**4.2.1 Mitochondria in obesity, insulin resistance and type 2 diabetes**

Recent evidence indicates that insulin resistance in skeletal muscle might develop due to the reduced capacity of mitochondria to oxidize lipids (Bjorntorp et al. 1967; Kelley et al. 2002; Petersen et al. 2004; Ukropcova et al. 2007) and reduced capacity for insulin-stimulated ATPsynthesis (Petersen et al. 2005). Obese individuals and subjects with type 2 diabetes are characterized also by reduced adiponectin signaling (Kern et al. 2003; Civitarese et al. 2004; Rasmussen et al. 2006), lower rates of fasting lipid utilization and impaired switch to carbohydrate oxidation in response to insulin (Kelley et al. 1999; Kelley & Mandarino 2000; Ukropcova et al. 2005; Ukropcova et al. 2007). Recent studies using microarray expression analysis reported a decrease in the expression of genes involved in mitochondrial biogenesis in skeletal muscle of individuals with insulin resistance (Patti et al. 2003) and T2D (Mootha et al. 2003). Further studies in insulin resistant subjects and individuals with type 2 diabetes have shown reduced mitochondrial content, lower electron transport chain activity in total mitochondria and in intramyofibrilar and subsarcolemal mitochondrial fractions (Kelley et al. 2002; Ritov et al. 2005). Taken together, these data support the hypothesis that insulin resistance in human skeletal muscle arises from lowering mitochondrial number and functional capacity. Another hypothesis challenges this paradigm; it is supported by observations that increased fatty acid availability is associated with increased mitochondrial fat oxidation. However, mitochondrial overload with energy rich substrates highlights the pathophysiological role of ROS and that of products of incomplete mitochondrial oxidation rather than simple lowering of mitochondrial functional capacity (Koves et al. 2008; Holloszy 2009). The importance of mitochondria for energy homeostasis makes this organelle an exciting target for investigation and better understanding to regulation of mitochondrial biogenesis and function would help us to understand its putative role in the pathogenesis of obesity and insulin resistance.

#### **4.2.2 Exercise and ageing keep constant battle for healthy mitochondria**

Exercise is one of the two physiological stimuli known to increase production of new mitochondria and to improve mitochondrial efficiency. In our work, we have shown that caloric restriction, the only officially acknowledged physiological stimulus demonstrated to prolong lifespan, is also inducing mitochondrial biogenesis in human skeletal muscle (Civitarese et al. 2007). Many scientists are on a quest, pursuing the vision of exercise mimicking pill, capable of induction of mitochondrial biogenesis *in vivo*. "Exercise in a pill" (another option would be a pill mimicking caloric restriction) is by many considered a putatively great tool to combat obesity and civilization diseases. However, healthy lifestyle intervention, with sufficient physical activity and matching caloric intake still proves to be the most natural and effective way how to stay fit, healthy and with increased chances to live up to be a hundred.

#### **4.2.3 Adipose tissue and skeletal muscle interplay**

154 Dyslipidemia - From Prevention to Treatment

structure and function is orchestrated by a strict coordination of nuclear and mitochondrial genome. Of ~1.000 mitochondrial proteins, only 13 are encoded by the mitochondrial genome, remaining proteins are translated from nuclear genome and transported across the inner mitochondrial membrane (Lanza & Nair 2010). Mitochondria cover majority of energetic needs of cells by coupling substrate oxidation with ATP formation, the process known as oxidative phosphorylation. This process also generates reactive oxygen species (ROS). It has been estimated that 0.2 – 2% of oxygen taken up by the cell is converted into ROS (Harper et al. 2004). Mechanisms for detoxifying the ROS are quite well developed in a eukaryotic cell which is another reason for their long lasting partnership with "dangerous" mitochondria. Sustained excessive production may accumulate amount of ROS exceeding the antioxidant capacity of the specific cell, eventually leading to cell damage and death (Harman 1956). During recent years, mitochondria , though not only those found in skeletal muscle, were put on the spot as organelles involved in aging and associated chronic civilization diseases such as Alzheimer's disease (Reddy 2009), some forms of cancer,

Recent evidence indicates that insulin resistance in skeletal muscle might develop due to the reduced capacity of mitochondria to oxidize lipids (Bjorntorp et al. 1967; Kelley et al. 2002; Petersen et al. 2004; Ukropcova et al. 2007) and reduced capacity for insulin-stimulated ATPsynthesis (Petersen et al. 2005). Obese individuals and subjects with type 2 diabetes are characterized also by reduced adiponectin signaling (Kern et al. 2003; Civitarese et al. 2004; Rasmussen et al. 2006), lower rates of fasting lipid utilization and impaired switch to carbohydrate oxidation in response to insulin (Kelley et al. 1999; Kelley & Mandarino 2000; Ukropcova et al. 2005; Ukropcova et al. 2007). Recent studies using microarray expression analysis reported a decrease in the expression of genes involved in mitochondrial biogenesis in skeletal muscle of individuals with insulin resistance (Patti et al. 2003) and T2D (Mootha et al. 2003). Further studies in insulin resistant subjects and individuals with type 2 diabetes have shown reduced mitochondrial content, lower electron transport chain activity in total mitochondria and in intramyofibrilar and subsarcolemal mitochondrial fractions (Kelley et al. 2002; Ritov et al. 2005). Taken together, these data support the hypothesis that insulin resistance in human skeletal muscle arises from lowering mitochondrial number and functional capacity. Another hypothesis challenges this paradigm; it is supported by observations that increased fatty acid availability is associated with increased mitochondrial fat oxidation. However, mitochondrial overload with energy rich substrates highlights the pathophysiological role of ROS and that of products of incomplete mitochondrial oxidation rather than simple lowering of mitochondrial functional capacity (Koves et al. 2008; Holloszy 2009). The importance of mitochondria for energy homeostasis makes this organelle an exciting target for investigation and better understanding to regulation of mitochondrial biogenesis and function would help us to understand its putative role in the

obesity and type 2 diabetes (Johannsen & Ravussin 2009).

pathogenesis of obesity and insulin resistance.

**4.2.2 Exercise and ageing keep constant battle for healthy mitochondria** 

Exercise is one of the two physiological stimuli known to increase production of new mitochondria and to improve mitochondrial efficiency. In our work, we have shown that caloric restriction, the only officially acknowledged physiological stimulus demonstrated to

**4.2.1 Mitochondria in obesity, insulin resistance and type 2 diabetes** 

Our organism can be viewed as a very complex society of tissues that need to communicate with one another in order to maintain metabolic health. Tissue cross-talk plays the central role in the regulation of food intake, energy expenditure, oxidative capacity, adaptation to changes in physical activity, nutritional status etc. As mentioned above, adipose tissue (as well as many other tissues in our body) is (are) a (the) source of many biologically active substances with autocrine, paracrine and endocrine activities, exerting effects over many different neighboring as well as distant tissues and organs.

Adiponectin is the most studied adipocytokine which is in relatively high quantities secreted from adipose tissue into the bloodstream. Adiponectin has very positive effects on our metabolic health as it activates glucose and fatty acid metabolism and improves insulin sensitivity. Adiponectin levels are inversely correlated with body fat mass and positively with insulin sensitivity (Hara et al. 2005) and it also displays anti-atherogenic and antiinflammatory effects (Antoniades et al. 2009). This hormone was first characterized in mice as a transcript overexpressed in preadipocytes (precursors of fat cells) differentiating into adipocytes. The human homologue was identified as the most abundant transcript in adipose tissue. Contrary to expectations and despite being produced in adipose tissue, adiponectin was found to be decreased in obesity. The gene was localized to chromosome 3p27, a region highlighted as affecting genetic susceptibility to T2D and obesity. Supplementation by differing forms of adiponectin was able to improve insulin control, blood glucose and triglyceride levels in mouse models. The question remains what are the mechanisms underlying positive effects of adiponectin on metabolism?

The molecular mechanisms leading to mitochondrial dysfunction in obesity and T2D remain largely unknown. Bergeron et al (Bergeron et al. 2001) demonstrated that activation of cAMP-activated protein kinase (AMPK) increases both mitochondrial biogenesis and oxidative capacity in skeletal muscle of rodents. In animal models of T2D, the activation of AMPK by adiponectin increases muscle and hepatic fat oxidation and improves insulin sensitivity (Yamauchi et al. 2001). Studies in obese and diabetic rhesus monkey demonstrate that plasma adiponectin level declines in the early phases of obesity and in parallel to the progressive development of insulin resistance (Hotta et al. 2001). Furthermore, circulating plasma adiponectin levels and the expression of both adiponectin receptors are reduced in subjects with a family history of diabetes (Civitarese et al. 2004), while prospective studies in Pima Indians show that high concentrations of adiponectin is protective against the development of T2D (Lindsay et al. 2002). Collectively, these data define a pathway in skeletal muscle by which adiponectin contributes to energy homeostasis by modulating mitochondrial number and function (Civitarese et al. 2006). Early defects in the secretion of adiponectin or in adiponectin signaling might contribute to the lower mitochondrial content

Adipose Tissue and Skeletal Muscle Plasticity in Obesity and Metabolic Disease 157

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

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.

chronic diseases (Elbekai & El-Kadi 2005).

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 adiponectin derivatives a promising new treatment for T2D.

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 differentiation program.
