**4.2.4 Mitochondrial biogenesis in muscle cells – Lipids and exercise**

Fatty acids are known to be the ligands of various transcription factors involved in the regulation of metabolism and mitochondrial biogenesis (Gilde & Van Bilsen 2003). It has been shown previously that fatty acids as well as a diet with an increased fat content is capable of inducing mitochondrial biogenesis both in vitro and in vivo (Watt et al. 2006; Hancock et al. 2008). In our work, we have tested the effect of chronic, 4-day long exposure to palmitate on metabolic phenotypes of human primary skeletal muscle cells. We observed an increase in number of active mitochondria as measured by incorporation of mitotracker (fluorescencent dye selectively activated within respiring mitochondria) as well as increased expression of genes involved in mitochondrial biogenesis, increase in the capacity for fatty acid oxidation (Ukropcova et al. 2005, Ukropcova et al, unpublished observation). At this moment we can only speculate on the mechanisms behind this oxidation boosting effect of palmitate. However, it has been shown that fatty acids are capable of activating AMP activated protein kinase (AMPK) in skeletal muscle (Watt et al. 2006). AMPK signaling is activated in energy deficit states and it primarily saves the cell by inducing *de novo* mitochondrial biogenesis. It cooperates with transcription factor PGC1, overexpression of which has been demonstrated to enhance both lipid oxidation and synthesis (Espinoza et al. 2010). Another possibility is that palmitate is a ligand for the transcription factors involved in the regulation of cell´s oxidative capacity, such as PPARδ (Gilde & Van Bilsen 2003). Animal (Hancock et al. 2008) as well as clinical studies (Bajaj et al. 2007) also support the role of fatty acids for PGC1 regulation at the level of gene expression. We and others have indicated that saturated fatty acids (e.g. palmitate) contribute to the regulation of metabolism by self-promoting their utilization *via* increased oxidative capacity of the skeletal muscle cell.

In addition, dynamic interrelations of skeletal muscle and adipose tissue during exercise are necessary to support muscle performance and adipose tissue energy fluxes management. The transcriptional coactivator PGC1 has also been shown to regulate several exerciseassociated aspects of muscle function. It could be hypothesized that this protein controls muscle plasticity, suppresses a broad inflammatory response and mediates the beneficial effects of exercise on metabolic health (Handschin and Spiegelman 2008).

### **4.2.5 Caloric restriction induces mitochondrial biogenesis in skeletal muscle**

Caloric restriction is a non-genetic manipulation that results in the lifespan extension of many different species, from yeasts to dogs, and even primates, and it is accompanied by

and/or function in the prediabetic state. Interestingly and in accordance with our results, it has been recently demonstrated that an adiponectin-like molecule, a recombinant globular domain of adiponectin (rgAd110-244), has a significant therapeutic potential to treat insulin resistance in mice fed a high fat diet for 3 months (Sulpice et al. 2009). This makes

It appears that adiponectin is also produced by skeletal muscle and that globular adiponectin is capable of inducing the differentiation and fusion of muscle cells in vitro (Fiaschi et al. 2009). Mimicking of pro-inflammatory settings or exposure to oxidative stress strongly increases the production of adiponectin from differentiating primary muscle cells. These data suggest a novel function of adiponectin, coordinating the myogenic

Fatty acids are known to be the ligands of various transcription factors involved in the regulation of metabolism and mitochondrial biogenesis (Gilde & Van Bilsen 2003). It has been shown previously that fatty acids as well as a diet with an increased fat content is capable of inducing mitochondrial biogenesis both in vitro and in vivo (Watt et al. 2006; Hancock et al. 2008). In our work, we have tested the effect of chronic, 4-day long exposure to palmitate on metabolic phenotypes of human primary skeletal muscle cells. We observed an increase in number of active mitochondria as measured by incorporation of mitotracker (fluorescencent dye selectively activated within respiring mitochondria) as well as increased expression of genes involved in mitochondrial biogenesis, increase in the capacity for fatty acid oxidation (Ukropcova et al. 2005, Ukropcova et al, unpublished observation). At this moment we can only speculate on the mechanisms behind this oxidation boosting effect of palmitate. However, it has been shown that fatty acids are capable of activating AMP activated protein kinase (AMPK) in skeletal muscle (Watt et al. 2006). AMPK signaling is activated in energy deficit states and it primarily saves the cell by inducing *de novo* mitochondrial biogenesis. It cooperates with transcription factor PGC1, overexpression of which has been demonstrated to enhance both lipid oxidation and synthesis (Espinoza et al. 2010). Another possibility is that palmitate is a ligand for the transcription factors involved in the regulation of cell´s oxidative capacity, such as PPARδ (Gilde & Van Bilsen 2003). Animal (Hancock et al. 2008) as well as clinical studies (Bajaj et al. 2007) also support the role of fatty acids for PGC1 regulation at the level of gene expression. We and others have indicated that saturated fatty acids (e.g. palmitate) contribute to the regulation of metabolism by self-promoting their utilization *via* increased oxidative capacity of the

In addition, dynamic interrelations of skeletal muscle and adipose tissue during exercise are necessary to support muscle performance and adipose tissue energy fluxes management. The transcriptional coactivator PGC1 has also been shown to regulate several exerciseassociated aspects of muscle function. It could be hypothesized that this protein controls muscle plasticity, suppresses a broad inflammatory response and mediates the beneficial

Caloric restriction is a non-genetic manipulation that results in the lifespan extension of many different species, from yeasts to dogs, and even primates, and it is accompanied by

effects of exercise on metabolic health (Handschin and Spiegelman 2008).

**4.2.5 Caloric restriction induces mitochondrial biogenesis in skeletal muscle** 

adiponectin derivatives a promising new treatment for T2D.

**4.2.4 Mitochondrial biogenesis in muscle cells – Lipids and exercise** 

differentiation program.

skeletal muscle cell.

delayed onset of chronic civilization diseases (Ball et al. 1947; Anderson et al. 2009). There are also hints that people who eat a calorie-restricted diet might live longer than those who overeat. People living in Okinawa, Japan, have a lower energy intake than the rest of the Japanese population and an extremely long life span (Willcox et al. 2007). In addition, calorie-restricted diets beneficially affect several biomarkers of aging, including decreased insulin sensitivity. Based on combined favorable changes in lipid and blood pressure, caloric restriction with or without exercise induces weight loss and favorably reduces risk for cardiovascular disease even in healthy non-obese individuals (Lefevre et al. 2009) and ameliorates the age-related loss of muscle mass, sarcopenia, in a variety a species (Marzetti et al. 2009). But how might caloric restriction slow aging? Some of the theories behind the lifespan extending effect of caloric restriction include (i) decreased oxidative damage, (ii) altered glucose utilization, (iii) increased insulin sensitivity, (iv) neuroendocrine changes and (v) enhanced stress responsiveness (Allard et al. 2009). Reduction of oxidative damage to proteins, lipids, and DNA is one of the leading theories, although the underlying mechanisms of this process are unclear. Cellular nutrient sensing systems seem to mediate many of the metabolic responses to caloric restriction, including the regulation of free radical production and oxidative stress. Mitochondria are the major consumers of cellular oxygen (~85%) and the predominant production site of free radicals, a by-product of oxidative phosphorylation. Studies in mammals have shown that caloric restriction reduces the generation of free radicals by mitochondria, in parallel to reductions in mitochondrial proton leak and whole-body energy expenditure. Paradoxically, caloric restriction induces mitochondrial proliferation in rodents (Lanza & Nair 2010), and either lowers (Handschin & Spiegelman 2008) or does not affect mitochondrial oxygen consumption (Lanza & Nair 2010). Low mitochondrial content seems to contribute to increased ROS production. When mitochondrial mass is reduced, mitochondria have increased "workload," leading to higher membrane potential and increased ROS production (Handschin & Spiegelman 2008; Lanza & Nair 2009; Lanza & Nair 2010). It has also been demonstrated that caloric restriction is strongly associated with an increased level and activation of sirtuins, namely the Sir2 histone deacetylase and its mammalian ortholog Sirt1. Sirtuins are members of the silent information regulator 2 (Sir2) family, a family of Class III histone/protein deacetylases. The enzymatic activity of most sirtuins has been shown to be dependent on nicotinamide dinucleotide, suggesting that the activity of these enzymes is dependent on the nutritive state of the organism (Allard et al. 2009). Specific Sirt1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation (Feige et al. 2008). PGC1α is a transcriptional coactivator playing a pivotal role in the regulation of mitochondrial biogenesis, which is known to be induced in response to exercise and caloric restriction (Fig. 8.). Research strongly supports the health benefits of exercise in humans of all ages. Increased exercise in the absence of other behavioral changes prevents the onset of many chronic diseases (Elbekai & El-Kadi 2005).

In our study we showed that short-term caloric deficit (caloric restriction with or without exercise) coordinately up-regulated the expression of genes involved in mitochondrial biogenesis in skeletal muscle resulting in increased mitochondrial content, improved whole body energy efficiency, and decreased DNA fragmentation in non-obese humans (Civitarese et al. 2007). Our results suggest that caloric restriction induces biogenesis of "efficient" mitochondria as an adaptive mechanism, which in turn lowers oxidative stress.

Adipose Tissue and Skeletal Muscle Plasticity in Obesity and Metabolic Disease 159

phenotype, characteristic by extra-large unilocular adipocytes, further promotes tissue hypoxia and development of chronic persistent inflammation and metabolic stress. Pathogenic modification of the adipocyte modulates its metabolic, secretory and immunologic function leading to the development of metabolic disease (Fig. 10.). Inactive skeletal muscle, overloaded with fat could also contribute to metabolic imbalance by switching the fiber type towards less oxidative (less insulin sensitive) fibers, by lack of antiinflammatory and insulin-sensitizing myokine production as well as by chronic inflammation associated with mitochondrial stress and stress of endoplasmic reticulum (Fig. 10.). Our environment greatly modifies our metabolic health by means of dietary influences and exercise activity which together with pathologies associated with hyperlipidemia, chronic systemic hypoxia and tissue inflammation determines adipose tissue and skeletal

muscle metabolic and secretory phenotype and subsequently our metabolic health.

cardiovascular disorders.

**6. Acknowledgement** 

support.

It is generally accepted that regular physical activity prevents metabolic and cardiovascular disease development, and supports healthy aging. Skeletal muscle has been shown to produce and secrete several bioactive factors (hormones) termed "myokines". Different spectra of myokines originating from either active "trained" or inactive "sedentary" skeletal muscles elicit distinct adaptive changes in immune system, metabolic balance and processes of cellular growth and differentiation in order to maintain the whole body homeostasis. This requires extensive communication of skeletal muscle with many different cells and organs but the nature of mechanisms that tie muscle activity to metabolic health is not completely understood. It seems to be essential (i) to identify myokines differentially expressed and secreted from muscle cells derived from healthy and obese individuals, and individuals with type 2 diabetes; to (ii) determine basic principles of the muscle cross-communication with adipocytes (differentiation) and endothelial cells (angiogenesis) in fostering tissue plasticity necessary for adaptation to obesity and type 2 diabetes; and (iii) to discover novel myokines and to investigate their physiological significance in cell culture models and *in vivo* in genetically modified animal models as well as in humans. Myokines may be involved in mediating the health beneficial effects of exercise and play important roles in the protection against chronic diseases associated with low-grade inflammation, insulin resistance, hyperlipidemia, such as cardiovascular disease, type-2-diabetes, and cancer. Extension of the knowledge on the mechanisms whereby regular exercise offers protection against chronic diseases in combination with clinical research serves as a foundation for the development of public health guidelines with regard to exercise. Moreover, identification of new myokines and understanding basic principles and mechanisms of their action will potentially provide pharmacological targets for the treatment of metabolic and

We would like to express our cordial gratitude to everybody who contributed to this work. There is too many people to thank. Above all we would like to acknowledge our parents and our wonderfull children Kiki and Jakub, our inspiration in everyday life. We would also like to thank the funding agencies: APVV grant agency (APVV 0122-06), European Foundation to Study Diabetes (EFSD New Horizons Collaborative Research Grant) and the 7th FP EC 2007-2.1.1-6. grant entitled "LipidomicNET" for their generous

Fig. 9. The free radical theory of aging posits that a senescent phenotype is induced by accumulation of oxidative damage resulting from reactive oxygen species. Exercise and caloric restriction (CR) are two interventions that induce mitochondrial biogenesis through PGC-1α. Although exercise and CR increase average life expectancy by protecting against age-related comorbidities, only CR has been shown to increase maximal life span; an effect that seems to require the activation of sirtuins (Lanza & Nair 2010).

Fig. 10. Determinants of metabolic health and disease (Ukropec et al. 2008)
