Diabetes has reached epidemic proportions worldwide. Type 2 diabetes (T2D) accounts for about 90% of all diabetes cases and is characterized by insulin resistance (IR) in major metabolic tissues. The dramatic rise in T2D is associated with the increased occurrence of obesity and excessive ectopic lipid accumulation, in particular in skeletal muscle, due to excessive caloric intake and decreased physical activity. However, the exact processes leading to IR remain unresolved. One of the leading hypotheses in the research field of type 2 diabetes is that inherited or acquired skeletal muscle mitochondrial dysfunction, associated with a reduced mitochondrial capacity to oxidize fatty acids (FAs), leads to a lipid overload in muscle cells. High levels of lipid intermediates then induce IR by activating a protein kinase, which impairs insulin signaling by phosphorylation of serine/threonine sites on the insulin receptor. Recent studies have challenged this theory and link insulin resistance to an increased capacity to oxidize FAs rather than the reverse. The high ¿-oxidation rates observed in insulin-resistant muscle are associated with low rates of complete FA oxidation and elevated incomplete ¿-oxidation. It has been hypothesized that the accumulation of incompletely metabolized FAs in the mitochondria causes ‘mitochondrial stress’ leading to insulin resistance. To clarify this issue, the timing and nature of muscle mitochondrial adaptations during the development of IR and T2D have been investigated in this thesis using both in vivo and in vitro approaches. The ambiguous results on the role of muscle mitochondrial dysfunction in T2D may be due to the different measurement methods used to determine muscle mitochondrial function. 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. 31P magnetic resonance spectroscopy (MRS) provides a non-invasive 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). 31P MRS has been used to assess skeletal muscle mitochondrial function in vivo by measuring (1) resting ATP synthesis flux with saturation transfer (ST) or (2) the rate of PCr recovery after exercise. However, both methods measure completely different parameters, i.e. basal metabolic rate and maximal oxidative capacity, respectively, which might explain the contradictory results that have been obtained. In chapter 2, we compared both parameters in rats treated with the complex I inhibitor diphenyleneiodonium (DPI) with the aim of establishing the most appropriate method for the assessment of in vivo muscle mitochondrial function. In vivo 31P 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 post-exercise 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 the rate constant of PCr recovery measured with dynamic 31P MRS after exercise provides a more sensitive measure of skeletal muscle mitochondrial function than the ATP synthesis flux determined with 31P ST in the resting state. The ATP synthesis flux itself represents the ATP demand of the muscle and in order to interpret the data in terms of mitochondrial function it is necessary to take the error signals, i.e. the concentrations of ADP and Pi, into account. Moreover, the Pi ¿ ATP flux obtained from a 31P ST experiment in the resting state is dominated by glycolytic exchange flux. The second methodological chapter (chapter 3) describes the pH dependence of the PCr recovery time constant (31PCr, i.e. the inverse of the PCr recovery rate constant). It has been shown that muscle tissue acidification at the end of exercise severely prolongs PCr recovery, which is an important complication in the interpretation of post-exercise PCr recovery data. It has been proposed that tPCr can be normalized for the end-exercise pH, in order to use it as a measure for mitochondrial function. However, a general correction for pH can only be applied if there are no intersubject differences in the pH dependence of PCr recovery kinetics. In this chapter the pH dependence of tPCr was investigated in healthy human volunteers on a subject-by-subject basis and it turned out that the effect of acidosis on PCr recovery kinetics after exercise was different between subjects. The pH dependence of tPCr correlated with the proton efflux rate at the start of recovery, indicating that subjects with a smaller pH dependence of tPCr have a higher rate of pH recovery. Therefore, tPCr can only be used as a measure of mitochondrial function when end-exercise pH is close to resting values. Simply correcting tPCr for end-exercise pH is not adequate, in particular when comparing patients and controls, as certain disorders are characterized by altered proton efflux from muscle fibers. In chapters 4-6, 31P MRS was applied to investigate the role of muscle mitochondrial dysfunction in the development of IR and T2D. Chapter 4 describes a cross-sectional human study, in which we examined in vivo skeletal muscle mitochondrial function in healthy, normoglycaemic controls, subjects with early stage T2D and long-standing, insulin-treated T2D patients. It was shown that PCr recovery after exercise was not different between groups, indicating that in vivo oxidative capacity was not impaired in both early and late stages of T2D. These results imply that mitochondrial dysfunction does not necessarily represent either cause or consequence of IR and/or T2D. It was suggested that 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 signaling. A cross-sectional study, as presented in chapter 4, only provides correlative data and does not provide detailed information about the time course of events during the development of T2D. Therefore, the aim of the next study, described in chapter 5, was to gain more insight into the timing and nature of mitochondrial adaptations during the development of high-fat diet-induced insulin resistance in rats. Adult Wistar rats were fed a high-fat diet or normal chow for 2.5 and 25 weeks. Muscle oxidative capacity was assessed in vivo from 31P MRS measurements of PCr recovery and in vitro by measuring mitochondrial DNA copy number and oxygen consumption in isolated mitochondria. Muscle lipid status was determined by 1H MRS (intramyocellular lipids, IMCL) and tandem mass spectrometry (acylcarnitines). The short-term high-fat diet induced increases in IMCL content and muscle medium- and long-chain acylcarnitines, together with an increased in vivo oxidative capacity. The latter result could be fully accounted for by increased mitochondrial content. The long-term high-fat diet resulted in even higher IMCL and acylcarnitine levels, a further increase in the number of muscle mitochondria and an increased capacity to oxidize fat-derived substrates in vitro. Surprisingly, this did not result in a higher muscle oxidative capacity in vivo. These findings show that skeletal muscle in high-fat diet-induced IR requires a progressively larger mitochondrial pool size to maintain normal oxidative capacity in vivo. Comparable to the results of the 25 wk high-fat diet are the results observed in Wistar rats on a similar high-fat diet for 15 wk, as presented in chapter 6. The observed dissociation between in vivo and in vitro determinations of muscle mitochondrial function after long-term high-fat diet feeding in both chapter 5 and 6 suggests that in vivo mitochondrial function in insulin-resistant rat muscle is compromised by factors not taken into account in vitro. In both studies, muscle tissue free carnitine was significantly decreased, whereas muscle medium- and long-chain acylcarnitines were significantly increased. This finding indicates that in vivo muscle mitochondrial function might have been compromised by high concentrations of lipid metabolites in vivo. This was corroborated by the finding that mitochondria from high-fat diet fed rats were more susceptible to FA-induced mitochondrial uncoupling, which can explain the observed in vivo mitochondrial dysfunction. Next, it was investigated whether the observed carnitine insufficiency, a common feature of insulin-resistant states, contributes to the accumulation of muscle lipid intermediates and the lipid-induced impairment of skeletal muscle mitochondrial function in vivo. Carnitine supplementation might reduce toxic lipid intermediates by increasing FA oxidation and export of lipid metabolites out of the mitochondria and into the plasma, thereby relieving mitochondrial stress. Despite the normalization of muscle free carnitine content, carnitine supplementation did not induce improvements in muscle lipid status, in vivo mitochondrial function or insulin resistance. These results indicate that the decrease in muscle free carnitine as a result of high-fat diet feeding did not contribute to the observed impairment in in vivo muscle mitochondrial function. Conclusions Although it has been hypothesized that skeletal muscle mitochondrial dysfunction and an associated decreased capacity to oxidize FAs is the primary cause for muscle lipid accumulation and IR, the findings in this thesis are in agreement with recent studies which have shown that high-fat diets lead to increased rather than decreased FA oxidation, resulting in the accumulation of lipid intermediates causing mitochondrial dysfunction. Furthermore, from the results presented in this thesis it is concluded that it is essential to use a combination of several techniques, preferably both in vivo and in vitro, for a thorough investigation of muscle mitochondrial function. Compelling evidence for this is provided in chapters 5 and 6, where it was shown that a normal in vivo oxidative capacity, as measured by PCr recovery, can mask an impairment in intrinsic mitochondrial function when it is accompanied by increased mitochondrial content
