Nonequilibrium thermodynamics and mitochondrial protein content predict insulin sensitivity and fuel selection during exercise in human skeletal muscle.

Introduction: Many investigators have attempted to define the molecular nature of changes responsible for insulin resistance in muscle, but a molecular approach may not consider the overall physiological context of muscle. Because the energetic state of ATP (ΔGATP) could affect the rate of insulin-stimulated, energy-consuming processes, the present study was undertaken to determine whether the thermodynamic state of skeletal muscle can partially explain insulin sensitivity and fuel selection independently of molecular changes. Methods: 31P-MRS was used with glucose clamps, exercise studies, muscle biopsies and proteomics to measure insulin sensitivity, thermodynamic variables, mitochondrial protein content, and aerobic capacity in 16 volunteers. Results: After showing calibrated 31P-MRS measurements conformed to a linear electrical circuit model of muscle nonequilibrium thermodynamics, we used these measurements in multiple stepwise regression against rates of insulin-stimulated glucose disposal and fuel oxidation. Multiple linear regression analyses showed 53% of the variance in insulin sensitivity was explained by 1) VO2max (p = 0.001) and the 2) slope of the relationship of ΔGATP with the rate of oxidative phosphorylation (p = 0.007). This slope represents conductance in the linear model (functional content of mitochondria). Mitochondrial protein content from proteomics was an independent predictor of fractional fat oxidation during mild exercise (R2 = 0.55, p = 0.001). Conclusion: Higher mitochondrial functional content is related to the ability of skeletal muscle to maintain a greater ΔGATP, which may lead to faster rates of insulin-stimulated processes. Mitochondrial protein content per se can explain fractional fat oxidation during mild exercise.

Fuel Selection in Skeletal Muscle Exercising at Low Intensity; Reliance on Carbohydrate in Very Sedentary Individuals.

Background: Resting skeletal muscle in insulin resistance prefers to oxidize carbohydrate rather than lipid, exhibiting metabolic inflexibility. Although this is established in resting muscle, complexities involved in directly measuring fuel oxidation using indirect calorimetry across a muscle bed have limited studies of this phenomenon in working skeletal muscle. During mild exercise and at rest, whole-body indirect calorimetry imperfectly estimates muscle fuel oxidation. We provide evidence that a method termed "ΔRER" can reasonably estimate fuel oxidation in skeletal muscle activated by exercise. Methods: Completely sedentary volunteers (n = 20, age 31 ± 2 years, V̇O2peak 24.4 ± 1.5 mL O2 per min/kg) underwent glucose clamps to determine insulin sensitivity and graded exercise consisting of three periods of mild steady-state cycle ergometry (15, 30, 45 watts, or 10%, 20%, and 30% of maximum power) with measurements of whole-body gas exchange. ΔRER, the RER in working muscle, was calculated as (V̇CO2exercise -V̇CO2rest)/(V̇O2exercise - V̇O2rest), from which the fraction of fuel accounted for by lipid was estimated. Results: Lactate levels were low and stable during steady-state exercise. Muscle biopsies were used to estimate mitochondrial content. The rise of V̇O2 at onset of exercise followed a monoexponential function, with a time constant of 51 ± 7 sec, typical of skeletal muscle; the average O2 cost of work was about 12 mL O2/watt/min, representing a mechanical efficiency of about 24%. At work rates of 30 or 45 watts, active muscle relied predominantly on carbohydrate, independent of insulin sensitivity within this group of very sedentary volunteers. Conclusions: The fraction of muscle fuel oxidation from fat was predicted by power output (P < 0.001) and citrate synthase activity (P < 0.05), indicating that low mitochondrial content may be the main driver of fuel choice in sedentary people, independent of insulin sensitivity.

Site-specific acetylation of adenine nucleotide translocase 1 at lysine 23 in human muscle.

Evidence suggests acetylation of human adenine nucleotide translocase 1 (ANT1) at lysine 23 (Lys23) reduces binding of ADP. Lys23 contributes to the positive charge that facilitates this interaction. This study was undertaken to characterize ANT1 abundance and acetylation by a novel method using small amounts of human skeletal muscle biopsies. Lysates of whole muscle or mitochondria from the same tissue were prepared from needle biopsies of vastus lateralis muscle of healthy volunteers. Lysed proteins were resolved on gels, the section containing ANT1 (surrounding 30 Kd) was excised, digested with trypsin, spiked with labeled unacetylated and acetylated synthetic standard peptides and analyzed by mass spectrometry. Natural logarithm transformation of data linearized ion intensities over a 10-fold range of peptide mass. Coefficients of variation ranged from 7 to 30% for ANT1 abundance and Lys23 acetylation. In three volunteers, ANT1 content was 8.36 ± 0.33 nmol/g wet weight muscle and 0.64 ± 0.05 nmol/mg mitochondria, so mitochondrial content was 13.3 ± 2.4 mg mitochondria per gram muscle. Acetylation of Lys23 averaged 14.3 ± 4.2% and 4.87 ± 1.84% in whole muscle and mitochondria, respectively. This assay makes it possible to assess effects of acetylation on the function of ANT1 in human muscle.

Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes.

VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P < 0.01), and mitochondrial area was two-fold higher in the VWA8 null cells (P < 0.05). The results of this study allow us to conclude that despite sustained, higher ROS production in VWA8 null cells, a global mitochondrial compensatory response was maintained, resulting in overall higher mitochondrial oxidative capacity.

Mitochondrial lactate metabolism: history and implications for exercise and disease.

Mitochondrial structures were probably observed microscopically in the 1840s, but the idea of oxidative phosphorylation (OXPHOS) within mitochondria did not appear until the 1930s. The foundation for research into energetics arose from Meyerhof's experiments on oxidation of lactate in isolated muscles recovering from electrical contractions in an O2 atmosphere. Today, we know that mitochondria are actually reticula and that the energy released from electron pairs being passed along the electron transport chain from NADH to O2 generates a membrane potential and pH gradient of protons that can enter the molecular machine of ATP synthase to resynthesize ATP. Lactate stands at the crossroads of glycolytic and oxidative energy metabolism. Based on reported research and our own modelling in silico, we contend that lactate is not directly oxidized in the mitochondrial matrix. Instead, the interim glycolytic products (pyruvate and NADH) are held in cytosolic equilibrium with the products of the lactate dehydrogenase (LDH) reaction and the intermediates of the malate-aspartate and glycerol 3-phosphate shuttles. This equilibrium supplies the glycolytic products to the mitochondrial matrix for OXPHOS. LDH in the mitochondrial matrix is not compatible with the cytoplasmic/matrix redox gradient; its presence would drain matrix reducing power and substantially dissipate the proton motive force. OXPHOS requires O2 as the final electron acceptor, but O2 supply is sufficient in most situations, including exercise and often acute illness. Recent studies suggest that atmospheric normoxia may constitute a cellular hyperoxia in mitochondrial disease. As research proceeds appropriate oxygenation levels should be carefully considered.

Von Willebrand factor A domain-containing protein 8 (VWA8) localizes to the matrix side of the inner mitochondrial membrane.

VWA8 is a poorly characterized mitochondrial AAA + ATPase protein. The specific submitochondrial localization of VWA8 remains unclear. The purpose of this study was to determine the specific submitochondrial compartment within which VWA8 resides in order to provide more insight into the function of this protein. Bioinformatics analysis showed that VWA8 has a 34 amino acid N-terminal Matrix-Targeting Signal (MTS) that is similar to those in proteins known to localize to the mitochondrial matrix. Experiments in C2C12 mouse myoblasts using confocal microscopy showed that deletion of the VWA8 MTS (vMTS) resulted in cytosolic, rather than mitochondrial, localization of VWA8. Biochemical analysis using differential sub-fractionation of mitochondria isolated from rat liver showed that VWA8 localizes to the matrix side of inner mitochondrial membrane, similar to the inner mitochondrial membrane protein Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETFDH). The results of these experiments show that the vMTS is essential for localization to the mitochondrial matrix and that once there, VWA8 localizes to the matrix side of inner mitochondrial membrane.

Deletion of the Mitochondrial Protein VWA8 Induces Oxidative Stress and an HNF4α Compensatory Response in Hepatocytes.

von Willebrand A domain-containing protein 8 (VWA8) is a poorly characterized, mitochondrial matrix-targeted protein with an AAA ATPase domain and ATPase activity that increases in livers of mice fed a high-fat diet. This study was undertaken to use CRISPR/Cas9 to delete VWA8 in cultured mouse hepatocytes and gain insight into its function. Unbiased omics techniques and bioinformatics were used to guide subsequent assays, including the assessment of oxidative stress and the determination of bioenergetic capacity. Metabolomics analysis showed VWA8 null cells had higher levels of oxidative stress and protein degradation; assays of hydrogen peroxide production revealed higher levels of production of reactive oxygen species (ROS). Proteomics and transcriptomics analyses showed VWA8 null cells had higher levels of expression of mitochondrial proteins (electron transport-chain Complex I, ATP synthase), peroxisomal proteins, and lipid transport proteins. The pattern of higher protein abundance in the VWA8 null cells could be explained by a higher level of hepatocyte nuclear factor 4 α (HNF4α) expression. Bioenergetic assays showed higher rates of carbohydrate oxidation and mitochondrial and nonmitochondrial lipid oxidation in intact and permeabilized cells. Inhibitor assays localized sites of ROS production to peroxisomes and NOX1/4. The rescue of VWA8 protein restored the wild-type phenotype, and treatment with antioxidants decreased the level of HNF4α expression. Thus, loss of VWA8 produces a mitochondrial defect that may be sensed by NOX4, leading to an increase in the level of ROS that results in a higher level of HNF4α. The compensatory HNF4α response results in a higher oxidative capacity and an even higher level of ROS production. We hypothesize that VWA8 is an AAA ATPase protein that plays a role in mitochondrial protein quality.

Characterization of the novel protein KIAA0564 (Von Willebrand Domain-containing Protein 8).

The VWA8 gene was first identified by the Kazusa cDNA project and named KIAA0564. Based on the observation, by similarity, that the protein encoded by KIAA0564 contains a Von Willebrand Factor 8 domain, KIAA0564 was named Von Willebrand Domain-containing Protein 8 (VWA8). The function of VWA8 protein is almost unknown. The purpose of this study was to characterize the tissue distribution, cellular location, and function of VWA8. In mice VWA8 protein was mostly distributed in liver, kidney, heart, pancreas and skeletal muscle, and is present as a long isoform and a shorter splice variant (VWA8a and VWA8b). VWA8 protein and mRNA were elevated in mouse liver in response to high fat feeding. Sequence analysis suggests that VWA8 has a mitochondrial targeting sequence and domains responsible for ATPase activity. VWA8 protein was targeted exclusively to mitochondria in mouse AML12 liver cells, and this was prevented by deletion of the targeting sequence. Moreover, the VWA8 short isoform overexpressed in insect cells using a baculovirus construct had in vitro ATPase activity. Deletion of the Walker A motif or Walker B motif in VWA8 mostly blocked ATPase activity, suggesting Walker A motif or Walker B motif are essential to the ATPase activity of VWA8. Finally, homology modeling suggested that VWA8 may have a structure most confidently similar to dynein motor proteins.

Subsarcolemmal mitochondria isolated with the proteolytic enzyme nagarse exhibit greater protein specific activities and functional coupling.

Skeletal muscle mitochondria are arranged as a reticulum. Insight into the functional characteristics of such structure is achieved by viewing the network as consisting of "subsarcolemmal" (SS) and "intermyofibrillar" (IMF) regions. During the decades, most, but not all, published studies have reported higher (sometimes over 2-fold) enzyme and enzyme-pathway protein-specific activities in IMF compared to SS mitochondria. We tested the hypothesis that non-mitochondrial protein contamination might account for much of the apparently lower specific activities of isolated SS mitochondria. Mouse gastrocnemii (n = 6) were suspended in isolation medium, minced, and homogenized according to procedures typically used to isolate SS mitochondria. However, the supernatant fraction, collected after the first slow-speed (800×g) centrifugation, was divided equally: one sample was exposed to nagarse (MITO+), while the other was not (MITO-). Nagarse treatment reduced total protein yield by 25%, while it increased protein-specific respiration rates (nmol O2 min-1 mg-1), by 38% under "resting" (state 4) and by 84% under maximal (state 3) conditions. Nagarse therefore increased the respiratory control ratio (state 3/state 4) by 30%. In addition, the ADP/O ratio was increased by 9% and the activity of citrate synthase (U/mg) was 49% higher. Mass spectrometry analysis indicated that the MITO+ preparation contained less contamination from non-mitochondrial proteins. We conclude that nagarse treatment of SS mitochondria removes not only non-mitochondrial proteins but also the protein of damaged mitochondria, improves indices of functional integrity, and the resulting protein-specific activities.

A Simple Hydraulic Analog Model of Oxidative Phosphorylation.

Mitochondrial oxidative phosphorylation is the primary source of cellular energy transduction in mammals. This energy conversion involves dozens of enzymatic reactions, energetic intermediates, and the dynamic interactions among them. With the goal of providing greater insight into the complex thermodynamics and kinetics ("thermokinetics") of mitochondrial energy transduction, a simple hydraulic analog model of oxidative phosphorylation is presented. In the hydraulic model, water tanks represent the forward and back "pressures" exerted by thermodynamic driving forces: the matrix redox potential (ΔGredox), the electrochemical potential for protons across the mitochondrial inner membrane (ΔGH), and the free energy of adenosine 5'-triphosphate (ATP) (ΔGATP). Net water flow proceeds from tanks with higher water pressure to tanks with lower pressure through "enzyme pipes" whose diameters represent the conductances (effective activities) of the proteins that catalyze the energy transfer. These enzyme pipes include the reactions of dehydrogenase enzymes, the electron transport chain (ETC), and the combined action of ATP synthase plus the ATP-adenosine 5'-diphosphate exchanger that spans the inner membrane. In addition, reactive oxygen species production is included in the model as a leak that is driven out of the ETC pipe by high pressure (high ΔGredox) and a proton leak dependent on the ΔGH for both its driving force and the conductance of the leak pathway. Model water pressures and flows are shown to simulate thermodynamic forces and metabolic fluxes that have been experimentally observed in mammalian skeletal muscle in response to acute exercise, chronic endurance training, and reduced substrate availability, as well as account for the thermokinetic behavior of mitochondria from fast- and slow-twitch skeletal muscle and the metabolic capacitance of the creatine kinase reaction.

Adenine nucleotide translocase is acetylated in vivo in human muscle: Modeling predicts a decreased ADP affinity and altered control of oxidative phosphorylation.

Proteomics techniques have revealed that lysine acetylation is abundant in mitochondrial proteins. This study was undertaken (1) to determine the relationship between mitochondrial protein acetylation and insulin sensitivity in human skeletal muscle, identifying key acetylated proteins, and (2) to use molecular modeling techniques to understand the functional consequences of acetylation of adenine nucleotide translocase 1 (ANT1), which we found to be abundantly acetylated. Eight lean and eight obese nondiabetic subjects had euglycemic clamps and muscle biopsies for isolation of mitochondrial proteins and proteomics analysis. A number of acetylated mitochondrial proteins were identified in muscle biopsies. Overall, acetylation of mitochondrial proteins was correlated with insulin action (r = 0.60; P < 0.05). Of the acetylated proteins, ANT1, which catalyzes ADP-ATP exchange across the inner mitochondrial membrane, was acetylated at lysines 10, 23, and 92. The extent of acetylation of lysine 23 decreased following exercise, depending on insulin sensitivity. Molecular dynamics modeling and ensemble docking simulations predicted the ADP binding site of ANT1 to be a pocket of positively charged residues, including lysine 23. Calculated ADP-ANT1 binding affinities were physiologically relevant and predicted substantial reductions in affinity upon acetylation of lysine 23. Insertion of these derived binding affinities as parameters into a complete mathematical description of ANT1 kinetics predicted marked reductions in adenine nucleotide flux resulting from acetylation of lysine 23. Therefore, acetylation of ANT1 could have dramatic physiological effects on ADP-ATP exchange. Dysregulation of acetylation of mitochondrial proteins such as ANT1 therefore could be related to changes in mitochondrial function that are associated with insulin resistance.

Skeletal muscle fuel selection occurs at the mitochondrial level.

Mammals exponentially increase the rate of carbohydrate oxidation as exercise intensity rises, while birds combust lipid almost exclusively while flying at high percentages of aerobic capacity. The fuel oxidized by contracting muscle depends on many factors: whole-body fuel storage mass, mobilization, blood transport, cellular uptake, and substrate selection at the level of the mitochondrion. We examined the fuel preferences of mitochondria isolated from mammalian and avian locomotory muscles using two approaches. First, the influence of substrates on the kinetics of respiration (Km,ADP and Vmax) was evaluated. For all substrates and combinations, Km,ADP was generally twofold higher in avian mitochondria. Second, fuel competition between pyruvate, glutamate and/or palmitoyl-l-carnitine at three levels of ATP free energy was determined using the principle of mass balance and the measured rates of O2 consumption and metabolite accumulation/utilization. Avian mitochondria strongly spared pyruvate from oxidation when another substrate was available and fatty acid was the dominant substrate, regardless of energy state. Mammalian mitochondria exhibited some preference for fatty acid over pyruvate at lower flux (higher energy state), but exhibited a much greater tendency to select pyruvate and glutamate when available. Studies in sonicated mitochondria revealed twofold higher electron transport chain electron conductance in avian mitochondria. We conclude that substantial fuel selection occurs at the level of the mitochondrial matrix and that avian flight muscle mitochondria are particularly biased toward the selection of fatty acid, possibly by facilitating high ß-oxidation flux by maintaining a more oxidized matrix.

Effect of calcium on the oxidative phosphorylation cascade in skeletal muscle mitochondria.

Calcium is believed to regulate mitochondrial oxidative phosphorylation, thereby contributing to the maintenance of cellular energy homeostasis. Skeletal muscle, with an energy conversion dynamic range of up to 100-fold, is an extreme case for evaluating the cellular balance of ATP production and consumption. This study examined the role of Ca(2+) in the entire oxidative phosphorylation reaction network in isolated skeletal muscle mitochondria and attempted to extrapolate these results back to the muscle, in vivo. Kinetic analysis was conducted to evaluate the dose-response effect of Ca(2+) on the maximal velocity of oxidative phosphorylation (V(maxO)) and the ADP affinity. Force-flow analysis evaluated the interplay between energetic driving forces and flux to determine the conductance, or effective activity, of individual steps within oxidative phosphorylation. Measured driving forces [extramitochondrial phosphorylation potential (ΔG(ATP)), membrane potential, and redox states of NADH and cytochromes b(H), b(L), c(1), c, and a,a(3)] were compared with flux (oxygen consumption) at 37 °C; 840 nM Ca(2+) generated an ~2-fold increase in V(maxO) with no change in ADP affinity (~43 µM). Force-flow analysis revealed that Ca(2+) activation of V(maxO) was distributed throughout the oxidative phosphorylation reaction sequence. Specifically, Ca(2+) increased the conductance of Complex IV (2.3-fold), Complexes I and III (2.2-fold), ATP production/transport (2.4-fold), and fuel transport/dehydrogenases (1.7-fold). These data support the notion that Ca(2+) activates the entire muscle oxidative phosphorylation cascade, while extrapolation of these data to the exercising muscle predicts a significant role of Ca(2+) in maintaining cellular energy homeostasis.

Mitochondrial function in sparrow pectoralis muscle.

Flying birds couple a high daily energy turnover with double-digit millimolar blood glucose concentrations and insulin resistance. Unlike mammalian muscle, flight muscle predominantly relies on lipid oxidation during locomotion at high fractions of aerobic capacity, and birds outlive mammals of similar body mass by a factor of three or more. Despite these intriguing functional differences, few data are available comparing fuel oxidation and free radical production in avian and mammalian skeletal muscle mitochondria. Thus we isolated mitochondria from English sparrow pectoralis and rat mixed hindlimb muscles. Maximal O(2) consumption and net H(2)O(2) release were measured in the presence of several oxidative substrate combinations. Additionally, NAD- and FAD-linked electron transport chain (ETC) capacity was examined in sonicated mitochondria. Sparrow mitochondria oxidized palmitoyl-l-carnitine 1.9-fold faster than rat mitochondria and could not oxidize glycerol-3-phosphate, while both species oxidized pyruvate, glutamate and malate-aspartate shuttle substrates at similar rates. Net H(2)O(2) release was not significantly different between species and was highest when glycolytic substrates were oxidized. Sonicated sparrow mitochondria oxidized NADH and succinate over 1.8 times faster than rat mitochondria. The high ETC catalytic potential relative to matrix substrate dehydrogenases in sparrow mitochondria suggests a lower matrix redox potential is necessary to drive a given O(2) consumption rate. This may contribute to preferential reliance on lipid oxidation, which may result in lower in vivo reactive oxygen species production in birds compared with mammals.

Exercise protects cardiac mitochondria against ischemia-reperfusion injury.

PURPOSE: Three to five consecutive days of endurance exercise can protect the heart against an ischemia-reperfusion (IR) insult. However, the mechanisms responsible for this exercise-mediated cardioprotection remain unknown. Given the important role that mitochondria play in IR-induced cardiac myocyte injury, we hypothesized that exercise training promotes cardioprotection, at least in part, by increasing mitochondrial antioxidants, preventing mitochondrial release of reactive oxygen species, and protecting cardiac mitochondria against IR-induced oxidative damage and functional impairment. METHODS: To test our hypothesis, Sprague-Dawley rats were assigned to either sedentary (n = 16) or exercise-trained (n = 16) groups. Exercise-trained animals performed 5 d of treadmill running for 60 min·d(-1) at 30 m·s(-1). Hearts were excised from sedentary and exercised-trained animals and were either perfused for 80 min or exposed to 40 min of global ischemia followed by 45 min of reperfusion by using an ex vivo isolated working heart model. After the protocol, cardiac subsarcolemmal and intermyofibrillar mitochondria were isolated and used to determine respiratory control ratio, reactive oxygen species emission, and indices of oxidative stress and apoptosis. RESULTS: Our results support our hypothesis because exercise training protected both cardiac subsarcolemmal and intermyofibrillar mitochondria from IR-induced uncoupling and oxidative damage. Specifically, the levels of cardiac mitochondrial 4-hydroxynonenal-conjugated proteins were elevated in hearts from sedentary animals exposed to IR compared with cardiac mitochondria isolated from exercise-trained animals. Exercise also resulted in an increase in mitochondrial antioxidant enzymes (copper-zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase) and prevented the IR-induced release of proapoptotic proteins from the mitochondria. CONCLUSIONS: Collectively, these novel findings reveal that exercise-induced cardioprotection is mediated, at least in part, through mitochondrial adaptations resulting in a mitochondrial phenotype that resists IR-induced damage.

Fuel oxidation at the walk-to-run-transition in humans.

Multiple factors (including anthropometric, kinetic, mechanical, kinematic, perceptual, and energetic factors) are likely to play a role in the walk-to-run transition in humans. The primary purpose of the present study was to consider an additional factor, the metabolic fuel source. Indirect calorimetry was used to measure fuel oxidation, and perception of effort was recorded as 10 overnight-fasted adults locomoted on a level treadmill at speeds progressing from 1.56 to 2.46 m s(-1) in increments of 0.11 m s(-1) and 10.0 minutes under 3 conditions: (1) unconstrained choice of gait, (2) walking at all speeds, and (3) running at all speeds. The preferred transition speed was 2.08 ± 0.03 m s(-1). Gait transition from walking to running increased oxygen consumption rate, decreased the perception of effort, and decreased the rate of carbohydrate oxidation. We propose that, in an evolutionary context, gait transition, guided by the perception of effort, can be viewed as a carbohydrate-sparing strategy.

Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle.

OBJECTIVE: The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. RESEARCH DESIGN AND METHODS: Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. RESULTS: NADH- and FADH(2)-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-DL-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). CONCLUSIONS: We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS.

Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small samples of healthy and diseased skeletal muscle.