Metformin 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. The 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. The 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. The 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.

Amiodarone is a potent antiarrhythmic drug and displays substantial liver toxicity in humans. It has previously been demonstrated that amiodarone and its metabolite (desethylamiodarone, DEA) can inhibit mitochondrial function, particularly complexes I (CI) and II (CII) of the electron transport system in various animal tissues and cell types. The present study, performed in human peripheral blood cells, and one liver-derived human cell line, is primarily aimed at assessing the concentration-dependent effects of these drugs on mitochondrial function (respiration and cellular ATP levels). Furthermore, we explore the efficacy of a novel cell-permeable succinate prodrug in alleviating the drug-induced acute mitochondrial dysfunction. Amiodarone and DEA elicit a concentration-dependent impairment of mitochondrial respiration in both intact and permeabilized platelets via the inhibition of both CI- and CII-supported respiration. The inhibitory effect seen in human platelets is also confirmed in mononuclear cells (PBMCs) and HepG2 cells. Additionally, amiodarone elicits a severe concentration-dependent ATP depletion in PBMCs, which cannot be explained solely by mitochondrial inhibition. The succinate prodrug NV118 alleviates the respiratory deficit in platelets and HepG2 cells acutely exposed to amiodarone. In conclusion, amiodarone severely inhibits metabolism in primary human mitochondria, which can be counteracted by increasing mitochondrial function using intracellular delivery of succinate.

Although mitochondrial respiration is believed to explain a substantial part of the variation in resting metabolic rate (RMR), few studies have empirically studied the relationship between organismal and cellular metabolism. We therefore investigated the relationship between RMR and mitochondrial respiration of permeabilized blood cells in wild great tits (Parus major L.). We also studied the correlation between mitochondrial respiration traits and blood cell count, as normalizing mitochondrial respiration by the cell count is a method commonly used to study blood metabolism. In contrast to previous studies, our results show that there was no relationship between RMR and mitochondrial respiration in intact blood cells (i.e. with the ROUTINE respiration). However, when cells were permeabilized and interrelation re-assessed under saturating substrate availability, we found that RMR was positively related to phosphorylating respiration rates through complexes I and II (i.e. OXPHOS respiration) and to the mitochondrial efficiency to produce energy (i.e. net phosphorylation efficiency), though variation explained by the models was low (i.e. linear model: R2=0.14 to 0.21). However, unlike studies in mammals, LEAK respiration without [i.e. L(n)] and with [i.e. L(Omy)] adenylates was not significantly related to RMR. These results suggest that phosphorylating respiration in blood cells can potentially be used to predict RMR in wild birds, but that this relationship may have to be addressed in standardized conditions (permeabilized cells) and that the prediction risks being imprecise. We also showed that, in our conditions, there was no relationship between any mitochondrial respiration trait and blood cell count. Hence, we caution against normalising respiration rates using this parameter as is sometimes done. Future work should address the functional explanations for the observed relationships, and determine why these appear labile across space, time, taxon, and physiological state.

Statins are the cornerstone of lipid-lowering therapy. Although generally well tolerated, statin-associated muscle symptoms (SAMS) represent the main reason for treatment discontinuation. Mitochondrial dysfunction of complex I has been implicated in the pathophysiology of SAMS. The present study proposed to assess the concentration-dependent ex vivo effects of three statins on mitochondrial respiration in viable human platelets and to investigate whether a cell-permeable prodrug of succinate (complex II substrate) can compensate for statin-induced mitochondrial dysfunction. Mitochondrial respiration was assessed by high-resolution respirometry in human platelets, acutely exposed to statins in the presence/absence of the prodrug NV118. Statins concentration-dependently inhibited mitochondrial respiration in both intact and permeabilized cells. Further, statins caused an increase in non-ATP generating oxygen consumption (uncoupling), severely limiting the OXPHOS coupling efficiency, a measure of the ATP generating capacity. Cerivastatin (commercially withdrawn due to muscle toxicity) displayed a similar inhibitory capacity compared with the widely prescribed and tolerable atorvastatin, but did not elicit direct complex I inhibition. NV118 increased succinate-supported mitochondrial oxygen consumption in atorvastatin/cerivastatin-exposed platelets leading to normalization of coupled (ATP generating) respiration. The results acquired in isolated human platelets were validated in a limited set of experiments using atorvastatin in HepG2 cells, reinforcing the generalizability of the findings.

Many animals downregulate body temperature to save energy when resting (rest‐phase hypothermia). Small birds that winter at high latitudes have comparatively limited capacity for hypothermia and so pay large energy costs for thermoregulation during cold nights. Available evidence suggests this process is fueled by adenosine triphosphate (ATP)‐dependent mechanisms. Most ATP is produced by oxidative phosphorylation in the mitochondria, but mitochondrial respiration may be lower during hypothermia because of the temperature dependence of biological processes. This can create conflict between increased organismal ATP demand and a lower mitochondrial capacity to provide it. We studied this in blood cell mitochondria of wild great tits ( Parus major ) by simulating rest‐phase hypothermia via a 6°C reduction in assay temperature in vitro. The birds had spent the night preceding the experiment in thermoneutrality or in temperatures representing mild or very cold winter nights, but night temperatures never affected mitochondrial respiration. However, across temperature groups, endogenous respiration was 14% lower in hypothermia. This did not reflect general thermal suppression of mitochondrial function because phosphorylating respiration was unaffected by thermal state. Instead, hypothermia was associated with a threefold reduction of leak respiration, from 17% in normothermia to 4% in hypothermia. Thus, the coupling of total respiration to ATP production was 96% in hypothermia, compared to 83% in normothermia. Our study shows that the thermal insensitivity of phosphorylation combined with short‐term plasticity of leak respiration may safeguard ATP production when endogenous respiration is suppressed. This casts new light on the process by which small birds endure harsh winter cold and warrants future tests across tissues in vivo.

Understanding mitochondrial biology and pathology is key to understanding the evolution of animal form and function. However, mitochondrial measurement often involves invasive, or even terminal, sampling, which can be difficult to reconcile in wild models or longitudinal studies. Non‐mammal vertebrates contain mitochondria in their red blood cells, which can be exploited for minimally invasive mitochondrial measurement. Several recent bird studies have measured mitochondrial function using isolated blood cells. Isolation adds time in the laboratory and might be associated with physiological complications. We developed and validated a protocol to measure mitochondrial respiration in bird whole blood. Endogenous respiration was comparable between isolated blood cells and whole blood. However, respiration towards oxidative phosphorylation was higher in whole blood, and whole blood mitochondria were better coupled and had higher maximum working capacity. Whole blood measurement was also more reproducible than measurement on isolated cells for all traits considered. Measurements were feasible over a 10‐fold range of sample volumes, although both small and large volumes were associated with changes to respiratory traits. The protocol was compatible with long‐term storage: after 24 h at 5°C without agitation, all respiration traits but maximum working capacity remained unchanged, the latter decreasing by 14%. Our study suggests that whole blood measurement provides faster, more reproducible, and more biologically and physiologically relevant (mitochondrial integrity) assessment of mitochondrial respiration. We recommend future studies to take a whole blood approach unless specific circumstances require the use of isolated blood cells.