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DOI: 10.1530/EJE-07-0756
European Journal of Endocrinology, Vol 158, Issue 5, 643-653
Copyright © 2008 by European Society of Endocrinology
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CLINICAL STUDIES

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

H M De Feyter, N M A van den Broek, S F E Praet1, K Nicolay, L J C van Loon1 and J J Prompers

Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands1 Department of Movement Sciences, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands

(Correspondence should be addressed to J J Prompers; Email: j.j.prompers{at}tue.nl)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 31P magnetic resonance spectroscopy (MRS). Intramyocellular lipid (IMCL) content was assessed in the same muscle using single-voxel 1H MRS.

Results: IMCL content tended to be higher in the type 2 diabetes patients when compared with normoglycaemic controls (P=0.06). The31P 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.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Insulin resistance is an early event in the pathogenesis of type 2 diabetes. However, the exact processes leading to insulin resistance remain unresolved. Previous studies have reported a strong correlation between intramyocellular lipid (IMCL) content and insulin resistance (1, 2). However, the proposed relationship between IMCL accumulation and skeletal muscle insulin resistance is not unambiguous, as it is strongly influenced by training status and/or habitual physical activity (3, 4). Nonetheless, several lines of evidence indicate that mitochondrial dysfunction, presumably associated with a reduced capacity to oxidize fatty acids, might stimulate IMCL accretion and, as such, contribute to the development of skeletal muscle insulin resistance (5).

Data to support the proposed role of skeletal muscle mitochondrial dysfunction in the development of insulin resistance and/or type 2 diabetes have been obtained with various in vitro methods, including measurements of oxidative enzyme activities (6, 7, 8, 9, 10), mRNA and/or protein expression of oxidative phosphorylation genes (10, 11, 12, 13, 14) as well as mitochondrial content, morphology and respiration (8, 9, 14, 15). Furthermore, in vivo magnetic resonance spectroscopy (MRS) measurements of basal mitochondrial ATP synthesis rates (16, 17, 18) and post-exercise phosphocreatine (PCr) (19) and ADP (20) recovery kinetics also point towards a potential role for mitochondrial dysfunction in the aetiology of insulin resistance and/or type 2 diabetes. However, it was recently shown that in vitro mitochondrial respiration of permeabilized muscle fibres from biopsies of the type 2 diabetes patients and healthy controls did not differ between groups when the data were normalized for mitochondrial DNA content or citrate synthase activity (21). In other words, the type 2 diabetes patients showed normal intrinsic mitochondrial function, but an impaired oxidative capacity that was entirely attributed to a lower mitochondrial content. Environmental factors play an important role in regulating skeletal muscle oxidative capacity, and the lower mitochondrial content in the type 2 diabetes patients might simply be the result of a reduced habitual physical activity level (21, 22, 23). Furthermore, mitochondrial dysfunction in type 2 diabetes might be secondary to impaired insulin signalling (24, 25, 26) and/or abnormal blood glucose, insulin (27, 28) and non-esterified fatty acid (NEFA) (26) levels. Therefore, the debate continues as to whether mitochondrial dysfunction represents either cause or consequence of insulin resistance and/or type 2 diabetes.

The aim of this study was to assess whether in vivo mitochondrial function is impaired in the early and/or overt diabetes state. Therefore, we examined in vivo muscle mitochondrial function in three groups of subjects, representative for different stages in the development of type 2 diabetes: long-standing, insulin-treated type 2 diabetes patients, subjects with impaired fasting glucose, impaired glucose tolerance and/or recently diagnosed type 2 diabetes, and healthy, normoglycaemic controls, all matched for age and body composition and with low habitual physical activity levels. In vivo mitochondrial function was determined from post-exercise PCr recovery kinetics measured with 31P MRS (29, 30, 31).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Subjects

Ten long-standing, insulin-treated type 2 diabetes patients (Diabetes group), 11 subjects with impaired fasting glucose, impaired glucose tolerance and/or recently diagnosed type 2 diabetes (pre-Diabetes group), and 12 healthy, normoglycaemic controls (Control group) were selected to participate in this study. All subjects were male. The diabetes patients had been diagnosed with type 2 diabetes for over 5 years, established by a fasting plasma glucose concentration larger than or equal to 7.0 mmol/l at the time of diagnosis as defined by the World Health Organization (WHO) (32). Subjects in the Control and pre-Diabetes groups had no family history of diabetes and were selected based on an oral glucose tolerance test (OGTT) according to the WHO criteria (32). Control subjects showed normal fasting glucose concentrations and normal glucose tolerance (fasting glucose <6.1 mmol/l and 2-h glucose <7.8 mmol/l). Subjects in the pre-Diabetes group had either elevated fasting plasma glucose concentrations (fasting glucose ≥6.1 and <7.0 mmol/l, and 2-h glucose <7.8 mmol/l; n=5), or impaired glucose tolerance (fasting glucose <7.0 mmol/l, and 2-h glucose ≥7.8 and <11.1 mmol/l; n=2), or recently diagnosed type 2 diabetes (<1 month; fasting glucose ≥7.0 mmol/l or 2-h glucose ≥11.1 mmol/l; n=4).

All diabetes patients were on exogenous insulin treatment and had been on a stable regimen of diabetes medication over the last 3 months before being recruited. Out of the ten participating diabetes patients, seven patients were treated with short (Novorapid®, n=6) or rapid acting insulin (Humulin®, n=1) before each meal either in combination with NPH insulin (Insulatard®, n=5), premixed biphasic isophane insulin (Mixtard 30/70® in combination with metformin, n=1), or a very long-acting insulin analogue (insulin glargine, n=1), all administered before bedtime. Two patients were treated with premixed biphasic isophane insulin (Mixtard 30/70®) twice a day in combination with metformin. One patient used NPH insulin (Humulin NPH®) once a day before breakfast in combination with metformin and a sulphonylurea (glimepiride). Patients using thiazolidinediones were excluded from participation. None of the subjects in the pre-Diabetes group used blood glucose-lowering medication and all showed glycosylated haemoglobin (HbA1c) contents below 6%. Subjects using β-blockers for less than 6 months and subjects with impaired liver function, renal failure, severe retinopathy or a history of severe cardiovascular problems were excluded from participation. Subjects with clinical signs of peripheral vascular disease (Fontaine stage II or higher) were excluded based on a thorough history considering signs of intermittent claudication, as well as a physical examination of the peripheral vascular system (no arterial bruits over the abdominal aorta, iliac artery and/or diminished pulses of femoral artery, dorsalis pedis and posterior tibial artery). The nature and the risks of the experimental procedures were explained to the subjects and all gave their written informed consent to participate in the study, which was approved by the local Medical Ethical Committee of the Máxima Medical Centre, Veldhoven, The Netherlands. This study is part of a larger project that studies mitochondrial function in chronic metabolic disease (33, 34).

Body composition

Body mass index (BMI) and waist circumference were measured using an analogue weight scale and standard measuring tape. Whole-body fat-free mass (FFM) and truncal fat mass were determined using whole-body dual energy X-ray absorptiometry (Hologic QDR-4500 Discovery A, software version 12.3.3, Hologic Inc., Bedford, MA, USA).

Habitual physical activity level

Habitual physical activity level was assessed with the Tecumseh and Minnesota occupational and leisure time activity questionnaire (35). The activity level was expressed in metabolic equivalents (MET), which is a scale of the energy cost of various physical activities in multiples of the resting metabolic rate.

Whole-body oxygen uptake capacity

Maximal whole-body oxygen uptake capacity (VO2peak) and maximal workload capacity (Wmax) were measured during an incremental exercise test until exhaustion, performed on a cycle ergometer (Medifit Ergometer, Medifit systems, Maarn, The Netherlands) using a ramp protocol (36). Gas exchange measurements were performed continuously (Ergostar II, PMS Professional Medical Systems, Basel, Switzerland). Maximal whole-body oxygen uptake capacity was defined as the VO2 value remaining unchanged or increasing at <1 ml/min per kg for 30 s or more despite an increment in workload (37). Cardiac function was monitored using a 12-lead electrocardiogram with heart rate being recorded continuously (Polar Electro, Kempele, Finland). The age-predicted maximal heart rate was calculated according to the prediction model of Gellish et al. (38).

Blood sampling and analyses

Subjects reported at the laboratory at 0800 h after an overnight fast. After 5–10 min of supine rest, fasting samples of venous blood were collected from an antecubital vein. Subsequently, a standard OGTT was performed for all subjects, except for the diabetes patients, and blood samples were collected 2 h after ingestion of the glucose load. Blood plasma samples were collected into EDTA-containing tubes and centrifuged for 10 min at 4 °C. Aliquots of plasma were frozen immediately in liquid nitrogen and stored at –80 °C until further analyses. Plasma concentrations of glucose (Roche) and NEFA (Wako Chemicals, Neuss, Germany) were analysed with a COBAS semi-automatic analyzer (Roche). Plasma insulin was determined in duplicate by RIA (Linco, St Charles, MO, USA) for Control and pre-Diabetes groups. Cross sensitivity of exogenously administered insulin with this RIA prohibited the detection of endogenously produced insulin in the insulin-treated diabetes patients. The blood HbA1c content was analysed by HPLC (Bio-Rad Diamat). For the Control and pre-Diabetes groups, the homeostasis model assessment (HOMA) index (39) was calculated.

MRS measurements

MRS measurements were performed with an 1.5 T whole-body scanner (Gyroscan S15/ACS, Philips Medical Systems, Best, The Netherlands) during two sessions. On the evening before the first MRS session, subjects received a standardized meal (mean±S.D.: 41.0±15.1 kJ per kg body weight, containing 40.7 energy% (En%) fat, 14.6 En% protein and 44.7 En% carbohydrate) after which subjects remained fasted and were allowed to drink water only. Subjects travelled by car or public transport and reported at the laboratory at 0830 h, where they received a standardized breakfast. First, 1H MRS measurements were performed for the quantification of IMCL. After a short break, the 31P MRS protocol was carried out to familiarize the subjects to the in-magnet exercise and to determine the optimal exercise intensity for the second visit. During the second MRS session, scheduled within 1 week, 31P MRS measurements were performed to assess skeletal muscle mitochondrial function. For the diabetes patients, blood glucose-lowering medication was not withdrawn before the MRS measurements.

1H MRS

IMCL was measured in the Musculus vastus lateralis with image-guided single-voxel 1H MRS using the body coil for transmission and an 8 cm diameter surface coil for signal reception. For each subject, five voxels with a size of 10x10x15 mm3 were measured at different positions within the M. vastus lateralis. The voxels were carefully placed to avoid subcutaneous and visible interstitial fat using standard T1-weighted images (Fig. 1A). Spectra were recorded with a point-resolved spectroscopy (PRESS) sequence (repetition time, 1500 ms; echo time, 35 ms; spectral width, 2000 Hz; number of data points, 2048; 128 averages) using chemical-shift-selective saturation for water suppression. Unsuppressed water spectra (32 averages) were recorded from the same voxels and used as an internal reference.


Figure 1
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  		Figure 1 (A) Transversal T1-weighted spin-echo image of the upper leg of a type 2 diabetes patient with voxel positioning within the M. vastus lateralis for single-voxel 1H MRS. The size of the voxel was 10x10x15 mm3. The arrow points at a phantom that was located in the centre of the 8 cm diameter surface coil. (B) 1H MR spectrum from the voxel depicted in (A). The spectrum was processed with 1 Hz line broadening. Peak annotations: tCr CH2/CH3, CH2/CH3 protons of total creatine; TMA, CH3 protons of trimethyl ammonium groups; EMCL CH2/CH3, CH2/CH3 protons of extramyocellular lipid, IMCL CH2/CH3, CH2/CH3 protons of intramyocellular lipid.

 
1H MRS data analysis

All spectra were fitted in the time domain by using a non-linear least-squares algorithm (AMARES) in the jMRUI software package (40) without further post-processing, except for manual phasing. The unsuppressed water spectrum was phased and fitted to a Lorentzian line shape. The zero-order phase correction from the water spectrum was applied to the corresponding water-suppressed spectrum and the total creatine (tCr) CH3 peak was referenced to 3.02 ppm. In the water-suppressed spectrum, peaks from trimethyl ammonium and tCr CH3, extramyocellular lipid (EMCL) and IMCL CH2, and EMCL and IMCL CH3 protons (see Fig. 1B) were fitted to the Gaussian line shapes. The positions and areas of the EMCL and IMCL CH3 peaks were constrained with respect to the positions and areas of the EMCL and the IMCL CH2 peaks respectively (41). To increase the reliability of the fit, the linewidth of the IMCL CH2 peak (LWIMCL) was constrained with respect to the linewidth of the water peak Formula according to


Formula

This constraint was derived from previously recorded data sets with a well-resolved IMCL peak (number of data sets=20, R=0.821, P<0.0001). A soft constraint that was empirically determined from the previously recorded data sets, was applied to the linewidth of the EMCL CH2 peak (LWEMCL), i.e. LWEMCL≤LWIMCL+3.33 Hz. When the resonance frequency ({delta}) of the IMCL CH2 peak could not be accurately fitted (i.e. {delta}≤1.25 ppm or {delta}≥1.31 ppm), the position of the IMCL CH2 peak was constrained with respect to the position of the tCr CH3 peak (chemical shift difference=3.02–1.28 ppm). IMCL was expressed as a percentage of the water signal measured in the same voxel without water suppression. The IMCL levels determined from different voxels of one subject were averaged.

31P MRS

31P MRS was performed as described previously (33). In short, 31P signals were collected using a 6 cm diameter surface coil placed over the M. vastus lateralis (spectral width, 2000 Hz; number of data points, 1024). A fully relaxed spectrum was measured at rest with a repetition time of 30 s and 24 scans. Then, spectra were acquired during a rest–exercise–recovery protocol with a repetition time of 3 s and two scans yielding a time resolution of 6 s. The first 20 spectra (2 min) were measured at rest, after which the subjects started the exercise. Subjects performed a dynamic incremental single-leg extension exercise in the supine position using a home-built MR-compatible ergometer (33). The duration of the exercise varied per subject, but never exceeded 8 min, so that at least 5 min of recovery were recorded. During the first visit, subjects performed a test run and exercised until fatigued. During the second session, the duration of the exercise was chosen to deplete PCr by about 50%, while aiming to avoid the intracellular pH dropping below 6.8.

31P MRS data analysis

The 31P MRS data were analysed as described previously (33). In short, spectra were fitted in the time domain by using a non-linear least-squares algorithm (AMARES) in the jMRUI software package (40). PCr, inorganic phosphate (Pi), ATP and phosphodiester (PDE) signals were fitted to the Lorentzian line shapes. Absolute concentrations of the phosphorylated metabolites were calculated after correction for partial saturation and assuming that the ATP concentration is 8.2 mM at rest (42). Intracellular pH was calculated from the chemical shift difference between the Pi and PCr resonances (43). The free cytosolic ADP concentration was calculated from the PCr concentration and pH using a creatine kinase equilibrium constant (Keq) of 1.66x109/M (44). Recoveries of PCr and ADP were fitted to mono-exponential functions using Matlab (version 7.30., Mathworks, Natick, MA, USA). Results are expressed as the metabolite's time constant of recovery, i.e. {tau}PCr and {tau}ADP , both of which are measures of the mitochondrial function (43, 45, 46). Calculation of the initial rate of PCr recovery (VPCr) was based on the PCr recovery rate (1/{tau}PCr) and the difference between the resting and end-exercise PCr concentrations (47). Calculation of the maximum aerobic capacity (Qmax) was based on the ADP-control model (48), in which VPCr has a hyperbolic dependence on the end-exercise ADP concentration according to Michaelis–Menten kinetics with a Km of 30 µM (47).

Statistical analysis

All data are expressed as means±S.D. Statistical analyses were performed using the SPSS 14.0 software package (SPSS Inc., Chicago, IL, USA). An ANOVA test was performed to test for overall differences between the Control, pre-Diabetes and Diabetes groups and a Bonferroni post hoc test was applied for pairwise comparisons between the three groups. For comparisons between two groups, differences were determined with an unpaired t-test. To evaluate the relationships between variables, Pearson correlation coefficients were calculated. All tests were carried out in a two-sided way and the level of significance was set at P<0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Subjects' characteristics

The subjects' characteristics are shown in Table 1. Subjects were carefully matched for age and BMI. In accordance, FFM and truncal fat mass did not differ between the Control, pre-Diabetes and Diabetes groups (P=0.29 and P=0.87 respectively). Wmax and VO2peak were similar for the Control and pre-Diabetes groups (P=1.00), but significantly lower in the Diabetes group. The latter was paralleled by a lower maximal heart rate and a lower habitual physical activity level, but the physical activity levels were not significantly different between groups (P=0.12). Wmax and VO2peak correlated with both maximal heart rate (Pearson's R between 0.59 and 0.68, P<0.01) and percentage of the age-predicted maximal heart rate (Pearson's R between 0.56 and 0.62, P<0.01).


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  		Table 1 Subjects' characteristics.

 
The fasting plasma glucose and insulin levels were not significantly different for the Control and pre-Diabetes groups (P=0.39 and P=0.14 respectively), but the HOMA index was significantly higher for the pre-Diabetes group than for the Control group. Also, the glucose and insulin concentrations measured 2 h after ingestion of the glucose load during the OGTT were significantly higher for the pre-Diabetes group than for the Control group. The HbA1c contents were similar for the Control and pre-Diabetes groups (P=1.00) and all subjects had HbA1c contents below 6%. The fasting glucose concentrations and HbA1c contents were significantly higher in the Diabetes group (glucose ≥8 mmol/l and HbA1c ≥6.3%) when compared with the Control and pre-Diabetes groups. Diabetes patients had been diagnosed with type 2 diabetes for 12±7 years and had been on exogenous insulin therapy for 7±8 years. Plasma NEFA concentrations did not differ between groups (P=0.16).

Wmax and VO2peak correlated negatively with both fasting plasma glucose (Pearson’s R between –0.48 and –0.57, P<0.01) and HbA1c (Pearson's R between –0.58 and –0.70, P<0.01).

IMCL content

Due to excessive EMCL contamination in the spectra, IMCL content could not be determined for two subjects in the Control group and one subject in the Diabetes group. For all other subjects, spectra from at least two out of the five voxels were quantified. IMCL content tended to be higher for the Diabetes group when compared with the Control group (1.3±0.4 (n=10), 1.6±0.7 (n=11) and 2.0±0.7 (n=9) % of the water signal in Control, pre-Diabetes and Diabetes groups respectively, P=0.06; Fig. 2).


Figure 2
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  		Figure 2 IMCL content in Control, pre-Diabetes and Diabetes groups. Bars indicate the mean values for the three groups and filled circles represent the individual data points. IMCL content tended to be higher for the Diabetes group when compared with the Control group (ANOVA, P=0.06).

 
Skeletal muscle mitochondrial function

Figure 3A and B show typical examples of 31P MR spectra from a subject's vastus lateralis muscle at rest and at the end of exercise respectively. Table 2 summarizes the baseline, end-exercise and recovery 31P MRS results for the Control, pre-Diabetes and Diabetes groups. At rest, PCr, Pi, ADP and PDE concentrations and intracellular pH were not significantly different for the Control, pre-Diabetes and Diabetes groups (P=0.93, P=0.50, P=0.09, P=0.42 and P=0.10 respectively). The end-exercise status has to be taken into account when analysing the recovery data. The end-exercise metabolite concentrations were similar for the three groups, except for the end-exercise ADP concentration, which was lower in the Diabetes group. However, for all groups, the end-exercise ADP concentration was well above the accepted Km value of 30 µM for oxidative ATP synthesis. The average PCr depletion was 54±10, 56±7 and 46±8% for Control, pre-Diabetes and Diabetes groups respectively. For none of the subjects did the end-exercise pH drop below 6.75. The average end-exercise pH was not different for the three groups (P=0.72), which is a prerequisite for comparing {tau}PCr.


Figure 3
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  		Figure 3 Typical M. vastus lateralis 31P MR spectra for a normoglycaemic control subject at rest (A, number of scans=60) and at the end of exercise (B, number of scans=2). Spectra were processed with 5 Hz line broadening. Pi, inorganic phosphate; PDE, phosphodiesters; PCr, phosphocreatine; {alpha}, β and {gamma}, the three phosphate groups of ATP. For this subject, the PCr depletion at the end of exercise (B) was 61% and the corresponding end-exercise pH was 6.96.

 

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  		Table 2 31P MRS parameters during rest, end of exercise and recovery.

 
The PCr and ADP recoveries could be satisfactorily described by mono-exponential functions (average R2 values for the mono-exponential fits were 0.96±0.02 and 0.91±0.06 for PCr and ADP recovery data respectively). Figure 4 illustrates both the raw data and mono-exponential fits of the PCr and ADP recoveries for one subject. Figure 5 shows the mean values and the distribution of {tau}PCr and Qmax for the Control, pre-Diabetes and Diabetes groups. The 31P MRS parameters for mitochondrial function, i.e. {tau}PCr , {tau}ADP and Qmax, did not differ between the Control, pre-Diabetes and Diabetes groups (P=0.62, P=0.29 and P=0.24 respectively; Table 2). VPCr was significantly lower for the Diabetes group when compared with the pre-Diabetes group as a result of the lower end-exercise ADP concentration.


Figure 4
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  		Figure 4 (A) PCr and (B) ADP recovery curves from the data set that were also used in Fig. 3. Mono-exponential functions (dark lines) were fit to the actual data (filled circles) obtained every 6 s. The time constants for PCr and ADP recovery were 41.8 and 19.8 s respectively.

 

Figure 5
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  		Figure 5 (A) {tau}PCr and (B) Qmax in Control, pre-Diabetes and Diabetes groups. Bars indicate the mean values for the three groups and filled circles represent the individual data points. {tau}PCr and Qmax were not significantly different between Control, pre-Diabetes and Diabetes groups (ANOVA, P=0.62 and P=0.24 respectively).

 
All 31P MRS parameters for mitochondrial function correlated significantly with both Wmax (absolute Pearson's R between 0.45 and 0.60, P<0.01) and VO2peak (absolute Pearson's R between 0.35 and 0.59, P<0.05). However, {tau}PCr, {tau}ADP and Qmax did not correlate with fasting plasma glucose or HbA1c.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study, it was shown that in vivo skeletal muscle oxidative capacity, as determined from post-exercise PCr recovery kinetics using 31P MRS, does not differ between long-standing, insulin-treated type 2 diabetes patients, subjects with early stage type 2 diabetes and healthy, normoglycaemic controls, all matched for age and body composition and with low habitual physical activity levels. Therefore, our results suggest that skeletal muscle mitochondrial dysfunction does not necessarily represent either cause or consequence of type 2 diabetes.

Maximal workload capacity and maximal whole-body oxygen uptake capacity were significantly lower in the type 2 diabetes patients and correlated negatively with both plasma glucose and blood HbA1c contents. The 31P MRS recovery parameters, {tau}PCr, {tau}ADP and Qmax, correlated significantly with Wmax and VO2peak, but not with plasma glucose or blood HbA1c levels. Therefore, Wmax and VO2peak seem to represent markers of the disease status, whereas {tau}PCr, {tau}ADP and Qmax report only on local muscle mitochondrial function. The Pearson correlation coefficient for the correlation between Qmax and VO2peak per kg FFM (VO2peak per kg FFM) was 0.47 (R2=0.22). Therefore, only 22% of the variance in VO2peak per kg FFM can be explained by Qmax. Qmax was measured locally in the M. vastus lateralis during a dynamic single-leg extension exercise, while whole-body VO2peak was determined on a cycle ergometer. The remaining variance in VO2peak per kg FFM could be accounted for by differences in cardiovascular capacity at peak work rates that are not expected to play a significant role during the local exercise in the MR scanner. The apparent discrepancy between the results for Qmax and VO2peak per kg FFM might therefore be explained by differences in cardiac output. The latter concept seems to be supported by a significant correlation between the maximal heart rate and VO2peak per kg FFM (Pearson's R=0.64, P<0.01).

In this study, 31P MRS was applied to assess in vivo skeletal muscle mitochondrial function from measurements during recovery from exercise. During recovery from exercise, PCr is resynthesized purely as a consequence of oxidative ATP synthesis (43, 45). Therefore, {tau}PCr provides information about mitochondrial function. Recently, Schrauwen-Hinderling et al. applied the same technique to study mitochondrial function in the M. vastus lateralis in overweight type 2 diabetes patients and BMI-matched control subjects (19). Contrary to our results, {tau}PCr was 45% longer in the type 2 diabetes group when compared with the control group (i.e. 39.4±17.5 vs 27.0±3.9 s), suggestive of impaired in vivo mitochondrial function in the type 2 diabetes patients. It should be noted that different exercise protocols were chosen for the 31P MRS measurements in the two studies. We applied an exercise protocol that progressively increased work to deplete PCr by about 50% without severe acidification. In the previous study, exercise was performed with a single load, until a steady state was reached with an average PCr depletion of 28%. The larger PCr depletion in the current study was accompanied by a slightly larger drop in pH (change in pH was on average 0.16 in this study versus 0.04 in (19)). Several studies have shown that PCr recovery is slowed down in the presence of intracellular acidosis (30, 49, 50). However, we carefully matched the end-exercise pH for the Control, pre-Diabetes and Diabetes groups, to allow a direct comparison of {tau}PCr between groups. As an alternative to {tau}PCr, both {tau}ADP and Qmax can also be used to assess in vivo mitochondrial function and these parameters have been shown to be independent of pH (30, 50, 51, 52, 53). In accordance with the {tau}PCr data matched for end-exercise pH, {tau}ADP and Qmax also did not differ between groups.

As suggested in the literature, {tau}PCr can be normalized for pH using a correction factor of –46 s per pH unit (49). Recalculating the values from Schrauwen-Hinderling et al. to a 0.12 lower end-exercise pH results in {tau}PCr's of about 45 and 32 s for the type 2 diabetes and control group respectively. This implies that the mitochondrial function in our Control, pre-Diabetes and Diabetes groups is similar to that of the type 2 diabetes patients in the study of Schrauwen-Hinderling et al., but that the controls in the latter study show a greater mitochondrial oxidative capacity. For other studies in healthy, elderly subjects previously reported in the literature (54, 55, 56), average values for {tau}PCr (recalculated at an end-exercise pH of 6.9 if necessary) have been shown to range between 43 and 46 s, which is well in line with the average {tau}PCr in our Control group. As such, muscle mitochondrial function in these overweight normoglycaemic control subjects does not seem to be substantially impaired.

As an alternative to measuring muscle oxidative capacity from dynamic 31P MRS experiments after exercise, Petersen et al. applied 31P MRS saturation transfer experiments to measure mitochondrial ATP synthesis rates in resting skeletal muscle of healthy, young, lean, insulin-resistant offspring of type 2 diabetes patients and insulin-sensitive control subjects matched for age, height, weight and physical activity (17). ATP synthesis rates were ~30% lower in insulin-resistant subjects than in controls and it was concluded that the insulin-resistant offspring might have an inherited defect in mitochondrial phosphorylation. However, as commented by Short et al. (24) and Wagenmakers (25), the lower ATP synthesis rates in insulin-resistant subjects could actually be caused by the impaired insulin signalling, jeopardizing insulin-dependent mitochondrial processes, rather than the reverse. This view is supported by studies that show that high-dose insulin infusions increase mRNA transcript levels of genes involved in mitochondrial function, mitochondrial protein synthesis and mitochondrial ATP production rates in healthy people, but not in the type 2 diabetes patients (28, 57, 58). In summary, it seems likely that the decreased basal ATP synthesis rates in insulin resistant subjects are a result of the decreased insulin sensitivity and do not necessarily reflect any intrinsic mitochondrial defect.

The diabetes patients in the current study were on exogenous insulin treatment for more than 5 years and continued their medication during the study. As blood glucose and insulin levels can affect measurements of mitochondrial function (27, 28), the higher plasma insulin levels in the Diabetes group as a result of exogenous insulin treatment might be a confounding factor in this study. Despite the insulin treatment, our diabetes patients were still hyperglycaemic (fasting plasma glucose concentration: 11.0±2.5 mmol/l). Therefore, hyperglycaemia might also have affected the measurement of in vivo muscle mitochondrial function in the diabetes patients.

Rabøl et al. recently reviewed the experimental data on mitochondrial dysfunction in type 2 diabetes and concluded that evidence of an intrinsic defect in the mitochondria of type 2 diabetes patients is far from convincing (23). Considering that type 2 diabetes patients are generally physically inactive, the impairments in oxidative metabolism in type 2 diabetes patients might simply be attributed to their sedentary lifestyle. In this regard, it is important to note that in most studies physical activity has not been (strictly) controlled for. For studies in which physical activity was taken into account, the results suggest that the abnormalities in oxidative metabolism in type 2 diabetes patients can at least partly be attributed to physical inactivity (14, 28, 59, 60). In accordance, recent data from respiration measurements on permeabilized muscle fibres show that when O2 flux is being normalized for mitochondrial DNA content or citrate synthase activity, no differences in mitochondrial respiration rate are observed between type 2 diabetes patients and healthy controls (21). These results imply that type 2 diabetes patients have normal intrinsic mitochondrial function, but an impaired oxidative capacity due to a reduced mitochondrial content, most likely secondary to lower habitual physical activity levels (21, 22). More recently, Turner et al. examined markers of muscle mitochondrial fatty acid oxidative capacity in rodent models of lipid-induced insulin resistance (61). Surprisingly, fatty acid oxidative capacity and protein expression of peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} and mitochondrial respiratory chain subunits appeared to be upregulated. As such, the authors concluded that, at least in these rodent models, high lipid availability does not lead to intramuscular lipid accumulation and insulin resistance by decreasing muscle mitochondrial fatty acid oxidative capacity. In accordance, in the current study we observed no differences in in vivo muscle mitochondrial function between long-standing type 2 diabetes patients, subjects with early stage type 2 diabetes and sedentary, normoglycaemic controls.

In conclusion, subjects with early stage type 2 diabetes as well as long-standing, insulin-treated type 2 diabetes patients do not show signs of in vivo skeletal muscle mitochondrial dysfunction. The latter implies that mitochondrial dysfunction does not necessarily represent either cause or consequence of insulin resistance and/or type 2 diabetes. Impairments in oxidative metabolism in type 2 diabetes patients observed in previous studies are likely to be secondary to a less active lifestyle and/or impaired insulin signalling.


   Acknowledgements
 
We are very grateful to Larry de Graaf for his technical assistance with the MR scanner.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received 29 January 2008
Accepted 4 February 2008




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