ptosis, epilepsy and psychiatric symptoms. The estimated carrier frequency of the p.R722H mutation was 1:135 in the Finnish population. No mutations in POLG2 , ANT1 and Twinkle genes were found. Analysis of the POLG1 sequence by homology modeling supported the notion that the p.R722H mutation is pathogenic. Conclusions The recessive c.2447G>A (p.R722H) mutation in the linker region of the POLG1 gene is pathogenic for multiple mtDNA deletions in muscle and is associated with a late-onset neurological phenotype as a homozygous state. The onset of the disease can be earlier in compound heterozygotes.