Comparison of in vivo postexercise phosphocreatine recovery and resting ATP synthesis flux for the assessment of skeletal muscle mitochondrial function

P-31 magnetic resonance spectroscopy (MRS) has been used to assess skeletal muscle mitochondrial function in vivo by measuring 1) phosphocreatine (PCr) recovery after exercise or 2) resting ATP synthesis flux with saturation transfer (ST). In this study, we compared both parameters in a rat model of mitochondrial dysfunction with the aim of establishing the most appropriate method for the assessment of in vivo muscle mitochondrial function. Mitochondrial dysfunction was induced in adult Wistar rats by daily subcutaneous injections with the complex I inhibitor diphenyleneiodonium (DPI) for 2 wk. In vivo P-31 MRS measurements were supplemented by in vitro measurements of oxygen consumption in isolated mitochondria. Two weeks of DPI treatment induced mitochondrial dysfunction, as evidenced by a 20% lower maximal ADP-stimulated oxygen consumption rate in isolated mitochondria from DPI-treated rats oxidizing pyruvate plus malate. This was paralleled by a 46% decrease in in vivo oxidative capacity, determined from postexercise PCr recovery. Interestingly, no significant difference in resting, ST-based ATP synthesis flux was observed between DPI-treated rats and controls. These results show that PCr recovery after exercise has a more direct relationship with skeletal muscle mitochondrial function than the ATP synthesis flux measured with P-31 ST MRS in the resting state. P-31 magnetic resonance spectroscopy saturation transfer high-resolution respirometry complex I inhibition diphenyleneiodonium mitochondria play a pivotal role in many cellular processes, the most important function being the production of energy in the form of ATP through a process termed oxidative phosphorylation. In the last decade, mitochondria gained interest in the field of insulin resistance (IR) and type 2 diabetes (T2D) (21, 34, 43, 49, 53, 55). Based on the in vivo observation that ATP synthesis flux in resting skeletal muscle is lower in insulin-resistant subjects and offspring of T2D patients compared with healthy controls (38, 39), it has been hypothesized that skeletal muscle mitochondrial dysfunction is a predisposing factor for IR and/or T2D. The proposed mechanism links muscle mitochondrial dysfunction to impaired fatty acid metabolism, which subsequently leads to the accumulation of intramyocellular lipids and lipid intermediates (e. g., diacylglycerol and ceramides) that interfere with the insulin signaling cascade (68). The role of skeletal muscle mitochondrial dysfunction in the development of IR and/or T2D has been investigated using a variety of techniques (12, 16-18, 24, 31-33, 37-40, 45, 48, 52). In vitro methodologies, like the determination of gene expression levels, enzyme activities, mitochondrial content, morphology, and respiration, provide specific information on different aspects of mitochondrial energy production, but the results cannot be directly translated to in vivo mitochondrial function. P-31 magnetic resonance spectroscopy (MRS) provides a noninvasive tool to monitor the energetic status of the cell in vivo by measuring intracellular phosphorous containing metabolites; i.e., phosphocreatine (PCr), ATP, and inorganic phosphate (Pi). P-31 MR spectra of resting skeletal muscle are relatively constant, even in diseased states, and to assess impairments in mitochondrial energy production one needs to perturb either the chemical or the magnetic equilibrium as described below. The resting P-i -> ATP flux (V-ATP) can be determined by saturating the gamma-ATP peak and monitoring the effect of this perturbation on the P-i magnetization in a so-called P-31 saturation transfer (ST) experiment (3, 5). Assuming that V-ATP is predominantly reflecting oxidative ATP synthesis by the F1F0-ATP synthase in the mitochondria, V-ATP has been taken as a measure for mitochondrial function (39, 40). However, the interpretation of P-31 ST data is not straightforward. The lower ATP synthesis rates in resting muscle of insulin-resistant subjects (38, 39) could actually reflect a normal regulatory response to a lower energy demand caused by impaired insulin signaling rather than an impairment of intrinsic mitochondrial function (1, 26, 51, 65). Moreover, V-ATP obtained from P-31 ST measurements is composed of both mitochondrial ATP synthase flux and glycolytic exchange flux, with the latter contributing by as much as 80% at rest (3, 4, 6, 26, 27). Therefore, decreased resting V-ATP does not necessarily reflect a mitochondrial defect. As an alternative to the resting state ST experiment described above, the metabolic steady state of the muscle; i.e., the chemical equilibrium, can be disturbed during exercise. During recovery from exercise, the PCr pool is replenished purely through oxidative ATP synthesis (42, 46, 58). Because the creatine kinase reaction is much faster than oxidative ATP production (64), the measurement of PCr recovery using dynamic P-31 MRS after exercise provides an alternative method to determine the rate of oxidative ATP synthesis. The PCr recovery rate constant (k(PCr)) thus reflects in vivo muscle mitochondrial oxidative capacity; i.e., the maximal capacity for oxidative ATP production, which is typically one order of magnitude higher than the ATP synthesis rate at rest. Therefore, the postexercise PCr recovery rate constant might be a more suitable measure for in vivo mitochondrial function compared with the resting ATP synthesis flux. In this study, we compared in vivo P-31 MRS postexercise PCr recovery and resting ATP synthesis flux in a rat model of mitochondrial dysfunction with the aim of establishing the most appropriate method for the assessment of in vivo skeletal muscle mitochondrial function. Mitochondrial dysfunction was induced by daily subcutaneous injections with diphenyleneiodonium (DPI), which irreversibly inhibits complex I (NADH-ubiquinone reductase) of the respiratory chain (9, 10, 30, 44). In vivo measurements were supplemented by in vitro measurements of oxygen consumption in isolated mitochondria to confirm inhibition of complex I and to compare in vivo and in vitro measurements of mitochondrial function.