Wayne Tyrus Willis
- Associate Professor, Medicine
My research and teaching interest focus on vertebrate energy metabolism, primarily in skeletal muscle during locomotion, but also in liver, kidney, and heart. Of particular interest are the thermodynamic and kinetic mechanisms underlying the control of metabolic flux and fuel selection and the influence of nutritional status, for example overnutrition and fasting. Computational modeling of metabolism at the mitochondrial and cellular level is becoming the primary focus of these studies.
- Ph.D. Physiology of Exercise
- University of California, Berkeley, California, United States
- Physiological and Biochemical Correlates of Increased Exercise Capacity in Trained Iron-Deficient Rats
- Department of Medicine, College of Medicine, University of Arizona (2016 - Ongoing)
- Department of Kinesiology and School of Life Sciences, Arizona State University (1989 - 2016)
- The Distinguished Arizona Lecture
- The Arizona Physiological Society, Fall 2016 (Award Finalist)
Licensure & Certification
- Bioenergetics Master Class Graduate, Buck Institute for Aging Research, Novato, California (2017)
- Advanced Techniques in Mitochondrial Physiology, Buck Institute for Aging Research, Novato, California (2001)
Metabolic Physiology, Mitochondrial Bioenergetics, Exercise Physiology
Control of Oxidative Metabolism in Skeletal Muscle, Liver, Heart, and Kidney. Fasting/starvation. Energy metabolism of locomotion. Computational modeling.
No activities entered.
- Kras, K. A., Langlais, P. R., Hoffman, N., Roust, L. R., Benjamin, T. R., De Filippis, E. A., Dinu, V., & Katsanos, C. S. (2018). Obesity modifies the stoichiometry of mitochondrial proteins in a way that is distinct to the subcellular localization of the mitochondria in skeletal muscle.. Metabolism.
- Willis, W. T. (2018). Dominant and sensitive control of oxidative flux by the ATP-ADP Carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.. Archives of Biochemistry and Biophysics, 647, 93-103.More infoCoauthors: Miranda-Grandjean D, Hudgens J, Willis EA, Finlayson J, De Filippis EA, Zapata Bustos R, Langlais PR, Mielke C, Mandarino LJ
- Luo, M., Mengos, A. E., Ma, W., Finlayson, J., Bustos, R. Z., Xiao Zhu, Y., Shi, C. X., Stubblefield, T. M., Willis, W. T., & Mandarino, L. J. (2017). Characterization of the novel protein KIAA0564 (Von Willebrand Domain-containing Protein 8). Biochemical and biophysical research communications, 487(3), 545-551.More infoThe 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.
- Willis, W. T. (2017). Role of adipocyte mitochondria in inflammation, lipemia and insulin sensitivity in humans: effects of pioglitazone treatment.. International Journal of Obesity (Nature), 42, 675-681, 2017..More infoAll authors and reference:Xie X, Sinha S, Yi Z, Langlais PR, Madan M, Bowen BP, Willis W, Meyer C. Role of adipocyte mitochondria in inflammation, lipemia and insulin sensitivity in humans: effects of pioglitazone treatment. Int J Obes 42:675-681, 2017.
- Kras, K. A., Willis, W. T., Barker, N., Czyzyk, T., Langlais, P. R., & Katsanos, C. S. (2016). Subsarcolemmal mitochondria isolated with the proteolytic enzyme nagarse exhibit greater protein specific activities and functional coupling. Biochemistry and biophysics reports, 6, 101-107.More infoSkeletal 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.
- Willis, W. T., Jackman, M. R., Messer, J. I., Kuzmiak-Glancy, S., & Glancy, B. (2016). A Simple Hydraulic Analog Model of Oxidative Phosphorylation. Medicine and science in sports and exercise, 48(6), 990-1000.More infoMitochondrial 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.
- Kras, K., Willis, W., Barker, N., Czyzyk, T., & Katsanos, C. (2014). The enzyme nagarse modifies parameters associated with function of mechanically-liberated mitochondria. FASEB JOURNAL, 28(1).
- Kuzmiak-Glancy, S., & Willis, W. T. (2014). Skeletal muscle fuel selection occurs at the mitochondrial level. JOURNAL OF EXPERIMENTAL BIOLOGY, 217(11), 1993-2003.
- Kuzmiak-Glancy, S., & Willis, W. T. (2014). Skeletal muscle fuel selection occurs at the mitochondrial level. The Journal of experimental biology, 217(Pt 11), 1993-2003.More infoMammals 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 β-oxidation flux by maintaining a more oxidized matrix.
- Mielke, C., Lefort, N., McLean, C. G., Cordova, J. M., Langlais, P. R., Bordner, A. J., Te, J. A., Ozkan, S. B., Willis, W. T., & Mandarino, L. J. (2014). Adenine Nucleotide Translocase Is Acetylated in Vivo in Human Muscle: Modeling Predicts a Decreased ADP Affinity and Altered Control of Oxidative Phosphorylation. BIOCHEMISTRY, 53(23), 3817-3829.
- Mielke, C., Lefort, N., McLean, C. G., Cordova, J. M., Langlais, P. R., Bordner, A. J., Te, J. A., Ozkan, S. B., Willis, W. T., & Mandarino, L. J. (2014). Adenine nucleotide translocase is acetylated in vivo in human muscle: Modeling predicts a decreased ADP affinity and altered control of oxidative phosphorylation. Biochemistry, 53(23), 3817-29.More infoProteomics 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.
- Glancy, B., Willis, W. T., Chess, D. J., & Balaban, R. S. (2013). Effect of Calcium on the Oxidative Phosphorylation Cascade in Skeletal Muscle Mitochondria. BIOCHEMISTRY, 52(16), 2793-2809.
- Glancy, B., Willis, W. T., Chess, D. J., & Balaban, R. S. (2013). Effect of calcium on the oxidative phosphorylation cascade in skeletal muscle mitochondria. Biochemistry, 52(16), 2793-809.More infoCalcium 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.
- Luo, M., Mengos, A. E., Stubblefield, T. M., Langlais, P. R., Willis, W. T., & Mandarino, L. J. (2013). Clustered Mitochondria Phenotype (Clu)-1 and Lipolysis-Stimulated Lipoprotein Receptor (LSR) Integrate Triglyceride (TG) Uptake and Mitochondrial Content in Liver. DIABETES, 62, A484-A484.
- Mielke, C., Ozkan, B., Bordner, A., Te, J., Willis, W. T., & Mandarino, L. J. (2013). Molecular Modeling Reveals Acetylation of Adenine Nucleotide Translocase (ANT) 1 Regulates Affinity for ADP. DIABETES, 62, A7-A8.
- Gardner, D. L., Kuzmiak, S., & Willis, W. T. (2012). NAD-Linked Isocitrate Dehydrogenase Activity is Phosphate Dependent in Sparrow Mitochondria. MEDICINE AND SCIENCE IN SPORTS AND EXERCISE, 44, 356-356.
- Kuzmiak, S., & Willis, W. T. (2012). Electron Conductance in Rat and Sparrow Skeletal Muscle Mitochondrial Electron Transport Chain. MEDICINE AND SCIENCE IN SPORTS AND EXERCISE, 44, 356-356.
- Kuzmiak, S., & Willis, W. T. (2012). Electron Conductance in Rat and Sparrow Skeletal Muscle Mitochondrial Electron Transport Chain. MEDICINE AND SCIENCE IN SPORTS AND EXERCISE, 44, 701-701.
- Kuzmiak, S., Glancy, B., Sweazea, K. L., & Willis, W. T. (2012). Mitochondrial function in sparrow pectoralis muscle. JOURNAL OF EXPERIMENTAL BIOLOGY, 215(12), 2039-2050.
- Kuzmiak, S., Glancy, B., Sweazea, K. L., & Willis, W. T. (2012). Mitochondrial function in sparrow pectoralis muscle. The Journal of experimental biology, 215(Pt 12), 2039-50.More infoFlying 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.
- Lee, Y., Min, K., Talbert, E. E., Kavazis, A. N., Smuder, A. J., Willis, W. T., & Powers, S. K. (2012). Exercise Protects Cardiac Mitochondria against Ischemia-Reperfusion Injury. MEDICINE AND SCIENCE IN SPORTS AND EXERCISE, 44(3), 397-405.
- Lee, Y., Min, K., Talbert, E. E., Kavazis, A. N., Smuder, A. J., Willis, W. T., & Powers, S. K. (2012). Exercise protects cardiac mitochondria against ischemia-reperfusion injury. Medicine and science in sports and exercise, 44(3), 397-405.More infoThree 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.
- Ganley, K. J., Stock, A., Herman, R. M., Santello, M., & Willis, W. T. (2011). Fuel oxidation at the walk-to-run-transition in humans. METABOLISM-CLINICAL AND EXPERIMENTAL, 60(5), 609-616.
- Ganley, K. J., Stock, A., Herman, R. M., Santello, M., & Willis, W. T. (2011). Fuel oxidation at the walk-to-run-transition in humans. Metabolism: clinical and experimental, 60(5), 609-16.More infoMultiple 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.
- Kuzmiak, S., & Willis, W. T. (2011). Pyruvate sparing in avian and mammalian muscle mitochondria. FASEB JOURNAL, 25.
- Kuzmiak, S., Glancy, B., Sweazea, K., & Willis, W. (2010). Mitochondrial function in sparrow pectoralis muscle. FASEB JOURNAL, 24.
- Lefort, N., Glancy, B., Bowen, B., Willis, W. T., Bailowitz, Z., De Filippis, E. A., Brophy, C., Meyer, C., Højlund, K., Yi, Z., & Mandarino, L. J. (2010). 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. Diabetes, 59(10), 2444-52.More infoThe 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.
- Lefort, N., Glancy, B., Bowen, B., Willis, W. T., Bailowitz, Z., De, F., Brophy, C., Meyer, C., Hojlund, K., Yi, Z., & Mandarino, L. J. (2010). 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. DIABETES, 59(10), 2444-2452.
- Lefort, N., Yi, Z., Bowen, B., Glancy, B., De Filippis, E. A., Mapes, R., Hwang, H., Flynn, C. R., Willis, W. T., Civitarese, A., Højlund, K., & Mandarino, L. J. (2009). Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. Journal of proteomics, 72(6), 1046-60.More infoMitochondria 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.
- Lefort, N., Yi, Z., Bowen, B., Glancy, B., De, F., Mapes, R., Hwang, H., Flynn, C. R., Willis, W. T., Civitarese, A., Hojlund, K., & Mandarino, L. J. (2009). Proteome profile of functional mitochondria from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. JOURNAL OF PROTEOMICS, 72(6), 1046-1060.
- Ganley, K. J., Herman, R. M., & Willis, W. T. (2008). Muscle metabolism during overground walking in persons with poststroke hemiparesis. TOPICS IN STROKE REHABILITATION, 15(3), 218-226.
- Glancy, B., & Willis, W. (2008). Superoxide Production by Rat Skeletal Muscle Mitochondria: Influence of High Fat Diet. FASEB JOURNAL, 22.
- Glancy, B., Barstow, T., & Willis, W. T. (2008). Linear relation between time constant of oxygen uptake kinetics, total creatine, and mitochondrial content in vitro. AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 294(1), C79-C87.
- Glancy, B., Barstow, T., & Willis, W. T. (2008). Linear relation between time constant of oxygen uptake kinetics, total creatine, and mitochondrial content in vitro. American journal of physiology. Cell physiology, 294(1), C79-87.More infoFollowing the onset of moderate aerobic exercise, the rate of oxygen consumption (J(o)) rises monoexponentially toward the new steady state with a time constant (tau) in the vicinity of 30 s. The mechanisms underlying this delay have been studied over several decades. Meyer's electrical analog model proposed the concept that the tau is given by tau = R(m) x C, where R(m) is mitochondrial resistance to energy transfer, and C is metabolic capacitance, determined primarily by the cellular total creatine pool (TCr = phosphocreatine + creatine). The purpose of this study was to evaluate in vitro the J(o) kinetics of isolated rat skeletal muscle mitochondria at various levels of TCr and mitochondrial protein. Mitochondria were incubated in a medium containing 5.0 mM ATP, TCr pools of 0-1.5 mM, excess creatine kinase, and an ATP-splitting system of glucose + hexokinase (HK). Pyruvate and malate (1 mM each) were present as oxidative substrates. J(o) was measured across time after HK was added to elicit one of two levels of J(o) (40 and 60% of state 3). At TCr levels (in mM) of 0.1, 0.2, 0.3, 0.75, and 1.5, the corresponding tau values (s, means +/- SE) were 22.2 +/- 3.0, 36.3 +/- 2.2, 65.7 +/- 4.3, 168.1 +/- 22.2, and 287.3 +/- 25.9. Thus tau increased linearly with TCr (R(2) = 0.916). Furthermore, the experimentally observed tau varied linearly and inversely with the mitochondrial protein added. These in vitro results consistently conform to the predictions of Meyer's electrical analog model.
- Lefort, N., Yi, Z., Bowen, B., Glancy, B., De, F., Hwang, H., Willis, W. T., Hojlund, K., & Mandarino, L. J. (2008). Mitochondrial proteome profile from human skeletal muscle using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS. DIABETES, 57, A429-A429.
- Willis, W. T., Glancy, B., & Barstow, T. J. (2008). Reply to "Letter to the editor: 'Physiological implications of linear kinetics of mitochondrial respiration in vitro'". AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 295(3), C847-C848.
- Ganley, K. J., Herman, R. M., & Willis, W. T. (2007). Fuel oxidation during walking in persons with central nervous system pathologies. FASEB JOURNAL, 21(6), A928-A928.
- Willis, W. T., Ganley, K. J., & Herman, R. M. (2005). Fuel oxidation during human walking. Metabolism: clinical and experimental, 54(6), 793-9.More infoHuman adults walk at a characteristic speed, but the mechanisms responsible for this ubiquitous and reproducible behavior remain unknown. In this study, preferred walking speed (PWS) was 4.7 +/- 0.1 km h -1 in 12 overnight-fasted adults, mean age 30.0 +/- 2.6 years. Indirect calorimetry was used to measure fuel oxidation during level treadmill walking from 3.2 to 7.2 km h -1 progressively increased at increments of 0.8 km h -1 and 10.0-min intervals. Corroborating many previous reports, the O2 cost of transport (mL O2 kg -1 km -1 ) was numerically lowest at 4.8 km h -1 , near PWS, but was not significantly different than 5.6 km h -1 . The impact of walking speed on the fuel selection of skeletal muscle was much more dramatic. At speeds less than or equal to PWS, muscle carbohydrate (CHO) oxidation rates were quite low, in the range that could be matched by gluconeogenesis. Above 4.8 km h -1 , CHO oxidation rate increased abruptly and tracked the perception of effort (RPE). Stepwise linear regression revealed that CHO oxidation explained 70% of the variance in RPE, and speed provided an additional 4%. In contrast, the other variables included in the analysis, fat oxidation rate, heart rate, and O2 cost of transport, contributed no additional explained variance in RPE. We conclude that PWS is just below a threshold speed, above which CHO oxidation abruptly increases. The central nervous system may be guided by the perception of effort in selecting a PWS that minimizes dependence on CHO oxidation. We further conclude that skeletal muscle metabolic control is an important factor to be taken into account by the central nervous system motor control of human locomotion.
- Carhart, M. R., He, J., Herman, R., D'Luzansky, S., & Willis, W. T. (2004). Epidural spinal-cord stimulation facilitates recovery of functional walking following incomplete spinal-cord injury. IEEE transactions on neural systems and rehabilitation engineering : a publication of the IEEE Engineering in Medicine and Biology Society, 12(1), 32-42.More infoWe investigated a novel treatment paradigm for developing functional ambulation in wheelchair-dependent individuals with chronic, incomplete spinal-cord injury. By coordinating epidural stimulation of the dorsal structures of the spinal cord with partial weight bearing treadmill therapy, we observed improvement in treadmill and over-ground ambulation in an individual with chronic incomplete tetraplegia. The application of partial weight-bearing therapy alone was not sufficient to achieve functional ambulation over ground, though treadmill ambulation improved significantly. Combining epidural spinal-cord stimulation (ESCS, T10-T12 vertebral levels) with partial weight-bearing therapy resulted in further improvement during treadmill ambulation. Moreover, the combination of therapies facilitated the transfer of the learned gait into over ground ambulation. Performance improvements were elicited by applying continuous, charge-balanced, monophasic pulse trains at a frequency of 40-60 Hz, a pulse duration of 800 micros, and an amplitude determined by the midpoint (50%) between the sensory and motor threshold values. The participant initially reported a reduction in sense of effort for over ground walking from 8/10 to 3/10 (Borg scale), and was able to double his walking speed. After several weeks of over ground training, he reached maximum walking speeds of 0.35 m/s, and was able to ambulate over 325 m. We propose that ESCS facilitated locomotor recovery in this patient by augmenting the use-dependent plasticity created by partial weight bearing therapy. Confirmation of these promising results in a controlled study of groups of spinal-cord-injured subjects is warranted.
- Messer, J. I., Jackman, M. R., & Willis, W. T. (2004). Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria. American journal of physiology. Cell physiology, 286(3), C565-72.More infoCarbohydrate depletion precipitates fatigue in skeletal muscle, but, because pyruvate provides both acetyl-CoA for mainline oxidation and anaplerotic carbon to the citric acid cycle (CAC), the mechanism remains obscure. Thus pyruvate and CAC kinetic parameters were independently quantified in mitochondria isolated from rat mixed skeletal muscle. Mitochondrial oxygen consumption rate (Jo) was measured polarographically while either pyruvate or malate was added stepwise in the presence of a saturating concentration of the other substrate. These substrate titrations were carried out across a physiological range of fixed extramitochondrial ATP free energy states (DeltaGP), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent Km,malate for mitochondrial Jo ranged from 21 to 32 microM, and the apparent Km,pyruvate ranged from 12 to 26 microM, with both substrate Km values increasing as DeltaGP declined. Vmax for both substrates also increased as DeltaGP fell, reflecting thermodynamic control of Jo. Reported in vivo skeletal muscle [malate] are >10-fold greater than the Km,malate determined in this study. In marked contrast, the K(m,pyruvate) determined is near the [pyruvate] reported in muscle approaching exhaustion associated with glycogen depletion. When data were evaluated in the context of a linear thermodynamic force-flow (DeltaGP-Jo) relationship, the DeltaGP-Jo slope was essentially insensitive to changes in [malate] in the range observed in vivo but decreased markedly with declining [pyruvate] across the physiological range. Mitochondrial respiration is particularly sensitive to variations in [pyruvate] in the physiological range. In contrast, physiological [malate] exerts very little, if any, influence on mitochondrial pyruvate oxidation measured in vitro.
- Commerford, S. R., Ferniza, J. B., Bizeau, M. E., Thresher, J. S., Willis, W. T., & Pagliassotti, M. J. (2002). Diets enriched in sucrose or fat increase gluconeogenesis and G-6-Pase but not basal glucose production in rats. American journal of physiology. Endocrinology and metabolism, 283(3), E545-55.More infoHigh-fat (HFD) and high-sucrose diets (HSD) reduce insulin suppression of glucose production in vivo, increase the capacity for gluconeogenesis in vitro, and increase glucose-6-phosphatase (G-6-Pase) activity in whole cell homogenates. The present study examined the effects of HSD and HFD on in vivo gluconeogenesis, the catalytic and glucose-6-phosphate translocase subunits of G-6-Pase, glucokinase (GK) translocation, and glucose cycling. Rats were fed a high-starch control diet (STD; 68% cornstarch), HSD (68% sucrose), or HFD (45% fat) for 7-13 days. The ratio of 3H in C6:C2 of glucose after 3H2O injection into 6- to 8-h-fasted rats was significantly increased in HSD (0.68 +/- 0.07) and HFD (0.71 +/- 0.08) vs. STD (0.40 +/- 0.10). G-6-Pase activity was significantly higher in HSD and HFD vs. STD in both intact and disrupted liver microsomes. HSD and HFD significantly increased the amount of the p36 catalytic subunit protein, whereas the p46 glucose-6-phosphate translocase protein was increased in HSD only. Despite increased nonglycerol gluconeogenesis and increased G-6-Pase, basal glucose and insulin levels as well as glucose production were not significantly different among groups. Hepatocyte cell suspensions were used to ascertain whether diet-induced adaptations in glucose phosphorylation and GK might serve to compensate for upregulation of G-6-Pase. Tracer-estimated glucose phosphorylation and glucose cycling (glucose glucose 6-phosphate) were significantly higher in cells isolated from HSD only. After incubation with either 5 or 20 mM glucose and no insulin, GK activity (nmol. mg protein(-1). min(-1)) in digitonin-treated eluates (translocated GK) was significantly higher in HSD (32 +/- 4 and 146 +/- 6) vs. HFD (4 +/- 1 and 83 +/- 10) and STD (9 +/- 2 and 87 +/- 9). Thus short-term, chronic exposure to HSD and HFD increase in vivo gluconeogenesis and the G-6-Pase catalytic subunit. Exposure to HSD diet also leads to adaptations in glucose phosphorylation and GK translocation.
- Willis, W. T., Kras, K. A., Mandarino, L. J., & Barakati, N. (2018, Fall). Assessment of skeletal muscle mitochondrial metabolic flux and control using polarographic and luciferase-based techniques. Arizona Physiological Society Annual Meeting. Tempe, Arizona.
- Willis, W. T., Willis, E. A., Hudgens, J., & Mandarino, L. J. (2018, Summer). KmADP For Oxidative Phosphorylation Depends On Substrate Oxidative Capacity. European Bioenergetics Conference 2018 (EBEC 2018). Budapest, Hungary: European Bioenergetics Society.
- Kras, K., Langlais, P. R., Willis, W. T., Mandarino, L., De Filippis, E., Roust, L., & Katsanos, C. (2016, June). Differential enrichment of Sub-sarcolemmal mitochondria with individual proteins between lean and obese, insulin-resistant subjects. American Diabetes Association Annual Meeting.
- Kras, K., Willis, W. T., De Filippis, E., Roust, L., Hoffman, N., & Katsanos, C. (2016, Fall). A single bout of aerobic exercise increases citrate synthase specific activity and ATP production rate in isolated mitochondria from human skeletal muscle.. American Physiological Society/American College of Sports Medicine Integrative Biology of Exercise Meeting. Phoenix, Arizona: American Physiological Society.
- Willis, W. T. (2016, Fall). Heeding The Wisdom of Kelvin As We Study Mitochondrial Physiology. Arizona Physiological Society Annual Meeting. Tucson, Arizona: Arizona Physiological Society.
- Kras, K., Willis, W. T., Barker, N., Czyzyk, T., Langlais, P. R., & Katsanos, C. (2015, Fall). Subsarcolemmal mitochondria isolated with proteolytic enzyme exhibit greater protein specific activities and functional coupling.. Arizona Physiological Society Annual Meeting. Glendale, Arizona: Arizona Physiological Society.
- Willis, W. T., Gladden, L. B., & Glancy, B. (2019, September). THERMODYNAMIC BARRIERS TO MITOCHONDRIAL LACTATE OXIDATION: INSIGHTS FROM COMPUTATIONAL MODELING. The 2019 NHLBI Mitochondrial Biology Symposium. Bethesda, Maryland: National Institutes of Health.
- Willis, W. T., Ravasz, D., Kitayev, A., Greenwood, B., Hill, C., Komlodi, T., Doerrier, C., Ozohanics, O., Moore, A. L., Gnaiger, E., Kiebish, M., Kolev, K., Seyfried, T. N., Narain, N., Adam-Vizi, V., & Chinopoulos, C. (2019, September). Endogenous quinones sustain a moderate NADH oxidation by complex I during anoxia. Oroboros O2k workshop on high-resolution respirometry. Schroecken, Austria: Oroboros.
- Katsanos, C. S., Kras, K. A., Langlais, P. R., Willis, W. T., De Filippis, E. A., & Roust, L. R. (2017, June). Effects of Obesity on Skeletal Muscle Biokgical Pathways Associated with Subsarcolemal Versus Intermyofibrillar Mitochondria Revealed by Proteomic Analysis. American Diabetes Association Annual Meeting.
- Willis, W. T., Willis, E., Hudgens, J., Finlayson, J., Langlais, P., & Mandarino, L. (2016, Fall). Adenine nucleotide translocase (ANT) control of oxidative flux in human skeletal muscle mitochondria: Influence of lysine acetylation.. American Physiological Society/American College Sports Medicine Integrative Biology of Exercise Meeting. Phoenix, Arizona: American Physiological Society.