Quantification of skeletal muscle mitochondrial function by 31P magnetic resonance spectroscopy techniques: a quantitative review.

Magnetic resonance spectroscopy (MRS) can give information about cellular metabolism in vivo which is difficult to obtain in other ways. In skeletal muscle, non-invasive (31) P MRS measurements of the post-exercise recovery kinetics of pH, [PCr], [Pi] and [ADP] contain valuable information about muscle mitochondrial function and cellular pH homeostasis in vivo, but quantitative interpretation depends on understanding the underlying physiology. Here, by giving examples of the analysis of (31) P MRS recovery data, by some simple computational simulation, and by extensively comparing data from published studies using both (31) P MRS and invasive direct measurements of muscle O2 consumption in a common analytical framework, we consider what can be learnt quantitatively about mitochondrial metabolism in skeletal muscle using MRS-based methodology. We explore some technical and conceptual limitations of current methods, and point out some aspects of the physiology which are still incompletely understood.

Pioglitazone treatment restores in vivo muscle oxidative capacity in a rat model of diabetes.

AIM: To determine the effect of pioglitazone treatment on in vivo and ex vivo muscle mitochondrial function in a rat model of diabetes. METHODS: Both the lean, healthy rats and the obese, diabetic rats are Zucker Diabetic Fatty (ZDF) rats. The homozygous fa/fa ZDF rats are obese and diabetic. The heterozygous fa/+ ZDF rats are lean and healthy. Diabetic Zucker Diabetic Fatty rats were treated with either pioglitazone (30 mg/kg/day) or water as a control (n = 6 per group), for 2 weeks. In vivo ¹H and ³¹P magnetic resonance spectroscopy was performed on skeletal muscle to assess intramyocellular lipid (IMCL) content and muscle oxidative capacity, respectively. Ex vivo muscle mitochondrial respiratory capacity was evaluated using high-resolution respirometry. In addition, several markers of mitochondrial content were determined. RESULTS: IMCL content was 14-fold higher and in vivo muscle oxidative capacity was 26% lower in diabetic rats compared with lean rats, which was, however, not caused by impairments of ex vivo mitochondrial respiratory capacity or a lower mitochondrial content. Pioglitazone treatment restored in vivo muscle oxidative capacity in diabetic rats to the level of lean controls. This amelioration was not accompanied by an increase in mitochondrial content or ex vivo mitochondrial respiratory capacity, but rather was paralleled by an improvement in lipid homeostasis, that is lowering of plasma triglycerides and muscle lipid and long-chain acylcarnitine content. CONCLUSION: Diminished in vivo muscle oxidative capacity in diabetic rats results from mitochondrial lipid overload and can be alleviated by redirecting the lipids from the muscle into adipose tissue using pioglitazone treatment.

Combined in vivo and in silico investigations of activation of glycolysis in contracting skeletal muscle.

The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calcium-calmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ((31)P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983, 2010) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100-200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions.

In vivo postprandial lipid partitioning in liver and skeletal muscle in prediabetic and diabetic rats.

AIMS/HYPOTHESIS: Insulin resistance and type 2 diabetes have been associated with ectopic lipid deposition. This study investigates the derangements in postprandial lipid handling in liver and skeletal muscle tissue at different stages during the pathogenesis of type 2 diabetes in a rat model. METHODS: Four groups (n = 6) of male Zucker diabetic fatty rats were used for this study: prediabetic fa/fa rats and healthy fa/+ littermates at the age of 6 weeks, and diabetic fa/fa rats and healthy fa/+ littermates at the age of 12 weeks. In vivo (1)H-[(13)C] magnetic resonance spectroscopy measurements were performed in liver and tibialis anterior muscle at baseline and 4, 24 and 48 h after oral administration of 1.5 g [U-(13)C]algal lipid mixture per kilogram body weight. Total and (13)C-labelled intracellular lipid contents were determined from the magnetic resonance spectra. RESULTS: In both prediabetic and diabetic rats, total lipid contents in muscle and liver were substantially higher than in healthy controls and this was accompanied by a 2.3-fold greater postprandial lipid uptake in the liver (p < 0.001). Interestingly, in prediabetic rats, skeletal muscle appeared to be protected from excess lipid uptake whereas after developing overt diabetes muscle lipid uptake was 3.4-fold higher than in controls (p < 0.05). Muscle lipid use was significantly lower in prediabetic and diabetic muscle, indicative of impairments in lipid oxidation. CONCLUSIONS/INTERPRETATION: In vivo postprandial lipid handling is disturbed in both liver and skeletal muscle tissue in prediabetic and diabetic rats, but the uptake of dietary lipids in muscle is only increased after the development of overt diabetes.

Physical activity is the key determinant of skeletal muscle mitochondrial function in type 2 diabetes.

CONTEXT: Conflicting data exist on mitochondrial function and physical activity in type 2 diabetes mellitus (T2DM) development. OBJECTIVE: The aim was to assess mitochondrial function at different stages during T2DM development in combination with physical exercise in longstanding T2DM patients. DESIGN AND METHODS: We performed cross-sectional analysis of skeletal muscle from 12 prediabetic 11 longstanding T2DM male subjects and 12 male controls matched by age and body mass index. INTERVENTION: One-year intrasubject controlled supervised exercise training intervention was done in longstanding T2DM patients. MAIN OUTCOME MEASUREMENTS: Extensive ex vivo analyses of mitochondrial quality, quantity, and function were collected and combined with global gene expression analysis and in vivo ATP production capacity after 1 yr of training. RESULTS: Mitochondrial density, complex I activity, and the expression of Krebs cycle and oxidative phosphorylation system-related genes were lower in longstanding T2DM subjects but not in prediabetic subjects compared with controls. This indicated a reduced capacity to generate ATP in longstanding T2DM patients only. Gene expression analysis in prediabetic subjects suggested a switch from carbohydrate toward lipid as an energy source. One year of exercise training raised in vivo skeletal muscle ATP production capacity by 21 ± 2% with an increased trend in mitochondrial density and complex I activity. In addition, expression levels of ß-oxidation, Krebs cycle, and oxidative phosphorylation system-related genes were higher after exercise training. CONCLUSIONS: Mitochondrial dysfunction is apparent only in inactive longstanding T2DM patients, which suggests that mitochondrial function and insulin resistance do not depend on each other. Prolonged exercise training can, at least partly, reverse the mitochondrial impairments associated with the longstanding diabetic state.

Efficacy of positive contrast imaging techniques for molecular MRI of tumor angiogenesis.

Superparamagnetic iron oxide particles (SPIOs) are promising contrast agents for molecular MRI. To improve the in vivo detection of iron-based contrast media, positive contrast imaging techniques have been developed. Here, the efficacy of two positive contrast techniques, white marker and susceptibility gradient mapping (SGM), were evaluated for molecular MRI of tumor angiogenesis and compared with conventional negative contrast gradient echo (GE) imaging. In vitro, cylindrical phantoms containing varying iron oxide concentrations were used to measure the response of positive contrast techniques. In vivo, tumor bearing mice were used as a model for tumor angiogenesis. Mice were injected with unlabeled SPIOs (n = 5) or SPIOs labeled with cyclic NGR peptide (cNGR) (n = 5), which homes specifically to angiogenic microvessels. Pre- and post-contrast GE and white marker images were acquired. Subsequently, SGM images and R(2)(*) maps were calculated. For image analysis, the contrast-to-noise ratio (CNR) and the percentage of enhanced voxels (EVs) in the tumor rim and core were calculated. In vitro, the linear increases in MRI signal response for increasing iron oxide concentration were much stronger for SGM than white marker. In vivo, the CNR of GE, white marker and SGM imaging was 5.7, 1.2 and 6.2, respectively, with equal acquisition times. Significant differences in the percentage of EVs between the tumor rim and core were found using R(2)(*) mapping, GE and SGM (p < 0.05). The two contrast agents had significantly different percentages of EVs by R(2)(*) mapping and SGM in the rim (p < 0.001). The in vivo efficacy of white marker and SGM was evaluated for molecular MRI relative to GE imaging and R(2)(*) mapping. Only SGM, and not white marker, can be used to transfer the negative contrast from targeted SPIOs in a positive contrast signal without loss of CNR.

Unchanged muscle fiber conduction velocity relates to mild acidosis during exhaustive bicycling.

Muscle fiber conduction velocity (MFCV) has often been shown to decrease during standardized fatiguing isometric contractions. However, several studies have indicated that the MFCV may remain constant during fatiguing dynamic exercise. It was investigated if these observations can be related to the absence of a large decrease in pH and if MFCV can be considered as a good indicator of acidosis, also during dynamic bicycle exercise. High-density surface electromyography (HDsEMG) was combined with read-outs of muscle energetics recorded by in vivo (31)P magnetic resonance spectroscopy (MRS). Measurements were performed during serial exhausting bouts of bicycle exercise at three different workloads. The HDsEMG recordings revealed a small and incoherent variation of MFCV during all high-intensity exercise bouts. (31)P MRS spectra revealed a moderate decrease in pH at the end of exercise (~0.3 units down to 6.8) and a rapid ancillary drop to pH 6.5 during recovery 30 s post-exercise. This additional degree of acidification caused a significant decrease in MFCV during cycling immediately after the rest period. From the data a significant correlation between MFCV and [H(+)] ([H(+)] = 10(-pH)) was calculated (p < 0.001, Pearson's R = -0.87). Our results confirmed the previous observations of MFCV remaining constant during fatiguing dynamic exercise. A constant MFCV is in line with a low degree of acidification, considering the presence of a correlation between pH and MFCV after further increasing acidification.

The effects of deformation, ischemia, and reperfusion on the development of muscle damage during prolonged loading.

Deep tissue injury (DTI) is a severe form of pressure ulcer where tissue damage starts in deep tissues underneath intact skin. In the present study, the contributions of deformation, ischemia, and reperfusion to skeletal muscle damage development were examined in a rat model during a 6-h period. Magnetic resonance imaging (MRI) was used to study perfusion (contrast-enhanced MRI) and tissue integrity (T2-weighted MRI). The levels of tissue deformation were estimated using finite element models. Complete ischemia caused a gradual homogeneous increase in T2 (∼20% during the 6-h period). The effect of reperfusion on T2 was highly variable, depending on the anatomical location. In experiments involving deformation, inevitably associated with partial ischemia, a variable T2 increase (17-66% during the 6-h period) was observed reflecting the significant variation in deformation (with two-dimensional strain energies of 0.60-1.51 J/mm) and ischemia (50.8-99.8% of the leg) between experiments. These results imply that deformation, ischemia, and reperfusion all contribute to the damage process during prolonged loading, although their importance varies with time. The critical deformation threshold and period of ischemia that cause muscle damage will certainly vary between individuals. These variations are related to intrinsic factors, such as pathological state, which partly explain the individual susceptibility to the development of DTI and highlight the need for regular assessments of individual subjects.

Ischemia-reperfusion injury in rat skeletal muscle assessed with T2-weighted and dynamic contrast-enhanced MRI.

Pressure ulcers are localized areas of soft tissue breakdown due to mechanical loading. Susceptible individuals are subjected to pressure relief strategies to prevent long loading periods. Therefore, ischemia-reperfusion injury may play an important role in the etiology of pressure ulcers. To investigate the inter-relation between postischemic perfusion and changes in skeletal muscle integrity, the hindlimbs of Brown Norway rats were subjected to 4-h ischemia followed by 2-h reperfusion. Dynamic contrast-enhanced MRI was used to examine perfusion, and changes in skeletal muscle integrity were monitored with T2-weighted MRI. The dynamic contrast-enhanced MRI data showed a heterogeneous postischemic profile in the hindlimb, consisting of areas with increased contrast enhancement (14-76% of the hindlimb) and regions with no-reflow (5-77%). For T2, a gradual increase in the complete leg was observed during the 4-h ischemic period (from 34 to 41 msec). During the reperfusion phase, a heterogeneous distribution of T2 was observed. Areas with increased contrast enhancement were associated with a decrease in T2 (to 38 msec) toward preischemic levels, whereas no-reflow areas exhibited a further increase in T2 (to 42 msec). These results show that reperfusion after prolonged ischemia may not be complete, thereby continuing the ischemic condition and aggravating tissue damage.

Influence of cell-internalization on relaxometric, optical and compositional properties of targeted paramagnetic quantum dot micelles.

Quantum dot micelles (pQDs) with a paramagnetic coating are promising nanoparticles for bimodal molecular imaging. Their bright fluorescence allows for optical detection, while their Gd payload enables visualization with contrast-enhanced MRI. A popular approach in molecular MRI is the targeting of abundantly expressed cell surface receptors. Ligand-receptor binding often results in cell internalization of the targeted contrast agent. The interpretation of molecular imaging with pQDs therefore requires knowledge about the consequences of cellular internalization for their relaxometric, optical and compositional properties. To study these, Cd-containing core-shell-shell QDs coated with a monolayer of lipids, of which 50 mol% was a Gd-containing lipid, were incubated with human umbilical vein-derived endothelial cells (HUVECs) for up to 24 h. α(ν) ß(3)-integrin targeted (RGD) and non-targeted (NT) pQDs were compared. pQDs uptake was monitored by fluorescence microscopy, FACS, ICP-MS, relaxometry and MRI. Cell-associated pQDs displayed longitudinal relaxation rates and fluorescent intensities which were linear with the cell-associated Gd and Cd concentrations, implying that the Gd and Cd uptake by HUVECs can be quantified using relaxometric and optical measurements, respectively. However, the Gd-to-Cd molar ratio in pellets of pQD-incubated cells was consistently higher than the Gd-to-Cd molar ratio of the pQDs as prepared. It is proposed that this increase in Gd-to-Cd molar ratio was due to non-specific lipid-transfer between the pQDs and the cellular membranes. These findings show that, in the case of contrast agents that are formed by non-covalent interactions, experimental procedures are needed with which representative components of the probes can be monitored.

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

(31)P 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 (31)P 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 (31)P ST MRS in the resting state.

Temporal effects of mechanical loading on deformation-induced damage in skeletal muscle tissue.

Mechanical loading of soft tissues covering bony prominences can cause skeletal muscle damage, ultimately resulting in a severe pressure ulcer termed deep tissue injury. Recently, by means of an experimental-numerical approach, it was shown that local tissue deformations cause tissue damage once a deformation threshold is exceeded. In the present study, the effects of load exposure time and intermittent load relief on the development of deformation-induced muscle damage were investigated. The data showed that a 2 h loading period caused more damage than 10 min loading. Intermittent load reliefs of 2 min during a 2 h loading period had minimal effect on the evolution of skeletal muscle damage. In addition, a local deformation threshold for damage was found, which was similar for each of the loading regimes applied in this study. For short loading periods, these results imply that local tissue deformations determine whether muscle damage will develop and the exposure time influences the amount of tissue damage. Temporary load reliefs were inefficient in reducing deformation-induced damage, but may still influence the development of ischemia-induced damage during longer loading periods.

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 (1)H magnetic resonance spectroscopy (MRS). Muscle oxidative capacity was assessed in vivo using (31)P 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.

Morphology, binding behavior and MR-properties of paramagnetic collagen-binding liposomes.

Collagen is an important component of the extracellular matrix (ECM) and plays an important role in normal tissue maturation and in pathological processes such as atherosclerosis and myocardial infarction. The diagnostics of the latter diseases using MRI could strongly benefit from the use of collagen-specific contrast agents. The current study aimed to develop a bimodal liposomal MR contrast agent that was functionalized with CNA35, a collagen adhesion protein of the Staphylococcus aureus bacterium. The liposomes were characterized in terms of CNA35 protein conjugation and loading. The overall morphology was assessed with DLS and cryo-TEM, while cryo-TEM tomography was used to visualize the protein coverage of the liposomes. The binding properties of the contrast agent were investigated using a fluorescence assay based on the rhodamine content of the liposomes. The bulk relaxivity was determined using regular relaxometry while the MR-properties of liposomes in their bound state were studied using NMR depth profiling. This CNA35 functionalized contrast agent and the set of in vitro experiments we performed indicate the potential of this technology for in vivo molecular imaging of collagen.

Validation of a numerical model of skeletal muscle compression with MR tagging: a contribution to pressure ulcer research.

Sustained tissue compression can lead to pressure ulcers, which can either start superficially or within deeper tissue layers. The latter type includes deep tissue injury, starting in skeletal muscle underneath an intact skin. Since the underlying damage mechanisms are poorly understood, prevention and early detection are difficult. Recent in vitro studies and in vivo animal studies have suggested that tissue deformation per se can lead to damage. In order to conclusively couple damage to deformation, experiments are required in which internal tissue deformation and damage are both known. Magnetic resonance (MR) tagging and T2-weighted MR imaging can be used to measure tissue deformation and damage, respectively, but they cannot be combined in a protocol for measuring damage after prolonged loading. Therefore, a dedicated finite element model was developed to calculate strains in damage experiments. In the present study, this model, which describes the compression of rat skeletal muscles, was validated with MR tagging. Displacements from both the tagging experiments and the model were interpolated on a grid and subsequently processed to obtain maximum shear strains. A correlation analysis revealed a linear correlation between experimental and numerical strains. It was further found that the accuracy of the numerical prediction decreased for increasing strains, but the positive predictive value remained reasonable. It was concluded that the model was suitable for calculating strains in skeletal muscle tissues in which damage is measured due to compression.

Computational modelling identifies the impact of subtle anatomical variations between amphibian and mammalian skeletal muscle on spatiotemporal calcium dynamics.

The physical sites of calcium entry and exit in the skeletal muscle cell are distinct and highly organised in space. It was investigated whether the highly structured spatial organisation of sites of Ca(2+) release, uptake and action in skeletal muscle cells substantially impacts the dynamics of cytosolic Ca(2+) handling and thereby the physiology of the cell. Hereto, the spatiotemporal dynamics of the free calcium distribution in a fast-twitch (FT) muscle sarcomere was studied using a reaction-diffusion computational model for two genotypes with known anatomical differences. A computational model of a murine FT muscle sarcomere is developed, de novo including a closed calcium mass balance to simulate spatiotemporal high stimulation frequency calcium dynamics at 35 degrees C. Literature data on high-frequency calcium dye measurements were used as a first step towards model validation. The murine and amphibian sarcomere models were phenotypically distinct to capture known differences in positions of troponin C, actin-myosin overlap and calcium release within the sarcomere between frog and mouse. The models predicted large calcium gradients throughout the myoplasm as well as differences in calcium concentrations near the mitochondria of frog and mouse. Furthermore, the predicted Ca(2+) concentration was high at positions where Ca(2+) has a regulatory function, close to the mitochondria and troponin C.

Compression-induced damage and internal tissue strains are related.

Prolonged mechanical loading of soft tissues adjacent to bony prominences can lead to degeneration of muscle tissue, resulting in a condition termed pressure-related deep tissue injury. This type of deep pressure ulcers can develop into a severe wound, associated with problematic healing and a variable prognosis. Limited knowledge of the underlying damage pathways impedes effective preventive strategies and early detection. Traditionally, pressure-induced ischaemia has been thought to be the main aetiological factor for initiating damage. Recent research, however, proposes tissue deformation per se as another candidate for initiating pressure-induced deep tissue injury. In this study, different strain parameters were evaluated on their suitability as a generic predictive indicator for deep tissue injury. With a combined animal-experimental numerical approach, we show that there is a reproducible monotonic increase in damage with increasing maximum shear strain once a strain threshold has been exceeded. This relationship between maximum shear strain and damage seems to reflect an intrinsic muscle property, as it applied across a considerable number of the experiments. This finding confirms that tissue deformation per se is important in the aetiology of deep tissue injury. Using dedicated finite element modeling, a considerable reduction in the inherent biological variation was obtained, leading to the proposal that muscle deformation can prove a generic predictive indicator of damage.

High-resolution NMR imaging of paramagnetic liposomes targeted to a functionalized surface.

A major application of molecular MR imaging is receptor mapping of cells lining blood vessels with targeted contrast agents. Since these agents accumulate at interfaces, knowledge of their influence on the relaxation process in this specific configuration is a prerequisite for understanding their working principle. A methodology is presented to study the influence of targeted contrast agents on surface relaxation in vitro. Paramagnetic liposomes attached to a functionalized surface were studied with high-resolution NMR imaging. The surface was prepared by covering a solid substrate with a layer of collagen. Paramagnetic liposomes were targeted to this surface by functionalizing the liposomes with collagen adhesion protein CNA-35. With a saturation-recovery sequence, 1D magnetization profiles with a resolution of 5 microm were measured in water in contact with the surface. Analytical predictions, obtained with the Bloch-Torrey equation, perfectly agreed with the experimental data. Therefore, the magnitude of the surface relaxation rate could be determined from the measurements without any assumption. By using the relaxivity of liposome solutions the surface coverage by liposomes could be estimated. With the presented methodology the behavior of Gd-based targeted contrast agents at biological interfaces can be studied in vitro. Their influence on relaxation processes can be characterized and quantified.

Early or advanced stage type 2 diabetes is not accompanied by in vivo skeletal muscle mitochondrial dysfunction.

OBJECTIVE: Several lines of evidence support a potential role of skeletal muscle mitochondrial dysfunction in the pathogenesis of insulin resistance and/or type 2 diabetes. However, it remains to be established whether mitochondrial dysfunction represents either cause or consequence of the disease. We examined in vivo skeletal muscle mitochondrial function in early and advanced stages of type 2 diabetes, with the aim to gain insight in the proposed role of mitochondrial dysfunction in the aetiology of insulin resistance and/or type 2 diabetes. METHODS: Ten long-standing, insulin-treated type 2 diabetes patients, 11 subjects with impaired fasting glucose, impaired glucose tolerance and/or recently diagnosed type 2 diabetes, and 12 healthy, normoglycaemic controls, matched for age and body composition and with low habitual physical activity levels were studied. In vivo mitochondrial function of the vastus lateralis muscle was evaluated from post-exercise phosphocreatine (PCr) recovery kinetics using (31)P magnetic resonance spectroscopy (MRS). Intramyocellular lipid (IMCL) content was assessed in the same muscle using single-voxel (1)H MRS. RESULTS: IMCL content tended to be higher in the type 2 diabetes patients when compared with normoglycaemic controls (P=0.06). The(31)P MRS parameters for mitochondrial function, i.e. PCr and ADP recovery time constants and maximum aerobic capacity, did not differ between groups. CONCLUSIONS: The finding that in vivo skeletal muscle oxidative capacity does not differ between long-standing, insulin-treated type 2 diabetes patients, subjects with early stage type 2 diabetes and sedentary, normoglycaemic controls suggests that mitochondrial dysfunction does not necessarily represent either cause or consequence of insulin resistance and/or type 2 diabetes.