In the present study we observed concomitantly with explicit insulin resistance an up- regulated PDK4 expression along with less prominent ERRα expression in response to the high-fat diet and/or to voluntary exercise. We also found that high-fat diet did not alter the oxidative capacity of isolated mitochondria or oxygen consumption in the muscle homogenate. Voluntary running exercise improved insulin sensitivity during the first 9 weeks of the high-fat diet, but no longer after 18 weeks, concomitantly with decreased running activity. The effects of exercise on the mitochondrial parameters were comparable or greater to those of the high-fat diet, but in most cases exercise and high-fat diet did not have additional/synergistic effects.
In addition to its ability to exert effects on oxidative metabolism in muscle , it has been suggested that PGC-1α controls skeletal muscle glucose metabolism by increasing the amount of PDK4 via a PGC-1α/ERRα-dependent mechanism . This is further supported by the finding that ERRα recruits PGC-1α to the PDK4 promoter [37, 38]. Our results show distinct effects of high-fat diet and voluntary running on PDK4 protein expression and, more elaborately, an additive effect of both HF diet and voluntary running on mRNA expression but not on protein expression. Although we did not measure the activity of PDC in our experiment, it is known that PDK4 negatively regulates the PDC, thus inhibiting the entry of pyruvate to the Krebs cycle . In addition, PDK4 has been found to be a contributor to lipid-induced changes of glucose metabolism in rodent and human studies [47–49]. We speculate that increased PDK4 expression after high-fat feeding and exercise is due to previous increase in PGC-1α and ERRα expression, which subsequently blunts cellular glucose oxidation. Our results suggest that in addition to molecular and cellular level in vitro, PGC- 1α/ERRα-dependent regulation of PDK4 expression also may operate in vivo in skeletal muscle.
Previous studies have demonstrated that a high-fat diet can increase the biogenesis of mitochondria and fatty acid oxidative capacity in skeletal muscle [12, 13]. It can be suggested that not only in the state of increased energy demand, such as exercise, but also in the case of constantly increased energy supply with high fatty acid availability, the oxidation of fatty acids can be intensified. This paradigm is supported by the present data, which shows improved total mitochondrial capacity in response to a high-fat diet for 19 weeks. These results contradict previous findings according to which a high-fat diet decreases the capacity of muscles to oxidize the accumulated lipids [1, 2], which would occur owing to the decreased number of mitochondria, as reported in the offspring of type 2 diabetic parents .
PGC-1α is considered the master regulator that coordinates the gene expression of oxidative metabolism as well as mitochondrial biogenesis in skeletal muscle [50, 51]. In our study the effect of chronic high-fat feeding for 19 weeks had no effect on the expression of PGC-1α. This is in contrast to a previous study that showed decreased PGC-1α expression in muscle after 1 week on a HF diet that persisted down-regulated over 11 weeks . In other studies a high-fat diet for 4–5 weeks has even increased muscle PGC-1α protein expression and the number of mitochondria [11, 12]. These discrepancies may be partly due to differences in the fatty acid compositions of the diets, since it has been shown that, depending on their chain length and saturation level, fatty acids have greatly varying effects on PGC-1α expression . PGC-1α mRNA and protein expression peak rapidly after a stimulus, such as an exercise bout [14, 26, 29] or an increase in the concentration of serum fatty acids . After a period of adaptation, no change or only slight changes in PGC-1α mRNA and protein levels have been observed, after 4 weeks of high-fat diet  or, as in the present study, after a high-fat diet and/or exercise for 19 weeks. In addition to PGC-1α, ERRα, acting downstream of PGC-1α, is also a critical transcriptional regulator of mitochondrial biogenesis and cellular energy metabolism [24, 28, 31]. Moreover, ERRα is expressed in tissues demonstrating a high capacity for fatty acid β-oxidation [54, 55]. In this study, we found a significant increase in ERRα mRNA expression after a high-fat diet combined with voluntary running, and in protein expression after a low-fat diet combined with running. We believe that the modest changes observed in PGC-1α and ERRα expression are remnants of previous high increases caused by every single exercise bout and/or dietary fatty acids. A limitation to our study is that we measured only the transcript levels of PGC-1α, but not alternative regulatory mechanisms. PGC-1α activity is also regulated by protein modifications, including phosphorylation, acetylation and ubiqitination .
High-fat feeding declines general physical activity in rodents [57, 58]. Similarly, in this study high-fat feeding induced consistent reduction of wheel-running after 12 weeks of diet, although at the end of the experiment cumulative running distances did not statistically differ between LF and HF mice. Access to running wheels increases general cage activity and affect several components of energy balance (reviewed in Novak et al. ) that may have effects to the regulation of muscle metabolism. However, it is not possible to dissect the effects of these factors in this study. In this study the HF mice with or without exercise were severely insulin resistant, as indicated by their increased levels of fasting insulin and glucose, suggesting that they had developed a metabolic condition resembling metabolic syndrome or type 2 diabetes . In our experiment, we found the plasma free fatty acid concentration to be significantly lower in the HF animals compared to LF animals. Conceivably, skeletal muscle had adapted to the chronic high-fat diet to be able to better extract and oxidize circulating lipids. The preference for fatty acids as an energy source is reflected in elevated blood glucose. Our data may suggest that both in chronic high-fat diet and in long-term exercise training, the switch of fuel usage from glucose to fatty acids is mediated by the elevated expression of PDK4. It is known that during long-term exercise or short-term fasting, the activity of PDC is attenuated in conjunction with increased fatty acid usage . Accordingly, the expression of PDK4 is increased in fasting, diabetes and other conditions associated with switching from glucose oxidation to fatty acid oxidation .
What is the mechanism behind high-fat diet-induced insulin resistance? It has been shown that chronic high-fat diet-induced insulin resistance, unlike insulin resistance induced by acute increase in plasma free fatty acids (i.e. Randle glucose fatty acid cycle), is not rapidly reversible . On the basis of our studies, we agree that most probably it is not the decrease in the amount or intrinsic function of mitochondria that leads to increased intramyocellular lipids [12, 13]. Our data on insulin resistance and normal mitochondrial function support the idea that lipids themselves or metabolites of lipid metabolism attribute to impaired response to insulin, e.g. via altered cell membrane properties  or by affecting IRS phosphorylation and GLUT4 translocation . Our data further suggest that the inhibition of pyruvate dehydrogenase by PDK4 is a possible contributor to insulin resistance. In this scenario high-fat diet-induced insulin resistance may be a consequence of the continuing regulatory process of PGC-1α/ERRα activated by chronic high fatty acid availability. Our data also show that voluntary running exercise improved insulin resistance only transiently during the 19-week high-fat diet, implying that the regulatory power of fatty acids is superior to exercise. On the other hand, the inability of exercise to improve insulin sensitivity after the 19 weeks of wheel running in the experiment might be due to the reduced amount of running during the latter half of the experiment. The role of fatty acids in insulin resistance is a complex process, with some fatty acids inducing and others reversing skeletal muscle insulin resistance , suggesting that a balanced fatty acid composition in the diet would be beneficial for optimal muscle cell metabolism and function.