Key points• Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects.• The most commonly used markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I-V protein, and complex I-IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres).• Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content.• mtDNA was found to be a poor biomarker of mitochondrial content.• Complex IV activity was closely associated with mitochondrial oxidative phosphorylation capacity.Abstract Skeletal muscle mitochondrial content varies extensively between human subjects. Biochemical measures of mitochondrial proteins, enzyme activities and lipids are often used as markers of mitochondrial content and muscle oxidative capacity (OXPHOS). The purpose of this study was to determine how closely associated these commonly used biochemical measures are to muscle mitochondrial content and OXPHOS. Sixteen young healthy male subjects were recruited for this study. Subjects completed a graded exercise test to determine maximal oxygen uptake (V O 2 peak ) and muscle biopsies were obtained from the vastus lateralis. Mitochondrial content was determined using transmission electron microscopy imaging and OXPHOS was determined as the maximal coupled respiration in permeabilized fibres. Biomarkers of interest were citrate synthase (CS) activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I-V protein content, and complex I-IV activity. Spearman correlation coefficient tests and Lin's concordance tests were applied to assess the absolute and relative association between the markers and mitochondrial content or OXPHOS. Subjects had a large range ofV O 2 peak (range 29.9-71.6 ml min −1 kg −1 ) and mitochondrial content (4-15% of cell volume). Cardiolipin content showed the strongest association with mitochondrial content followed by CS and complex I activities. mtDNA was not related to mitochondrial content. Complex IV activity showed the strongest association with muscle oxidative capacity followed by complex II activity. We conclude that cardiolipin content, and CS and complex I activities are the biomarkers that exhibit the strongest association with mitochondrial content, while complex IV activity is strongly associated with OXPHOS capacity in human skeletal muscle.

We investigated the genetic basis for a global and uniform decrease in mitochondrial translation in fibroblasts from patients in two unrelated pedigrees who developed Leigh syndrome, optic atrophy, and ophthalmoplegia. Analysis of the assembly of the oxidative phosphorylation complexes showed severe decreases of complexes I, IV, and V and a smaller decrease in complex III. The steady-state levels of mitochondrial mRNAs, tRNAs, and rRNAs were not reduced, nor were those of the mitochondrial translation elongation factors or the protein components of the mitochondrial ribosome. Using homozygosity mapping, we identified a 1 bp deletion in C12orf65 in one patient, and DNA sequence analysis showed a different 1 bp deletion in the second patient. Both mutations predict the same premature stop codon. C12orf65 belongs to a family of four mitochondrial class I peptide release factors, which also includes mtRF1a, mtRF1, and Ict1, all characterized by the presence of a GGQ motif at the active site. However, C12orf65 does not exhibit peptidyl-tRNA hydrolase activity in an in vitro assay with bacterial ribosomes. We suggest that it might play a role in recycling abortive peptidyl-tRNA species, released from the ribosome during the elongation phase of translation.

We have identified 12 patients with autosomal recessive mitochondrial encephalomyopathy with elevated methylmalonic acid. The disorder has a high incidence of 1 in 1700 in the Faroe Islands due to a founder effect, and a carrier frequency of 1 in 33. The symptoms comprise hypotonia, muscle atrophy, hyperkinesia, severe hearing impairment and postnatal growth retardation. Neuroimaging showed demyelination and central and cortical atrophy, including atrophy of the basal ganglia, and some of the patients fulfilled the criteria for Leigh syndrome. Urine and plasma methylmalonic acid were elevated. Homozygosity mapping with the Affymetrix 10 K array revealed a homozygous region on chromosome 13q14 harbouring the SUCLA2 gene. Mutations in SUCLA2 were recently shown to cause a similar disorder in a small Israeli family. Mutation analysis identified a novel splice site mutation in SUCLA2, IVS4 + 1G --> A, leading to skipping of exon 4. The SUCLA2 gene encodes the ATP-forming beta subunit of the Krebs cycle enzyme succinyl-CoA ligase. The hallmark of the condition, elevated methylmalonic acid, can be explained by an accumulation of the substrate of the enzyme, succinyl-CoA, which in turn leads to elevated methylmalonic acid, because the conversion of methylmalonyl-CoA to succinyl-CoA is inhibited.

Exercise intolerance is a prominent symptom in patients with mitochondrial myopathy (MM), but it is still unsettled whether exercise training is safe and beneficial for patients with MM. To address this, we studied the effect of 12 weeks cycle training on exercise capacity, quality of life and underlying molecular and cellular events in five patients with single large-scale deletions, one with a microdeletion and 14 with point mutations of mitochondrial DNA (mtDNA), and 13 healthy subjects. Each training session lasted 30 min, and was performed at an intensity of 70% of VO2max (maximal oxygen uptake). Each subject performed 50 training sessions in 12 weeks. All subjects were evaluated before and after training, and 13 MM patients were studied after 8 weeks of deconditioning. Evaluation included VO2max and mutation load and mtDNA quantity, mitochondrial enzymatic activity, and number of centrally nucleated, apoptotic, ragged red and cytochrome oxidase (COX)-negative fibres in muscle biopsies from the quadriceps muscle. After 12 weeks of training, VO2max and muscle citrate synthase increased in MM (26 and 67%) and healthy (17 and 65%) subjects, while mtDNA quantity in muscle only increased in the MM patients (81%). In the MM patients, training did not change mtDNA mutation load in muscle, mitochondrial enzyme complex activities, muscle morphology and plasma creatine kinase. After deconditioning, VO2max and citrate synthase activity returned to values before training, while muscle mtDNA mutation load decreased. These findings show that aerobic training efficiently improves oxidative capacity in MM patients. Based on unchanged levels of mutant load in muscle, morphological findings on muscle biopsy and plasma creatine kinase levels during training, the treatment appears to be safe. Regular, supervised aerobic exercise is therefore recommended in MM patients with the studied mutations.

Fatal infantile lactic acidosis is a severe metabolic disorder characterized by the onset of lactic acidosis within the 1st d of life and early death. We found a combined respiratory-chain enzyme deficiency associated with mitochondrial DNA (mtDNA) depletion in a small consanguineous family with this disorder. To identify the disease-causing gene, we performed single-nucleotide polymorphism homozygosity mapping and found homozygous regions on four chromosomes. DNA sequencing revealed a homozygous 2-bp deletion in SUCLG1, a gene that encodes the alpha subunit of the Krebs-cycle enzyme succinate-coenzyme A ligase (SUCL). The mtDNA depletion is likely explained by decreased mitochondrial nucleoside diphosphate kinase (NDPK) activity resulting from the inability of NDPK to form a complex with SUCL.

Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However, the CI, CI + CIII and CII + CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating that it originated from the respiratory electron transport chain. The p.278Y>C mutation enhanced superoxide production, as indicated by direct measurements in mitochondria and by the imbalance of glutathione homeostasis in intact cybrids. Remarkably, although the assembly of CI or CIII was not affected, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III2IV1 were reduced, whereas those of I1III2IVn slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I1III2IVn, in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.

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Patients with type 2 diabetes (T2D) and patients with nonalcoholic fatty liver disease (NAFLD) frequently exhibit elevated plasma concentrations of glucagon (hyperglucagonemia). Hyperglucagonemia and α-cell hyperplasia may result from elevated levels of plasma amino acids when glucagon's action on hepatic amino acid metabolism is disrupted. We therefore measured plasma levels of glucagon and individual amino acids in patients with and without biopsy-verified NAFLD and with and without type T2D. Fasting levels of amino acids and glucagon in plasma were measured, using validated ELISAs and high-performance liquid chromatography, in obese, middle-aged individuals with I) normal glucose tolerance (NGT) and NAFLD, II) T2D and NAFLD, III) T2D without liver disease, and IV) NGT and no liver disease. Elevated levels of total amino acids were observed in participants with NAFLD and NGT compared with NGT controls (1,310 ± 235 µM vs. 937 ± 281 µM, P = 0.03) and in T2D and NAFLD compared with T2D without liver disease (1,354 ± 329 µM vs. 511 ± 235 µM, P < 0.0001). Particularly amino acids with known glucagonotropic effects (e.g., glutamine) were increased. Plasma levels of total amino acids correlated to plasma levels of glucagon also when adjusting for body mass index (BMI), glycated hemoglobin (Hb), and cholesterol levels (β = 0.013 ± 0.007, P = 0.024). Elevated plasma levels of total amino acids associate with hyperglucagonemia in NAFLD patients independently of glycemic control, BMI or cholesterol - supporting the potential importance of a "liver-α-cell axis" in which glucagon regulates hepatic amino acid metabolism. Fasting hyperglucagonemia as seen in T2D may therefore represent impaired hepatic glucagon action with increasing amino acids levels. NEW & NOTEWORTHY Hypersecretion of glucagon (hyperglucagonemia) has been suggested to be linked to type 2 diabetes. Here, we show that levels of amino acids correlate with levels of glucagon. Hyperglucagonemia may depend on hepatic steatosis rather than type 2 diabetes.