High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H(2)O(2)-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H(2)O(2) emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H(2)O(2) emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.

Lactate (La) has long been at the center of controversy in research, clinical, and athletic settings. Since its discovery in 1780, La has often been erroneously viewed as simply a hypoxic waste product with multiple deleterious effects. Not until the 1980s, with the introduction of the cell-to-cell lactate shuttle did a paradigm shift in our understanding of the role of La in metabolism begin. The evidence for La as a major player in the coordination of whole-body metabolism has since grown rapidly. La is a readily combusted fuel that is shuttled throughout the body, and it is a potent signal for angiogenesis irrespective of oxygen tension. Despite this, many fundamental discoveries about La are still working their way into mainstream research, clinical care, and practice. The purpose of this review is to synthesize current understanding of La metabolism via an appraisal of its robust experimental history, particularly in exercise physiology. That La production increases during dysoxia is beyond debate, but this condition is the exception rather than the rule. Fluctuations in blood [La] in health and disease are not typically due to low oxygen tension, a principle first demonstrated with exercise and now understood to varying degrees across disciplines. From its role in coordinating whole-body metabolism as a fuel to its role as a signaling molecule in tumors, the study of La metabolism continues to expand and holds potential for multiple clinical applications. This review highlights La's central role in metabolism and amplifies our understanding of past research.

Assessment of mitochondrial ADP-stimulated respiratory kinetics in permeabilized skeletal myofibres (PmFB) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (∼20-300 μM) and tend to overestimate respiration at rest. Noting PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. Blebbistatin (BLEB), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >∼250 and ∼80 μM in red and white rat PmFB, respectively. In the absence of BLEB, PmFB contracted and the Km for ADP decreased by ∼2 to 10-fold in a temperature-dependent manner. PmFB were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30°C but not 37°C. In PmFB from humans, contraction elicited high sensitivity to ADP (m <100 μM) whereas blocking contraction (+BLEB) and including PCr:Cr = 2 to mimic the resting energetic state yielded a Km for ADP = ∼1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

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.

Statins, the widely prescribed cholesterol-lowering drugs for the treatment of cardiovascular disease, cause adverse skeletal muscle side effects ranging from fatigue to fatal rhabdomyolysis. The purpose of this study was to determine the effects of simvastatin on mitochondrial respiration, oxidative stress, and cell death in differentiated primary human skeletal muscle cells (i.e. myotubes). Simvastatin induced a dose dependent decrease in viability of proliferating and differentiating primary human muscle precursor cells, and a similar dose-dependent effect was noted in differentiated myoblasts and myotubes. Additionally, there were decreases in myotube number and size following 48 h of simvastatin treatment (5 µM). In permeabilized myotubes, maximal ADP-stimulated oxygen consumption, supported by palmitoyl-carnitine + malate (PCM, complex I and II substrates) and glutamate + malate (GM, complex I substrates), was 32–37% lower (P<0.05) in simvastatin treated (5 µM) vs. control myotubes, providing evidence of impaired respiration at complex I. Mitochondrial superoxide and hydrogen peroxide generation were significantly greater in the simvastatin treated human skeletal myotube cultures compared to control. In addition, simvastatin markedly increased protein levels of Bax (pro-apoptotic, +53%) and Bcl-2 (anti-apoptotic, +100%, P<0.05), mitochondrial PTP opening (+44%, P<0.05), and TUNEL-positive nuclei in human skeletal myotubes, demonstrating up-regulation of mitochondrial-mediated myonuclear apoptotic mechanisms. These data demonstrate that simvastatin induces myotube atrophy and cell loss associated with impaired ADP-stimulated maximal mitochondrial respiratory capacity, mitochondrial oxidative stress, and apoptosis in primary human skeletal myotubes, suggesting mitochondrial dysfunction may underlie human statin-induced myopathy.