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.

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.

SUMMARY Chronic metabolic diseases have been linked to molecular signatures of mitochondrial dysfunction. Nonetheless, molecular remodeling of the transcriptome, proteome, and/or metabolome does not necessarily translate to functional consequences that confer physiologic phenotypes. The work here aims to bridge the gap between molecular and functional phenomics by developing and validating a multiplexed assay platform for comprehensive assessment of mitochondrial energy transduction. The diagnostic power of the platform stems from a modified version of the creatine kinase energetic clamp technique, performed in parallel with multiplexed analyses of dehydrogenase activities and ATP synthesis rates. Together, these assays provide diagnostic coverage of the mitochondrial network at a level approaching that gained by molecular “-omics” technologies. Application of the platform to a comparison of skeletal muscle versus heart mitochondria reveals mechanistic insights into tissue-specific distinctions in energy transfer efficiency. This platform opens exciting opportunities to unravel the connection between mitochondrial bioenergetics and human disease.

Key pointsr In vivo skeletal muscle mitochondrial respiratory capacity was determined from the post-exercise recovery kinetics of muscle oxygen consumption (mV O 2 ) measured using near-infrared spectroscopy (NIRS) in humans.r NIRS recovery rates were compared with the in situ gold standard of high-resolution respirometry measured in permeabilized muscle fibre bundles prepared from muscle biopsies taken from the same participants.r NIRS-measured recovery kinetics of mV O 2 were well correlated with maximal ADP-stimulated mitochondrial respiration in permeabilized fibre bundles.r NIRS provides a cost-effective, non-invasive means of assessing in vivo mitochondrial respiratory capacity. AbstractThe present study aimed to compare in vivo measurements of skeletal muscle mitochondrial respiratory capacity made using near-infrared spectroscopy (NIRS) with the current gold standard, namely in situ measurements of high-resolution respirometry performed in permeabilized muscle fibres prepared from muscle biopsies. Mitochondrial respiratory capacity was determined in 21 healthy adults in vivo using NIRS to measure the recovery kinetics of muscle oxygen consumption following a ß15 s isometric contraction of the vastus lateralis muscle. Maximal ADP-stimulated (State 3) respiration was measured in permeabilized muscle fibres using high-resolution respirometry with sequential titrations of saturating concentrations of metabolic substrates. Overall, the in vivo and in situ measurements were strongly correlated (Pearson's r = 0.61-0.74, all P < 0.01). Bland-Altman plots also showed good agreement with no indication of bias. The results indicate that in vivo NIRS corresponds well with the current gold standard, in situ high-resolution respirometry, for assessing mitochondrial respiratory capacity.(Received 14 March 2014; accepted after revision 6 June 2014; first published online 20 June 2014) Corresponding author T. E. Ryan: 115 Heart Drive, Room 4100, East Carolina Heart Institute, Greenville, NC 27834, USA. Email: ryant@ecu.edu Abbreviations ETS, electron transport system; HHb, deoxygenated haemoglobin and myoglobin; mV O2 , muscle oxygen consumption; NIRS, near-infrared spectroscopy; PCr, phosphocreatine; PmFBs, permeabilized fibre bundles; 31 P-MRS, 31 phosphorus magnetic resonance spectroscopy; O 2 Hb, oxygenated haemoglobin and myoglobin; S tO2 , tissue saturation; tHB, total haemoglobin and myoglobin.

Menopause results in a progressive decline in 17β-estradiol (E2) levels, increased adiposity, decreased insulin sensitivity, and a higher risk for type 2 diabetes. Estrogen therapies can help reverse these effects, but the mechanism(s) by which E2 modulates susceptibility to metabolic disease is not well understood. In young C57BL/6N mice, short-term ovariectomy decreased-whereas E2 therapy restored-mitochondrial respiratory function, cellular redox state (GSH/GSSG), and insulin sensitivity in skeletal muscle. E2 was detected by liquid chromatography-mass spectrometry in mitochondrial membranes and varied according to whole-body E2 status independently of ERα. Loss of E2 increased mitochondrial membrane microviscosity and HO emitting potential, whereas E2 administration in vivo and in vitro restored membrane E2 content, microviscosity, complex I and I + III activities, HO emitting potential, and submaximal OXPHOS responsiveness. These findings demonstrate that E2 directly modulates membrane biophysical properties and bioenergetic function in mitochondria, offering a direct mechanism by which E2 status broadly influences energy homeostasis.