**2.2 Brown adipose tissue in human physiology**

144 Dyslipidemia - From Prevention to Treatment

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

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

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

hypertrophy (Kim et al. 2007).

obesity and metabolic disorders.

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

properties such as unfavorable secretory profile (Matsuzawa et al. 1999).

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 adipose tissue activity (Vijgen et al. 2011).

Fig. 3. Metabolically activated brown adipose tissue in supraclavicular region (arrows, B) in morbidly obese individuals after personalized cooling protocol (Vijgen et al. 2011).

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

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 adipocytes, giving them the genuine, thermogenic capacity.

Adipose Tissue and Skeletal Muscle Plasticity in Obesity and Metabolic Disease 147

Clearly not all obese individuals develop metabolic and cardiovascular complications; here we discuss several regulatory mechanisms representing a base for the strategies to prevent

d

tyrosine (AMPT) – competitive inhibitor of TH activity (Vargovic et al. 2011).

Fig. 5. Adipocytes have internal catecholamine production capacity. Adipose tissue contains mRNA and proteins specific for tyrosine hydroxylase (TH) and phenylethanolamine Nmethyltransferase PNMT as shown on histological sections probed with specific PNMT (**a,b**) and TH (**c**) antibodies, scale bar: 20 µm. (**d**) Adipocytes freshly isolated from mesenteric adipose tissue produce dopamine (DA), norepinephrine (NE) and epinephrine (EPI) into the media. Production of catecholamines is largely inhibited by addition of alpha-methyl-p-

Hypothesis that adipose tissue populated by large adipocytes contains the local microhypoxia suffering areas, which has a profound effect on the tissue metabolic and inflammatory phenotype, has been largely accepted. Hypoxia is one of the major triggers strongly inhibiting adipocyte differentiation. Tissue hypoxia in obesity is associated with the defects in the nutrient and oxygen supply into the tissue, related to a defective blood flow regulation which might be perpetuated by the increased fat cell size (Yun et al. 2002). This is happening in spite of the almost unlimited capacity of adipose tissue to expand in a nontransformed form, which is a very unique property of adipose tissue that cannot be seen in any other organ. To accomplish this adipose tissue requires potent mechanisms to remodel, acutely and chronically, as it can rapidly reach the diffusional limit of oxygen; molecular response to hypoxia is therefore an early determinant that limits healthy adipose tissue expansion. Proper expansion requires a highly coordinated response among many different cell types, including endothelial precursor cells, immune cells, and preadipocytes (Sun et al. 2011). It has also been demonstrated that mitochondrial oxidative apparatus is essential for the white fat adipocyte differentiation (De Pauw et al. 2009). Beside their key role in ATP

metabolic disease development.

**3.1 Hypoxia** 

Fig. 4. Metabolically activated "brite adipocytes" as found in inguinal white adipose tissue of mice lacking the UCP1-thermogenesis after gradual exposure to cold.

(**A**) histomorphology, (**B**) O2 consumption by oxymetry, (**C**) adipose tissue *ex vivo* palmitate oxidation, (**D**&**E**) evidence for AMPK activation (Ukropec et al. 2006).

In collaboration with laboratory of dr. Kvatnansky we have recently observed that catecholamines, important regulators of lipolysis in adipose tissue, could be produced within adipocytes. Adipocytes isolated from mesenteric adipose tissue expressed genes encoding the catecholamine biosynthetic enzymes and produced catecholamines *de novo*. Administration of tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, significantly reduced concentration of catecholamines in isolated adipocytes *in vitro* (Fig. 5.). We therefore hypothesized that the sympathetic innervation of adipose tissue is not the only source of catecholamines and that adipocyte-derived catecholamines could dynamically modulate metabolic or thermogenic properties of the white adipose tissue perhaps by enhancing "brite adipocyte" function (Vargovic et al. 2011).

#### **3. Adipocentric view on the pathophysiology of metabolic disease**

The prevalence of obesity and its consequent pathologies in modern society is of serious health concern. Although the expansion of adipose tissue mass during pathological obesity is in itself not a grave problem, rather it is the ensuing pathologies resultant of this state, including development of hypertension, type 2 diabetes and cardiac myopathies, that impacts peoples lives and health services worldwide.

Clearly not all obese individuals develop metabolic and cardiovascular complications; here we discuss several regulatory mechanisms representing a base for the strategies to prevent metabolic disease development.

Fig. 5. Adipocytes have internal catecholamine production capacity. Adipose tissue contains mRNA and proteins specific for tyrosine hydroxylase (TH) and phenylethanolamine Nmethyltransferase PNMT as shown on histological sections probed with specific PNMT (**a,b**) and TH (**c**) antibodies, scale bar: 20 µm. (**d**) Adipocytes freshly isolated from mesenteric adipose tissue produce dopamine (DA), norepinephrine (NE) and epinephrine (EPI) into the media. Production of catecholamines is largely inhibited by addition of alpha-methyl-ptyrosine (AMPT) – competitive inhibitor of TH activity (Vargovic et al. 2011).
