**2. Adipose tissue in metabolic health and disease**

Despite the fact that the primary role of adipose tissue is an effective lipid storage and timely regulation of its release and that these processes could, in a simplified adipocentric view, be the primary determinants of the dyslipidemia and metabolic disease progression, adipose tissue has a broad range of other regulatory functions exerted *via* its autocrine, paracrine and endocrine actions. Adipose tissue secretory products, "adipokines", could modulate food intake, energy expenditure, or tissue oxidative capacity (Trayhurn et al. 1998; Ukropec et al. 2001; Ahima & Lazar 2008; Henry & Clarke 2008; Friedman 2011). In addition, adipose tissue dynamically changes its structure (*tissue remodeling, lipid composition*), function (*lipid storage & lipolysis*) as well as endocrine action in response to different physiological (*fasting / refeeding, exercise, microgravity*) and pathophysiological (*obesity, prediabetes, diabetes, cachexia, lipodystrophy, growth hormone deficiency*) conditions (Ukropec et al. 2008; Itoh et al. 2011; Pietilainen et al. 2011). It is important to understand that adipose tissue is a mixture of very different cell-types. Apart from approximately 50% of mature lipid-laden adipocytes it contains various stromal cells including preadipocytes, endothelial cells, fibroblasts, pluripotent stem cells and immune cells which substantially influence its function (Bjorntorp 1974; Sethi & Vidal-Puig 2007; Divoux & Clement 2011). Extreme enlargement of the fat cell size, such as we have recently observed in individuals with growth hormone deficiency, is perhaps the best early marker of the obesity related metabolic disease development (Ukropec et al. 2008a) (Fig. 1.). Adipose tissue with enlarged adipocytes, expressing markers of the local tissue microhypoxia but not responding to it

Adipose Tissue and Skeletal Muscle Plasticity in Obesity and Metabolic Disease 143

A B

hypertrophy

hyperplasia

Fig. 2. (A) Graph depicts large variability in adipose tissue morphology in individuals with identical body fat mass (764 subjects). (B) Level of hyperplasia is represented by adequately lowered morphology value while hypertrophy is associated with parallel increase in morphology value defined as a difference between measured and expected adipocyte volume given by the curve-like fit , for given body fat mass (Arner, E. et al. 2010).

Insulin sensitivity could therefore govern adipose tissue morphology towards beneficial hyperplastic state at the population level. Conversely, defects in insulin action are interconnected with the hypertrophic adipocytes, and higher risk of lipid flooding the nonadipose tissues. Absolute numbers of adipocytes as well as their capacity to expand and store lipids are quite difficult to modulate. Morbid obesity and lipodystrophy represent the two medical conditions associated with excessive hypertriglyceridemia, hepatic steatosis, and disordered muscle glucose metabolism, due to defected ability of adipose tissue to store lipids or the selective loss of adipose tissue respectively. Activation of adipogenic programme by PPAR agonists or chronic leptin treatment improves insulin-stimulated hepatic and peripheral glucose metabolism in obese and lipodystrophic patients respectively (Petersen et al. 2002; Rieusset et al. 2002; Van Gaal et al. 2003). Adipogenesis is necessary to increase the adipose tissue cellularity (hyperplastic adaptive change) and lipid storage capacity; it is largely dependent on the signal transducers and activators of transcription (STAT) pathway. In brief, transcription factors C/EBP, C/EBP, and PPAR control adipogenesis by regulating STAT5B and STAT5A. Regulation of PPAR-STAT5 by C/EBP signaling seems to be the crucial adipogenesis - initiating cascade of the various adipogenic genes (Jung et al. 2011). Activation of adipogenic program should be paralleled with the extracellular matrix remodeling. As mentioned above, adipocytes are embedded in a unique extracellular matrix which main function is to provide mechanical support, in addition to participating in a variety of signaling events. Extracellular matrix requires remodeling to accommodate growing adipocytes in the expanding adipose tissue. We have recently participated in the research by Christian Wolfrum's laboratory investigating regulatory processes related to adipose tissue hyperplasia. In this work, the transcription factor retinoid-related orphan receptor γ (RORγ) was identified as an important regulator of adipocyte development through regulation of its newly identified target gene matrix metallopeptidase-3. RORγ might serve as a novel predictor for the risk of metabolic complications in obesity as well as a pharmaceutical target for the treatment of obesity-

associated diseases (Meissburger et al. 2011).

properly and attracting large amount of activated immunocompetent cells, has recently been termed "pathogenic adipose tissue" (Bays et al. 2008; Ukropec et al. 2008b).

Fig. 1. Fat cell size in obesity and in growth hormone deficiency (GHD), percentage of cumulative frequency (Y axis), (Ukropec et al. 2008a).

The tissue damage due to overwhelming adipocytes with lipids, largely exceeding their lipid storage capacity, changes the adipokine secretory profile and leads to uncontrolled release of a large amount of lipids into circulation while the capacity of the adipose tissue adaptive remodeling is compromised. This is followed by the accumulation of lipids in tissues not designed for the lipid storage such as liver, skeletal muscle, pancreatic beta cells or lung which largely interferes with their physiological functions and accelerates the development of metabolic disease (Bays et al. 2008; Foster et al. 2010; Unger et al. 2010). Limited expandability of the adipose tissue seems to determine individual propensity to the development of metabolic disease (Arner, P. et al. 2011). Adaptive expansion of the adipose tissue is enabled by combination of adipocyte hypertrophy and hyperplasia. Expansion of adipose tissue requires quite extensive tissue remodeling, in order to maintain adequate energy and oxygen supply, active neuronal network as well as integrity and functional properties of cellular membranes (Itoh et al. 2011; Pietilainen et al. 2011). Broader knowledge of the fat cell life-cycle dynamics is critical for our understanding the pathophysiological mechanisms limiting adipose tissue hyperplastic expansion. In 2008, Arner's group analyzed the adipocyte turnover by detecting the genomic DNA incorporation of atmospheric 14C derived from above-ground nuclear bomb tests in period between 1955 and 1963. This work revealed that approximately 10% of fat cells are renewed per year at all adult ages and levels of BMI and that neither adipocyte death nor generation rate is altered in early onset obesity. It seemed that the steady production of adipocytes in adults results in a stable size of the constantly turning over adipocyte population (Spalding et al. 2008). More recent work by these authors examining morphology of the subcutaneous adipose tissue from 764 individuals with broad range of BMI (18-60 kg.m2) defines hyperplasia and hypertrophy as a difference between measured adipocyte volume and volume predicted by the curve-like fit, for adipocyte volume and body fat mass (Fig. 2.). In this analysis occurrence of hyperplasia or hypertrophy correlated with fasting plasma insulin and insulin sensitivity. In addition, total adipocyte number was greatest in individuals with pronounced hyperplasia, and smallest in those with pronounced hypertrophy. The absolute number of new adipocytes generated each year was 70% lower in patients with hypertrophy than with hyperplasia. Whereas the relative death rate (~ 10% per year) or mean age of adipocytes (~ 10 years) was not correlated with adipocyte morphology (Arner, E. et al. 2010).

properly and attracting large amount of activated immunocompetent cells, has recently been

**lean control lean GHD obese control obese GHD** 

Fig. 1. Fat cell size in obesity and in growth hormone deficiency (GHD), percentage of

Adipocyte diameter (µm)

The tissue damage due to overwhelming adipocytes with lipids, largely exceeding their lipid storage capacity, changes the adipokine secretory profile and leads to uncontrolled release of a large amount of lipids into circulation while the capacity of the adipose tissue adaptive remodeling is compromised. This is followed by the accumulation of lipids in tissues not designed for the lipid storage such as liver, skeletal muscle, pancreatic beta cells or lung which largely interferes with their physiological functions and accelerates the development of metabolic disease (Bays et al. 2008; Foster et al. 2010; Unger et al. 2010). Limited expandability of the adipose tissue seems to determine individual propensity to the development of metabolic disease (Arner, P. et al. 2011). Adaptive expansion of the adipose tissue is enabled by combination of adipocyte hypertrophy and hyperplasia. Expansion of adipose tissue requires quite extensive tissue remodeling, in order to maintain adequate energy and oxygen supply, active neuronal network as well as integrity and functional properties of cellular membranes (Itoh et al. 2011; Pietilainen et al. 2011). Broader knowledge of the fat cell life-cycle dynamics is critical for our understanding the pathophysiological mechanisms limiting adipose tissue hyperplastic expansion. In 2008, Arner's group analyzed the adipocyte turnover by detecting the genomic DNA incorporation of atmospheric 14C derived from above-ground nuclear bomb tests in period between 1955 and 1963. This work revealed that approximately 10% of fat cells are renewed per year at all adult ages and levels of BMI and that neither adipocyte death nor generation rate is altered in early onset obesity. It seemed that the steady production of adipocytes in adults results in a stable size of the constantly turning over adipocyte population (Spalding et al. 2008). More recent work by these authors examining morphology of the subcutaneous adipose tissue from 764 individuals with broad range of BMI (18-60 kg.m2) defines hyperplasia and hypertrophy as a difference between measured adipocyte volume and volume predicted by the curve-like fit, for adipocyte volume and body fat mass (Fig. 2.). In this analysis occurrence of hyperplasia or hypertrophy correlated with fasting plasma insulin and insulin sensitivity. In addition, total adipocyte number was greatest in individuals with pronounced hyperplasia, and smallest in those with pronounced hypertrophy. The absolute number of new adipocytes generated each year was 70% lower in patients with hypertrophy than with hyperplasia. Whereas the relative death rate (~ 10% per year) or mean age of adipocytes (~ 10 years) was not correlated with adipocyte morphology (Arner, E. et al. 2010).

60 80 100 120 140 160 180 200 220

cumulative frequency (Y axis), (Ukropec et al. 2008a).

5% 10% 15% 20% 25%

B

termed "pathogenic adipose tissue" (Bays et al. 2008; Ukropec et al. 2008b).

Percentage of cumulative frequency

Fig. 2. (A) Graph depicts large variability in adipose tissue morphology in individuals with identical body fat mass (764 subjects). (B) Level of hyperplasia is represented by adequately lowered morphology value while hypertrophy is associated with parallel increase in morphology value defined as a difference between measured and expected adipocyte volume given by the curve-like fit , for given body fat mass (Arner, E. et al. 2010).

Insulin sensitivity could therefore govern adipose tissue morphology towards beneficial hyperplastic state at the population level. Conversely, defects in insulin action are interconnected with the hypertrophic adipocytes, and higher risk of lipid flooding the nonadipose tissues. Absolute numbers of adipocytes as well as their capacity to expand and store lipids are quite difficult to modulate. Morbid obesity and lipodystrophy represent the two medical conditions associated with excessive hypertriglyceridemia, hepatic steatosis, and disordered muscle glucose metabolism, due to defected ability of adipose tissue to store lipids or the selective loss of adipose tissue respectively. Activation of adipogenic programme by PPAR agonists or chronic leptin treatment improves insulin-stimulated hepatic and peripheral glucose metabolism in obese and lipodystrophic patients respectively (Petersen et al. 2002; Rieusset et al. 2002; Van Gaal et al. 2003). Adipogenesis is necessary to increase the adipose tissue cellularity (hyperplastic adaptive change) and lipid storage capacity; it is largely dependent on the signal transducers and activators of transcription (STAT) pathway. In brief, transcription factors C/EBP, C/EBP, and PPAR control adipogenesis by regulating STAT5B and STAT5A. Regulation of PPAR-STAT5 by C/EBP signaling seems to be the crucial adipogenesis - initiating cascade of the various adipogenic genes (Jung et al. 2011). Activation of adipogenic program should be paralleled with the extracellular matrix remodeling. As mentioned above, adipocytes are embedded in a unique extracellular matrix which main function is to provide mechanical support, in addition to participating in a variety of signaling events. Extracellular matrix requires remodeling to accommodate growing adipocytes in the expanding adipose tissue. We have recently participated in the research by Christian Wolfrum's laboratory investigating regulatory processes related to adipose tissue hyperplasia. In this work, the transcription factor retinoid-related orphan receptor γ (RORγ) was identified as an important regulator of adipocyte development through regulation of its newly identified target gene matrix metallopeptidase-3. RORγ might serve as a novel predictor for the risk of metabolic complications in obesity as well as a pharmaceutical target for the treatment of obesityassociated diseases (Meissburger et al. 2011).

Adipose Tissue and Skeletal Muscle Plasticity in Obesity and Metabolic Disease 145

Humans and other mammals have two types of adipose tissue that contribute to control of the whole body energy metabolism. The above discussed white adipose tissue, "the bad guy" associated with obesity, is necessary for energy storage. Brown adipose, "the good guy", contains a lots of mitochondria and is ready to burn energy to generate heat in response to cold or dietary intake, keeping the body warm and slim (Cannon & Nedergaard 2004). Until recently, physiologically relevant amount of brown fat was only found in newborns. However, accumulating evidence indicates that adult humans – or at least significant portion of us retain physiologically relevant amount of brown fat (van Marken Lichtenbelt et al. 2009; Vijgen et al. 2011; Virtanen & Nuutila 2011). This provides an exciting possibility to precisely regulate the adaptive thermogenic process in humans, which could dissipate energy and lower the obesity related metabolic burden. Brown adipose tissue activity in humans was determined with the aid of 18F-fluorodeoxyglucose positronemission tomography and computing tomography mainly in the supraclavicular region of cold-exposed individuals. Importantly, specimens of the adipose tissue from the supraclavicular region of adult humans with active brown adipose tissue were positive for UCP1 protein (Fig. 3.) (van Marken Lichtenbelt et al. 2009; Zingaretti et al. 2009). Vision of translating this knowledge into the clinical practice is quite reachable (Nedergaard & Cannon 2010; Tseng et al. 2010). Clinical importance could be significant, despite the fact that the volume of active brown adipose tissue tends to be lower in the overweight or obese than in the lean individuals (van Marken Lichtenbelt et al. 2009), and that it decreases with age (Cypess et al. 2009). Interestingly, applying the personalized cooling protocol for maximal nonshivering conditions to morbidly obese individuals could still increase brown

Fig. 3. Metabolically activated brown adipose tissue in supraclavicular region (arrows, B) in

It has recently been shown that brown adipocytes and muscle cells share the common origin and in this respect they are quite distinct from white adipocytes (Tseng et al. 2008; Seale & Lazar 2009). The question remains, what is the origin of "brown fat-like white (brite) " adipocytes containing UCP1 which could be induced in white fat depots under certain pohysiological (cold exposure) (Fig. 4.) or pharmacological (activation of SNS, agonists of PPAR) conditions (Granneman et al. 2005; Li et al. 2005; Ukropec et al. 2006). Nedegaard`s laboratory had recently reported that chronic treatment with the PPAR agonist rosiglitazone promotes not only the expression of PGC1 and mitochondriogenesis but also a catecholamine – inducible UCP1 gene expression in a significant subset of the white

morbidly obese individuals after personalized cooling protocol (Vijgen et al. 2011).

**2.2 Brown adipose tissue in human physiology** 

adipose tissue activity (Vijgen et al. 2011).

**2.3 Metabolic activation of the white adipose tissue** 

adipocytes, giving them the genuine, thermogenic capacity.

Khan et al., recently proposed that "adipose tissue fibrosis" is a hallmark of metabolically challenged adipocytes. Authors observed that the absence of collagen VI, the highly enriched extracellular matrix component of adipose tissue, results in the uninhibited expansion of individual adipocytes, which is paradoxically associated with substantial improvements in whole-body energy homeostasis, both with high-fat diet exposure and in the ob/ob background. Weakening the extracellular scaffold of adipocytes seems to enable their stressfree expansion during states of positive energy balance, which is consequently associated with an improved inflammatory profile (Khan et al. 2009). Further support to the notion that metabolic deregulation is rather due to lipid-leakage than the adipocyte hypertrophy *per se* comes from the experiment where mice lacking leptin were made to overexpress adiponectin. This led to the modest increase in circulating levels of full-length adiponectin and to subsequent normalization of glucose and insulin levels, dramatic improvement of glucose tolerance and positive effect on serum triglyceride levels. Adiponectin in fact completely rescued the diabetic phenotype in ob/ob mice. These mice displayed increased expression of PPAR target genes and a reduction in macrophage infiltration in adipose tissue and systemic inflammation. Adiponectin expressing ob/ob mice, however, were morbidly obese, with significantly higher levels of adiposity and adipocyte hypertrophy than their ob/ob littermates. Adiponectin seems to act as a peripheral "starvation" signal promoting the storage of triglycerides preferentially in adipose tissue. As a consequence, reduced triglyceride levels in the liver and muscle convey improved systemic insulin sensitivity despite adipocyte hypertrophy (Kim et al. 2007).

#### **2.1 Specificities of subcutaneous and visceral adipose tissue**

In contrast to visceral adipose tissue, which is often blamed from inducing detrimental metabolic effects, subcutaneous adipose tissue has the potential to benefit lipid and glucose metabolism. It has been repeatedly shown that differences in regional body fat distribution determine the propensity for the development of obesity related metabolic complications (Tchernof 2007). Accumulation of fat in the visceral region (mesentery, omentum, retroperitoneum), that in fact corresponds to central obesity (determined by increased waist circumference) is associated with cardiovascular disease and type 2 diabetes, independently on overall obesity (Wajchenberg 2000; Hamdy et al. 2006; Pischon et al. 2008). The amount of visceral fat increases with age in both genders but man in general have greater visceral adiposity than women (Wajchenberg 2000). Consistent with this notion, removal of visceral adipose tissue (omentectomy) decreases glucose and insulin levels in humans (Thorne et al. 2002). By contrast peripheral obesity – increased subcutaneous adipose tissue mass, mainly in the region of buttock and thighs seem to be associated with improved insulin sensitivity and lower risk for type 2 diabetes mellitus (Snijder et al. 2003; Koska et al. 2008). One possible explanation for the detrimental effect of visceral fat accumulation comes from its unfortunate anatomical location (Arner, P. 1998; Bergman et al. 2006), but second theory based on adipose tissue transplantation experiments blames rather the tissue internal properties such as unfavorable secretory profile (Matsuzawa et al. 1999).

Adipose tissue transplantation experiments have been primarily used as a tool to study physiology for human reconstructive surgery, but they provide important information on differences between visceral and subcutaneous adipose tissue which opens the vision of the adipose tissue or adipose tissue derived stem cells transplantation for the treatment of obesity and metabolic disorders.
