Jeroen A. L. Jeneson

The signal transduction function for oxidative phosphorylation is at least second order in ADP

To maintain ATP constant in the cell, mitochondria must sense cellular ATP utilization and transduce this demand to F0-F1-ATPase. In spite of a considerable research effort over the past three decades, no combination of signal(s) and kinetic function has emerged with the power to explain ATP homeostasis in all mammalian cells. We studied this signal transduction problem in intact human muscle using 31P NMR spectroscopy. We find that the apparent kinetic order of the transduction function of the signal cytosolic ADP concentration ([ADP]) is at least second order and not first order as has been assumed. We show that amplified mitochondrial sensitivity to cytosolic [ADP] harmonizes with in vitro kinetics of [ADP] stimulation of respiration and explains ATP homeostasis also in mouse liver and canine heart. This result may well be generalizable to all mammalian cells.

Increased mitochondrial content rescues in vivo muscle oxidative capacity in long-term high-fat-diet-fed rats

Mitochondria are thought to play a crucial role in the etiology of muscle insulin resistance (IR). The aim of this study was to gain more insight into the timing and nature of mitochondrial adaptations during the development of high‐fat‐diet (HFD)‐induced IR. Adult Wistar rats were fed HFD or normal chow for 2.5 and 25 wk. Intramyocellular lipids (IMCLs) were quantified in vivo using 1H magnetic resonance spectroscopy (MRS). Muscle oxidative capacity was assessed in vivo using 31P MRS and in vitro by measuring mitochondrial DNA copy number and oxygen consumption in isolated mitochondria. MRS in tibialis anterior muscle revealed 3.3‐fold higher IMCL content and 1.2‐fold increased oxidative capacity after 2.5 wk of HFD feeding. The latter result could be fully accounted for by increased mitochondrial content. After 25 wk of HFD, maximal ADP‐stimulated oxygen consumption in isolated mitochondria oxidizing pyruvate plus malate remained unaffected, while IMCL and mitochondrial content had further increased compared to controls (5.1‐fold and 1.4‐fold, respectively). Interestingly, in vivo oxidative capacity at this time point was identical to controls. These results show that skeletal muscle in HFD‐induced IR accompanied by IMCL accumulation requires a progressively larger mitochondrial pool size to maintain normal oxidative capacity in vivo. —Van den Broek, N. M. A., Ciapaite, J., De Feyter, H. M. M. L., Houten, S. M., Wanders, R. J. A., Jeneson, J. A. L., Nicolay, K., Prompers, J. J. Increased mitochondrial content rescues in vivo muscle oxidative capacity in long‐term high‐fat‐diet‐fed rats. FASEB J. 24, 1354–1364 (2010). www.fasebj.org

Differential effects of short- and long-term high-fat diet feeding on hepatic fatty acid metabolism in rats

Imbalance in the supply and utilization of fatty acids (FA) is thought to contribute to intrahepatic lipid (IHL) accumulation in obesity. The aim of this study was to determine the time course of changes in the liver capacity to oxidize and store FA in response to high-fat diet (HFD). Adult male Wistar rats were fed either normal chow or HFD for 2.5weeks (short-term) and 25weeks (long-term). Short-term HFD feeding led to a 10% higher palmitoyl-l-carnitine-driven ADP-stimulated (state 3) oxygen consumption rate in isolated liver mitochondria indicating up-regulation of β-oxidation. This adaptation was insufficient to cope with the dietary FA overload, as indicated by accumulation of long-chain acylcarnitines, depletion of free carnitine and increase in FA content in the liver, reflecting IHL accumulation. The latter was confirmed by in vivo((1))H magnetic resonance spectroscopy and Oil Red O staining. Long-term HFD feeding caused further up-regulation of mitochondrial β-oxidation (24% higher oxygen consumption rate in state 3 with palmitoyl-l-carnitine as substrate) and stimulation of mitochondrial biogenesis as indicated by 62% higher mitochondrial DNA copy number compared to controls. These adaptations were paralleled by a partial restoration of free carnitine levels and a decrease in long-chain acylcarnitine content. Nevertheless, there was a further increase in IHL content, accompanied by accumulation of lipid peroxidation and protein oxidation products. In conclusion, partially effective adaption of hepatic FA metabolism to long-term HFD feeding came at a price of increased oxidative stress, caused by a combination of higher FA oxidation capacity and oversupply of FA.

Magnitude and control of mitochondrial sensitivity to ADP

The transduction function for ADP stimulation of mitochondrial ATP synthesis in skeletal muscle was reconstructed in vivo and in silico to investigate the magnitude and origin of mitochondrial sensitivity to cytoplasmic ADP concentration changes. Dynamic in vivo measurements of human leg muscle phosphocreatine (PCr) content during metabolic recovery from contractions were performed by (31)P-NMR spectroscopy. The cytoplasmic ADP concentration ([ADP]) and rate of oxidative ATP synthesis (Jp) at each time point were calculated from creatine kinase equilibrium and the derivative of a monoexponential fit to the PCr recovery data, respectively. Reconstructed [ADP]-Jp relations for individual muscles containing more than 100 data points were kinetically characterized by nonlinear curve fitting yielding an apparent kinetic order and ADP affinity of 1.9 +/- 0.2 and 0.022 +/- 0.003 mM, respectively (means +/- SD; n = 6). Next, in silico [ADP]-Jp relations for skeletal muscle were generated using a computational model of muscle oxidative ATP metabolism whereby model parameters corresponding to mitochondrial enzymes were randomly changed by 50-150% to determine control of mitochondrial ADP sensitivity. The multiparametric sensitivity analysis showed that mitochondrial ADP ultrasensitivity is an emergent property of the integrated mitochondrial enzyme network controlled primarily by kinetic properties of the adenine nucleotide translocator.

In vivo31P MRS detection of an alkaline inorganic phosphate pool with short T1 in human resting skeletal muscle

Non‐invasive determination of mitochondrial content is an important objective in clinical and sports medicine. 31P MRS approaches to obtain information on this parameter at low field strength typically require in‐magnet exercise. Direct observation of the intra‐mitochondrial inorganic phosphate (Pi) pool in resting muscle would constitute an alternative, simpler method. In this study, we exploited the higher spectral resolution and signal‐to‐noise at 7T to investigate the MR visibility of this metabolite pool. 31P in vivo MR spectra of the resting soleus (SOL) muscle were obtained with 1H MR image‐guided surface coil localization (six volunteers) and of the SOL and tibialis anterior (TA) muscle using 2D CSI (five volunteers). A resonance at a frequency 0.38 ppm downfield from the cytosolic Pi resonance (Pi1; pH 7.0 ± 0.04) was reproducibly detected in the SOL muscle in all subjects and conditionally attributed to the intra‐mitochondrial Pi pool (Pi2; pH 7.3 ± 0.07). In the SOL muscle, the Pi2/Pi1 ratio was 1.6 times higher compared to the TA muscle in the same individual. Localized 3D CSI results showed that the Pi2 peak was present in voxels well away from blood vessels. Determination of the T1 of the two Pi pools in a single individual using adiabatic excitation of the spectral region around 5 ppm yielded estimates of 4.3 ± 0.4 s vs 1.4 ± 0.5 s for Pi1 and Pi2, respectively. Together, these results suggest that the intra‐mitochondrial Pi pool in resting human skeletal muscle may be visible with 31P MRS at high field. Copyright

An MR-compatible bicycle ergometer for in-magnet whole-body human exercise testing

An MR‐compatible ergometer was developed for in‐magnet whole‐body human exercise testing. Designed on the basis of conventional mechanically braked bicycle ergometers and constructed from nonferrous materials, the ergometer was implemented on a 1.5‐T whole‐body MR scanner. A spectrometer interface was constructed using standard scanner hardware, complemented with custom‐built parts and software to enable gated data acquisition during exercise. High‐quality 31P NMR spectra were reproducibly obtained from the medial head of the quadriceps muscle of the right leg of eight healthy subjects during two‐legged high‐frequency pedaling (80 revolutions per minute) at three incremental workloads, including maximal. Muscle phosphocreatine content dropped 82%, from 32.2 ± 1.0 mM at rest to 5.7 ± 1.1 mM at maximal workload (mean ± standard error; n = 8), indicating that the majority of quadriceps motor units were recruited. The cardiovascular load of the exercise was likewise significant, as evidenced by heart rates of 150 (±10%) beats per minute, measured immediately afterward. As such, the newly developed MR bicycling exercise equipment offers a powerful new tool for clinical musculoskeletal and cardiovascular MR investigation. The basic design of the ergometer is highly generic and adaptable for application on a wide selection of whole‐body MR scanners. Magn Reson Med, 2010.

Prediction of muscle energy states at low metabolic rates requires feedback control of mitochondrial respiratory chain activity by inorganic phosphate

The regulation of the 100-fold dynamic range of mitochondrial ATP synthesis flux in skeletal muscle was investigated. Hypotheses of key control mechanisms were included in a biophysical model of oxidative phosphorylation and tested against metabolite dynamics recorded by 31P nuclear magnetic resonance spectroscopy (31P MRS). Simulations of the initial model featuring only ADP and Pi feedback control of flux failed in reproducing the experimentally sampled relation between myoplasmic free energy of ATP hydrolysis (ΔGp = ΔGp o′+RT ln ([ADP][Pi]/[ATP]) and the rate of mitochondrial ATP synthesis at low fluxes (<0.2 mM/s). Model analyses including Monte Carlo simulation approaches and metabolic control analysis (MCA) showed that this problem could not be amended by model re-parameterization, but instead required reformulation of ADP and Pi feedback control or introduction of additional control mechanisms (feed forward activation), specifically at respiratory Complex III. Both hypotheses were implemented and tested against time course data of phosphocreatine (PCr), Pi and ATP dynamics during post-exercise recovery and validation data obtained by 31P MRS of sedentary subjects and track athletes. The results rejected the hypothesis of regulation by feed forward activation. Instead, it was concluded that feedback control of respiratory chain complexes by inorganic phosphate is essential to explain the regulation of mitochondrial ATP synthesis flux in skeletal muscle throughout its full dynamic range.

Non-invasive quantitative 31P MRS assay of mitochondrial function in skeletal muscle in situ

A method for non-invasive, quantitative 31P NMR spectroscopic investigation of mitochondrial function in human skeletal muscle in situ is described. High time resolution 31P NMR measurements of phosphocreatine, inorganic phosphate and ATP resonances were conducted on human forearm flexor muscle during involuntary twitch contraction at eight different frequencies. Mitochondrial and glyco(geno)lytic ATP synthesis fluxes, and the cytosolic free energy of ATP hydrolysis (ΔGp) were calculated at incremental steady-states of energy balance. The covariation of mitochondrial ATP synthesis flux, JpMOP, and ΔGp was quasi-linear over the physiological range of free energy values. Curve-fit analysis of the covariation yielded a maximal sustainable pMOPof 0.24 ± 0.06 mmol ATP l-1·s-1 and a midpoint potential, (ΔGp)0.5, of 58.1 ± 1.2 kJ/mole in the muscle cells. (Mol Cell Biochem 174: 17–22, 1997)

Silencing of glycolysis in muscle: experimental observation and numerical analysis

The longstanding problem of rapid inactivation of the glycolytic pathway in skeletal muscle after contraction was investigated using 31P NMR spectroscopy and computational modelling. Accumulation of phosphorylated glycolytic intermediates (hexose monophosphates) during cyclic contraction and subsequent turnover during metabolic recovery was measured in vivo in human quadriceps muscle using dynamic 31P NMR spectroscopy. The concentration of hexose monophosphates in muscle peaked 40 s into metabolic recovery from maximal contractile work at 6.9 ± 1.3 mm (mean ±s.d.; n= 8) and subsequently declined at a rate of 0.009 ± 0.001 mm s−1. It was next tested whether the current knowledge of the kinetic controls in the glycolytic pathway in muscle integrated in the Lambeth and Kushmerick computational model of skeletal muscle glycolysis explained the experimental data. It was found that the model underestimated the magnitude of deactivation of the glycolytic pathway in resting muscle, resulting in depletion of glycolytic intermediates and substrate for oxidative ATP synthesis. Numerical analysis of the model identified phosphofructokinase and pyruvate kinase as the kinetic control sites involved in deactivation of the glycolytic pathway. Ancillary 100‐fold inhibition of both phosphofructokinase and pyruvate kinase was found necessary to predict glycolytic intermediate and ADP concentrations correctly in resting human muscle. Incorporation of this information into the model resulted in highly improved agreement between predicted and measured in vivo dynamics of hexose monophosphates in muscle following contraction. We concluded that silencing of the glycolytic pathway in muscle following contraction is most likely to be mediated by phosphofructokinase and pyruvate kinase inactivation on a time scale of seconds and minutes, respectively, and is necessary to prevent depletion of vital cellular substrates.

Treadmill but not wheel running improves fatigue resistance of isolated extensor digitorum longus muscle in mice

Aim:  The present study is the first to compare the physiological impact of either forced treadmill or voluntary wheel running exercise on hindlimb muscle in mice.