A growing body of research is investigating the potential contribution of mitochondrial function to the etiology of type 2 diabetes. Numerous in vitro, in situ, and in vivo methodologies are available to examine various aspects of mitochondrial function, each requiring an understanding of their principles, advantages, and limitations. This review provides investigators with a critical overview of the strengths, limitations and critical experimental parameters to consider when selecting and conducting studies on mitochondrial function. In vitro (isolated mitochondria) and in situ (permeabilized cells/tissue) approaches provide direct access to the mitochondria, allowing for study of mitochondrial bioenergetics and redox function under defined substrate conditions. Several experimental parameters must be tightly controlled, including assay media, temperature, oxygen concentration, and in the case of permeabilized skeletal muscle, the contractile state of the fibers. Recently developed technology now offers the opportunity to measure oxygen consumption in intact cultured cells. Magnetic resonance spectroscopy provides the most direct way of assessing mitochondrial function in vivo with interpretations based on specific modeling approaches. The continuing rapid evolution of these technologies offers new and exciting opportunities for deciphering the potential role of mitochondrial function in the etiology and treatment of diabetes.

Background: Muscle wasting and weakness in Duchenne muscular dystrophy (DMD) causes severe locomotor limitations and early death due in part to respiratory muscle failure. Given that current clinical practice focuses on treating secondary complications in this genetic disease, there is a clear need to identify additional contributions in the aetiology of this myopathy for knowledge‐guided therapy development. Here, we address the unresolved question of whether the complex impairments observed in DMD are linked to elevated mitochondrial H₂O₂ emission in conjunction with impaired oxidative phosphorylation. This study performed a systematic evaluation of the nature and degree of mitochondrial‐derived H₂O₂ emission and mitochondrial oxidative dysfunction in a mouse model of DMD by designing in vitro bioenergetic assessments that attempt to mimic in vivo conditions known to be critical for the regulation of mitochondrial bioenergetics.Methods: Mitochondrial bioenergetics were compared with functional and histopathological indices of myopathy early in DMD (4 weeks) in D2.B10‐DMD^mdx/2J mice (D2.mdx)—a model that demonstrates severe muscle weakness. Adenosine diphosphate's (ADP's) central effect of attenuating H₂O₂ emission while stimulating respiration was compared under two models of mitochondrial‐cytoplasmic phosphate exchange (creatine independent and dependent) in muscles that stained positive for membrane damage (diaphragm, quadriceps, and white gastrocnemius).Results: Pathway‐specific analyses revealed that Complex I‐supported maximal H₂O₂ emission was elevated concurrent with a reduced ability of ADP to attenuate emission during respiration in all three muscles (mH₂O₂: +17 to +197% in D2.mdx vs. wild type). This was associated with an impaired ability of ADP to stimulate respiration at sub‐maximal and maximal kinetics (−17 to −72% in D2.mdx vs. wild type), as well as a loss of creatine‐dependent mitochondrial phosphate shuttling in diaphragm and quadriceps. These changes largely occurred independent of mitochondrial density or abundance of respiratory chain complexes, except for quadriceps. This muscle was also the only one exhibiting decreased calcium retention capacity, which indicates increased sensitivity to calcium‐induced permeability transition pore opening. Increased H₂O₂ emission was accompanied by a compensatory increase in total glutathione, while oxidative stress markers were unchanged. Mitochondrial bioenergetic dysfunctions were associated with induction of mitochondrial‐linked caspase 9, necrosis, and markers of atrophy in some muscles as well as reduced hindlimb torque and reduced respiratory muscle function.Conclusions: These results provide evidence that Complex I dysfunction and loss of central respiratory control by ADP and creatine cause elevated oxidant generation during impaired oxidative phosphorylation. These dysfunctions may contribute to early stage disease pathophysiology and support the growing notion that mitochondria are a potential therapeutic target in this disease.

We provide an overview of groundbreaking studies that laid the foundation for our current understanding of exercise-induced mitochondrial biogenesis and its contribution to human skeletal muscle fitness. We highlight the mechanisms by which skeletal muscle responds to the acute perturbations in cellular energy homeostasis evoked by a single bout of endurance-based exercise and the adaptations resulting from the repeated demands of exercise training that ultimately promote mitochondrial biogenesis through hormetic feedback loops. Despite intense research efforts to elucidate the cellular mechanisms underpinning mitochondrial biogenesis in skeletal muscle, translating this basic knowledge into improved metabolic health at the population level remains a future challenge.

• ATP transfer from mitochondria to the cytoplasm occurs mainly through phosphate transfer to creatine by mitochondrial creatine kinase (miCK) but also by transport and/or diffusion of ADP and ATP through specific mitochondrial transport protein complexes. • Determining the effect of exercise on phosphate shuttling may require contractile signals in situ and varying creatine concentrations to alter miCK activity. • Mitochondrial respiratory sensitivity to ADP was assessed in permeabilized muscle fibre bundles (PmFBs) before and after 2 h cycling exercise in human skeletal muscle. • In relaxed PmFBs, ADP sensitivity decreased post-exercise when miCK phosphate shuttling was low (no creatine) with no change in net ADP sensitivity in the presence of creatine, whereas in contracting fibres post-exercise ADP sensitivity was higher with creatine. • This shows miCK activity is increased post-exercise, especially during contraction in PmFBs, and suggests exercise regulates phosphate shuttling, which would improve maintenance of energy homeostasis during contraction. Abstract Energy transfer between mitochondrial and cytosolic compartments is predominantly achieved by creatine-dependent phosphate shuttling (PCr/Cr) involving mitochondrial creatine kinase (miCK). However, ADP/ATP diffusion through adenine nucleotide translocase (ANT) and voltage-dependent anion carriers (VDACs) is also involved in this process. To determine if exercise alters the regulation of this system, ADP-stimulated mitochondrial respiratory kinetics were assessed in permeabilized muscle fibre bundles (PmFBs) taken from biopsies before and after 2 h of cycling exercise (60%) in nine lean males. Concentrations of creatine (Cr) and phosphocreatine (PCr) as well as the contractile state of PmFBs were manipulated in situ. In the absence of contractile signals (relaxed PmFBs) and miCK activity (no Cr), post-exercise respiratory sensitivity to ADP was reduced in situ (up to 126% higher apparent K_m to ADP) suggesting inhibition of ADP/ATP diffusion between matrix and cytosolic compartments (possibly ANT and VDACs). However this effect was masked in the presence of saturating Cr (no effect of exercise on ADP sensitivity). Given that the role of ANT is thought to be independent of Cr, these findings suggest ADP/ATP, but not PCr/Cr, cycling through the outer mitochondrial membrane (VDACs) may be attenuated in resting muscle after exercise. In contrast, in contracted PmFBs, post-exercise respiratory sensitivity to ADP increased with miCK activation (saturating Cr; 33% lower apparent K_m to ADP), suggesting prior exercise increases miCK sensitivity in situ. These observations demonstrate that exercise increases miCK-dependent respiratory sensitivity to ADP, promoting mitochondrial–cytosolic energy exchange via PCr/Cr cycling, possibly through VDACs. This effect may mask an underlying inhibition of Cr-independent ADP/ATP diffusion. This enhanced regulation of miCK-dependent phosphate shuttling may improve energy homeostasis through more efficient coupling of oxidative phosphorylation to perturbations in cellular energy charge during subsequent bouts of contraction.

Muscle weakness and wasting are defining features of cancer-induced cachexia. Mitochondrial stress occurs before atrophy in certain muscles, but the possibility of heterogeneous responses between muscles and across time remains unclear. Using mice inoculated with Colon-26 cancer, we demonstrate that specific force production was reduced in quadriceps and diaphragm at 2 weeks in the absence of atrophy. At this time, pyruvate-supported mitochondrial respiration was lower in quadriceps while mitochondrial H ₂ O ₂ emission was elevated in diaphragm. By 4 weeks, atrophy occurred in both muscles, but specific force production increased to control levels in quadriceps such that reductions in absolute force were due entirely to atrophy. Specific force production remained reduced in diaphragm. Mitochondrial respiration increased and H ₂ O ₂ emission was unchanged in both muscles versus control while mitochondrial creatine sensitivity was reduced in quadriceps. These findings indicate muscle weakness precedes atrophy and is linked to heterogeneous mitochondrial alterations that could involve adaptive responses to metabolic stress. Eventual muscle-specific restorations in specific force and bioenergetics highlight how the effects of cancer on one muscle do not predict the response in another muscle. Exploring heterogeneous responses of muscle to cancer may reveal new mechanisms underlying distinct sensitivities, or resistance, to cancer cachexia.