Johannes K. Ehinger

Metformin induces lactate production in peripheral blood mononuclear cells and platelets through specific mitochondrial complex I inhibition.

Metformin is a widely used antidiabetic drug associated with the rare side effect of lactic acidosis which has been proposed to be linked to drug‐induced mitochondrial dysfunction. Using respirometry, the aim of this study was to evaluate mitochondrial toxicity of metformin to human blood cells in relation to that of phenformin, a biguanide analogue withdrawn in most countries due to a high incidence of lactic acidosis.

Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage.

Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further, we aimed to explore the limits of sample size and the impact of storage as well as to establish a wide range of reference data from different pediatric and adult cohorts. Our results indicate that platelet mitochondria are well suited for ex-vivo analysis with the need for minute sample amounts and excellent reproducibility and stability.

Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency

Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [13C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction.

Mitochondrial dysfunction in blood cells from amyotrophic lateral sclerosis patients

Mitochondrial dysfunction is implicated in amyotrophic lateral sclerosis, where the progressive degeneration of motor neurons results in muscle atrophy, paralysis and death. Abnormalities in both central nervous system and muscle mitochondria have previously been demonstrated in patient samples, indicating systemic disease. In this case–control study, venous blood samples were acquired from 24 amyotrophic lateral sclerosis patients and 21 age-matched controls. Platelets and peripheral blood mononuclear cells were isolated and mitochondrial oxygen consumption measured in intact and permeabilized cells with additions of mitochondrial substrates, inhibitors and titration of an uncoupler. Respiratory values were normalized to cell count and for two markers of cellular mitochondrial content, citrate synthase activity and mitochondrial DNA, respectively. Mitochondrial function was correlated with clinical staging of disease severity. Complex IV (cytochrome c-oxidase)-activity normalized to mitochondrial content was decreased in platelets from amyotrophic lateral sclerosis patients both when normalized to citrate synthase activity and mitochondrial DNA copy number. In mononuclear cells, complex IV-activity was decreased when normalized to citrate synthase activity. Mitochondrial content was increased in amyotrophic lateral sclerosis patient platelets. In mononuclear cells, complex I activity declined and mitochondrial content increased progressively with advancing disease stage. The findings are, however, based on small subsets of patients and need to be confirmed. We conclude that when normalized to mitochondria-specific content, complex IV-activity is reduced in blood cells from amyotrophic lateral sclerosis patients and that there is an apparent compensatory increase in cellular mitochondrial content. This supports systemic involvement in amyotrophic lateral sclerosis and suggests further study of mitochondrial function in blood cells as a future biomarker for the disease.

Mitochondrial Respiratory Function in Peripheral Blood Cells from Huntington's Disease Patients

Patients with Huntingtons disease display symptoms from both the central nervous system and peripheral tissues. Mitochondrial dysfunction has been implicated as part of the pathogenesis of the disease and has been reported in brain tissue and extracerebral tissues, such as muscle and blood cells, but the results are inconsistent. Therefore, the authors performed a refined evaluation of mitochondrial function in 2 types of peripheral blood cells from 14 patients with Huntingtons disease and 21 control subjects. Several hypotheses were predefined, including impaired mitochondrial complex II function (primary), complex I function (secondary), and maximum oxidative phosphorylation capacity (secondary) in patient cells.

Changes in energy metabolism due to acute rotenone-induced mitochondrial complex I dysfunction – An in vivo large animal model

Metabolic crisis is a clinical condition primarily affecting patients with inherent mitochondrial dysfunction in situations of augmented energy demand. To model this, ten pigs received an infusion of rotenone, a mitochondrial complex I inhibitor, or vehicle. Clinical parameters, blood gases, continuous indirect calorimetry, in vivo muscle oxygen tension, ex vivo mitochondrial respiration and metabolomics were assessed. Rotenone induced a progressive increase in blood lactate which was paralleled by an increase in oxygen tension in venous blood and skeletal muscle. There was an initial decrease in whole body oxygen utilization, and there was a trend towards inhibited mitochondrial respiration in platelets. While levels of succinate were decreased, other intermediates of glycolysis and the TCA cycle were increased. This model may be suited for evaluating pharmaceutical interventions aimed at counteracting metabolic changes due to complex I dysfunction.

Bioenergetic bypass using cell-permeable succinate, but not methylene blue, attenuates metformin-induced lactate production

BackgroundMetformin is the most common pharmacological treatment for type 2 diabetes. It is considered safe but has been associated with the development of lactic acidosis under circumstances where plasma concentrations exceed therapeutic levels. Metformin-induced lactic acidosis has been linked to the drug’s toxic effect on mitochondrial function. Current treatment strategies aim to remove the drug and correct for the acidosis. With a mortality of 20%, complementary treatment strategies are needed. In this study, it was investigated whether targeting mitochondria with pharmacological agents that bypass metformin-induced mitochondrial dysfunction can counteract the energetic deficit linked to toxic doses of metformin.MethodsThe redox agent methylene blue and the cell-permeable succinate prodrug NV118 were evaluated by measuring mitochondrial respiration and lactate production of human platelets exposed to metformin and co-treated with either of the two pharmacological bypass agents.ResultsThe cell-permeable succinate prodrug NV118 increased mitochondrial respiration which was linked to phosphorylation by the ATP-synthase and alleviated the increase in lactate production induced by toxic doses of metformin. The redox agent methylene blue, in contrast, failed to mitigate the metformin-induced changes in mitochondrial respiration and lactate generation.ConclusionsThe cell-permeable succinate prodrug NV118 bypassed the mitochondrial dysfunction and counteracted the energy deficit associated with toxic doses of metformin. If similar effects of NV118 prove translatable to an in vivo effect, this pharmacological strategy presents as a promising complementary treatment for patients with metformin-induced lactic acidosis.

Oxygen consumption in platelets as an adjunct diagnostic method for pediatric mitochondrial disease

BackgroundDiagnosing mitochondrial disease (MD) is a challenge. In addition to genetic analyses, clinical practice is to perform invasive procedures such as muscle biopsy for biochemical and histochemical analyses. Blood cell respirometry is rapid and noninvasive. Our aim was to explore its possible role in diagnosing MD.MethodsBlood samples were collected from 113 pediatric patients, for whom MD was a differential diagnosis. A respiratory analysis model based on ratios (independent of mitochondrial specific content) was derived from a group of healthy controls and tested on the patients. The diagnostic accuracy of platelet respirometry was evaluated against routine diagnostic investigation.ResultsMD prevalence in the cohort was 16%. A ratio based on the respiratory response to adenosine diphosphate in the presence of complex I substrates had 96% specificity for disease and a positive likelihood ratio of 5.3. None of the individual ratios had sensitivity above 50%, but a combined model had 72% sensitivity.ConclusionNormal findings of platelet respirometry are not able to rule out MD, but pathological results make the diagnosis more likely and could strengthen the clinical decision to perform further invasive analyses. Our results encourage further study into the role of blood respirometry as an adjunct diagnostic tool for MD.