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Introduction
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1. INTRODUCTION
1.1 Obesity
Overweight and obesity represent nowadays a health problem of global importance, affecting more than 50% of the adult population, especially in the developed Western countries. Obesity is one of the major health risk factors typical, but not exclusive of wealthy countries and is the cause of the increase in serious illness and mortality frequently associated with metabolic and cardiovascular disorders such as atherosclerosis, hepatic steatosis, insulin resistance, dyslipidemia and hypertension. The ensemble of obesity and the complications described above is defined by the term of "metabolic syndrome". The primary cause of this syndrome is debated.
An important role in the onset of these diseases certainly originates from insulin resistance that often accompanies obesity. Insulin resistance is one of the first symptoms observed in obese individuals, it is often associated with increased visceral fat mass, although the molecular mechanisms that determine its development are not yet fully clarified. The term "insulin resistance" defines a condition of resistance to insulin action in the uptake, metabolism and / or in the accumulation of glucose in the body. The causes of hyporesponsiveness to insulin may be different including: a decreased glucose transport, reduced metabolism of adipocytes or muscle cells due to enzymatic defects, liver abnormalities, impairment in post-receptor transduction pathways ("signaling") (Kahn B and Flyer JS, 2000).
Obesity consists in an abnormal development of the adipose tissue, caused by an imbalance in the homeostasis of body weight. Two types of obesity are described in literature: hypertrophic obesity when adipocytes increase in size and hyperplastic obesity when adipocytes increase in number. Adipocyte size is inversely related to insulin sensitivity: the larger ones are more prone to insulin resistance, whereas small adipocytes show increased insulin sensitivity (Roberts R et al., 2009). Fat cells that exceed a critical size become unable to maintain physiological homeostasis due to saturation of their depots; they respond by compensatory mechanisms that
3 can reduce both their ability to increase in size and accumulate triglycerides. Large adipocytes also express high levels of the inflammatory factor TNF-α, which inhibits the enzyme lipoprotein lipase (see section 1.2) and thus interferes with the normal anti-lipolytic action of insulin. This results in an effect of "spill-over" (release) of non-esterified free fatty acids (NEFA) and glycerol into the bloodstream, resulting in turn in increased serum levels of fatty acids and in the establishment of insulin resistance. The changes in the size of the fat depot undoubtedly lead to changes in the area surrounding the adipocyte and in the paracrine function of the cell.
1.2 Physiological role of white and brown adipose tissue
The adipose tissue is divided into white adipose tissue (WAT) and brown adipose tissue (BAT).
The white adipose tissue is composed of unilocular fat cells (Fig.1) and is the most widespread in the human organism. Its physiological functions are:
• mechanical function: it covers nerves, blood vessels, muscles and fills some interstices in the bone marrow.
• insulating function: the fat does not conduct and prevents dissipation of heat generated by the
body.
• reservoir function: the plasma membrane of the adipocyte contains the enzyme lipoprotein lipase (LPL), which undermines the lipids from their carrier proteins (hepatic lipoproteins VLDL or enteric chylomicrons) and splits them into glycerin and fatty acids; the latter then cross the membrane and enter the cytoplasm, where they are re-converted into triglycerides.
• it is an integral part of the regulation of appetite.
• it is an integral part of the regulation of metabolism and homeostasis of glucose.
• it is involved in the functions of human fertility.
• it significantly regulates the formation and differentiation of blood cells.
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• it contributes to the processes of blood coagulation.
• it plays a central role in several mechanisms of non-specific and specific cell-mediated and humoral immune defense.
• in case of infection, it releases factors that activate and stimulate the immune system.
The WAT is not constituted only by adipocytes: it is also composed by other cell types, including pre-adipocytes, fibroblasts, endothelial cells and macrophages that are contained mostly in the stromal vascular fraction (SVF).
Brown adipose tissue (BAT) is specialized for non-shivering thermogenesis, the process whereby the energy derived from fatty acids oxidation is used for the generation of heat due to mitochondrial uncoupling (Koppen A et al., 2010; Cannon B et al., 2004). In brown adipocytes, triglycerides are organized as multilocular adipocytes (multiple small lipid droplets, Fig.1), which are immunoreactive for the functional thermogenic protein uncoupling protein1 (UCP1).
(Koppen A et al., 2010). For a long time it was assumed that in humans BAT was mainly present in infants and relatively scarce if not absent in adults (Lean ME et al., 1986). In contrast, small mammals preserve BAT through adulthood and this provides an efficient defense against the cold. The ratio WAT / BAT varies with genetic background, sex, age, nutritional status and environmental conditions (Frontini A and Cinti S, 2010).
5 Figure 1. Unilocular adipocytes of white adipose tissue (left) and multilocular adipocytes of brown adipose tissue (right).
WAT and BAT are commonly held to occupy distinct anatomical sites in the body. However, it is reported in literature that WAT and BAT are found together in subcutaneous and visceral fat depots, collectively forming a multi-depot organ that is called "adipose organ" (Frontini A and Cinti S, 2010).
1.3 The evolution of the white adipose tissue concept
The white adipose tissue has been considered for a long time only a passive site of energy storage. This energy, accumulated as triacylglycerols during the periods of excessive consumption of food, is mobilized when the caloric intake decreases. However, studies emerging over the past several years have established an additional role for the this tissue, indicating the WAT as a true endocrine organ that secretes numerous proteins with a broad biological activity (Fig.2).
6 Figure 2. Evolution of concept of adipose tissue (Kahn B and Flier JS, The Journal of Clinical Investigation, 2000).
In addition to release free fatty acids into the bloodstream (Free Fatty Acids, FFA) and cholesterol, WAT synthesizes and secretes a number of molecules, named adipokines, which play an important role in the autocrine and paracrine physiology of the adipose organ (Trayhurn P and Beattie J, 2001). The adipokines are classified according to their structure and their functional role: they include classical cytokines (tumor necrosis factor TNF-α, interleukin 6 IL-6, IL-8, C-reactive protein CRP, pentraxin 3), chemokines (MIP1-α, MCP-1), growth factors (TGF- beta, transforming growth factor ß), proteins of the complement of the alternative system (adipsin, stimulating protein acetylation), the hormone leptin, proteins involved in the vascular homeostasis (PAI-1, plasminogen activator-inhibitor-1), regulating the blood pressure (angiotensinogen), of lipid metabolism (RBP, retinol-binding protein, apolipoprotein E), of
7 glucose homeostasis (adiponectin, resistin), involved in angiogenesis (VEGF, vascular endothelial growth factor), acute phase proteins and of inflammatory response to stress (metallothionein, haptoglobin) (Chiellini C et al., 2002). Adipose tissue can also produce active steroid hormones, including estrogen and cortisol (Bujalska IJ et al., 1997; Deslypere JP et al., 1985) (Fig.3).
Figure 3. Molecules secreted by the white adipose tissue.
Through such secreted products, adipocytes possess the capacity to influence local adipocyte biology, as well as systemic metabolism at sites as diverse as brain, liver, muscle, β cells, gonads, lymphoid organs and systemic vasculature.
This concept raises many possibilities for additional links between adipose function or mass and insulin resistance, independent of the adipocyte roles in energy storage and release.
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1.4 How obesity regulates the expression of key adipokines
A particular emphasis should be placed on changes in the production of the various factors released from the WAT of the obese. These changes of expression were, in some cases, correlated with the onset of diseases related to obesity. Among the wide range of molecules secreted by WAT, there are some that are over-expressed in obesity and others the levels of which are decreased. The physiological significance of these changes remain in most cases to be clarified.
• Leptin is the major secretory product of adipocytes and has both central (especially in the hypothalamus) and peripheral actions. It is defined "satiety hormone", because it leads to a reduction in food intake. Other actions include inhibition of insulin secretion and facilitation of glucose transport (Margetic S et al., 2002). Its expression in the WAT and its circulating levels are positively and strongly correlated with the degree of obesity (Maffei M et al., 1995).
• TNF-α is an inflammatory cytokine over-expressed by the WAT of the obese. It is secreted primarily by the cells of SVF and contributes to insulin resistance, since it inhibits the insulin signaling; it also modulates the production of PAI-1, leptin and regulates the function of BAT in obesity.
• IL-6, IL-8, TGF-β, MIP1-α and MCP-1 are other inflammatory factors the levels of which are increased in the WAT of obese humans and animal models.
• Adiponectin (also known as Acrp30) is a hormone capable of modulating the endothelial adhesion, to inhibit the inflammatory response, to increase insulin sensitivity, to decrease the hepatic gluconeogenesis and to increase the lipid oxidation in the muscle. It is probably involved
9 in the link between atherosclerosis and obesity; in contrast to many other proteins secreted by WAT, the expression of adiponectin and its circulating levels decrease in obesity (Ouchi N et al., 2011).
1.5 Obesity and inflammation
Obesity has been recently defined as a "chronic low inflammatory state" and the rational underlying this new concept is the increased production by the WAT of some pro-inflammatory markers, pro-coagulant factors, cytokines, chemokines and acute phase proteins leading to the activation of the inflammatory pathways NF-kB and JNK. It is unclear whether the increased release of these molecules by the WAT is responsible for a local or systemic (autocrine and / or paracrine) inflammatory state. It is currently believed that the production of these cytokines upon obesity is typical of localized events within the expanding fat cell, maybe due to the state of hypoxia induced by the increased fat mass, that leads in turn to the establishment of various mechanisms of compensation, such as angiogenesis. As being now widely recognized, the increased inflammatory state importantly contributes to the development of insulin resistance and of the other disorders associated with the metabolic syndrome; in this regard a direct link has been identified in the TNF-α, an inflammatory molecule over-expressed by the WAT of the obese, which induces insulin resistance because it interferes specifically with the insulin pathway. According to some schools of thought, obesity is not only the cause of the inflammation, but also to some extent a consequence of it (Kahn B and Flyer JS, 2000).
The molecular and cytological alterations taking place in WAT upon obesity play a determinant role in this phenomenon. It is in fact known that obesity is associated with a massive infiltration of macrophages in WAT, but not in liver and muscle (Weisberg SP et al., 2003). This accumulation of macrophages certainly contributes to the inflammatory-like gene expression pattern displayed by the WAT of the obese (Fig.4). The mechanisms underlying macrophages
10 recruitment are still a matter of investigation, and likely involve increased secretion of chemotactic molecules by the adipocytes.
Figure 4. Macrophage infiltration in the obese white adipose tissue (Wellen KE and Hotamisligil GS, The Journal of Clinical Investigation, 2003).
Monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor 2 (CCR2) have been so far considered the main players in this process (Kanda H et al., 2006;
Weisberg SP et al., 2006). Moreover, these studies reported that obese mice deficient for MCP-1 or CCR2 exhibit a lower WAT accumulation of macrophages as compared to obese wild type controls. Adipose tissue macrophages (ATM) in these obese models was not however reduced to that of the lean mice, thus suggesting that other factors induced by obesity concur to the active recruitment of inflammatory cells.
A new area of investigation concerns in fact the role of adipocytes in the recruitment of immune cells in the blood and in the onset of inflammation. It has also been shown that adipocytes and some types of immune cells (T lymphocytes and macrophages) have similar roles in processes such as complement activation and production of inflammatory cytokines. Furthermore, the
11 precursors of adipocytes have a potent activity as phagocytic cells and can be transformed into macrophage-like cells in response to appropriate stimuli (i.e. TNF-α). These data add convincing evidence on the existence of a link between obesity, inflammation and immune response.
1.6 Haptoglobin in the adipose tissue
Among the genes over-expressed in the WAT of the obese, haptoglobin (Hp), a classical marker of inflammation, has been recently identified in our laboratory (Chiellini C et al., 2004): the expression of Hp is in fact increased of 5 / 6 times in the WAT of animals genetically obese or made obese experimentally by means high-calorie diet feeding.
The data from this study also indicate that the expression of this glycoprotein in the WAT is induced by TNF-α: obese animals that are knockout for TNF-α or for its receptor do not exhibit the typical increase of Hp mRNA observed in obese controls with an intact TNF-α pathway (Chiellini C et al., 2002).
It has been recently demonstrated that Hp is also present in the human WAT, where its expression increases upon obesity (Chiellini C et al., 2002) and that its plasma levels are strongly correlated with the percentage of body fat (Chiellini C et al., 2004). These data are in perfect agreement with the observation reported by Fain and colleagues that human WAT explants are able to release Hp, thus contributing to its concentration in the blood (Fain JN et al., 2004). By using proteomic analysis, it has been suggested that Hp is the primary protein secreted by 3T3- L1 murine adipocytes (do Nascimento CO et al., 2004) and, according to other studies, Hp is secreted by adipocytes isolated from human adipose tissue matrix, but not by cells of the stromal vascular fraction (Fain JN et al., 2004), this being in line with the concept that Hp is one of the few inflammatory molecules secreted specifically by adipocytes and not by the SVF fraction (do Nascimento CO et al., 2004). Serum Hp is also directly correlated with the body mass index (BMI), with the levels of leptin, of C-reactive protein (CRP) and age (Chiellini C et al., 2004).
12 The so far depicted scenario led to consider Hp a novel adipokine and a new marker of adiposity.
Hp belongs to the group of those molecules the expression of which is regulated both by obesity and inflammation and which likely orchestrates the relationship between these two conditions.
1.7 What is Haptoglobin
Before being identified as a marker of obesity, Hp has been characterized with regard to its structure and function in various contexts that will be briefly described in the following paragraphs.
1.7.1 Expression and regulation
Hp is a glycoprotein present in the plasma or serum of all mammals; it is involved in the acute inflammatory response and is primarily synthesized in the liver. It is present at a concentration of 30-300 mg / dl in the serum. The hepatic synthesis of Hp is induced by IL-6, IL-1 and TNF-α.
Hp is also expressed in specific cells of the lung. Furthermore, Hp is highly expressed in the arteries after substantial changes of flow induced by "shear stress" and by NO, which influences in turn the expression of IL-6. The presence of Hp in the arteries plays likely an important role in cell migration and in arterial restructuring. Plasma levels of Hp are reduced in intravascular hemolysis, renal impairment, administration of estrogens (Dobryszycka W et al., 1997); besides obesity, increased Hp plasma levels are found instead in acute and chronic inflammation, cancer metastasizing, malignant lymphomas, tissue necrosis (myocardial infarction), collagen and burns (Quaye IK., 2008).
1.7.2 Genetics and Structure
In humans, there are two common alleles for Hp, named 1 and 2, and consequently three possible different genotypes / phenotypes: Hp 1-1, Hp 2-1 and Hp 2-2. The human Hp is synthesized initially as a single polypeptide chain; it is cut successively in an α-amino terminal chain and ß-
13 carboxy terminal chain, linked by disulfide bridges. The ß chain (40 kDa) is identical in both alleles, whereas the α chain exists in two forms: α1 and α2.
The chain α1 (see Fig.5) binds always to ß chain at the C-terminal to form a unit (α-ß) and the N- terminus binds a unit (α1-ß), forming a homodimer (α1-ß )2 such as for example in the molecule Hp 1-1 (86 kDa). The α2 chain instead binds an additional unit (α-ß). The α2 chains can then form complex polymers, for example (α1-ß) 2 - (α2-ß)n in the molecule Hp 2-1 (86-300 kDa) and (α2-ß) in Hp 2-2 (170-900 kDa).
Figure 5. A) Schematic structure of human α1 andα2 chain.
B) Hp phenotypes (From: Lai IH et al., FEBS Journal, 2008)
1.8 Currently known functions of Haptoglobin
Diversified functions have been attributed to Hp: the most widely described and characterized function of this circulating protein is to bind with high affinity, specifically and
14 stechiometrically, with free hemoglobin (Hb) released by haemolysis (Hb-binding capacity: 38- 230 mg / dl), so preventing, within certain limits, the appearance of hemoglobinuria.
Hb released into the plasma after intravascular hemolytic processes and bound to Hp (irreversible Hp-Hb complex) is recognized by the "scavenger" receptor CD163. The CD163 receptor is a membrane protein expressed exclusively on the surface of macrophages / monocytes of the reticulo-endothelial system and hepatocytes, and its expression is induced by pro-inflammatory stimuli (Moestrup SK and Møller HJ, 2004). This specific receptor-ligand interaction leads to the secretion of cytokines (i.e. IL-10) by macrophages, with the consequent induction of the enzyme heme-oxygenase 1 expression. Thus, there is the removal by endocytosis of the complex Hb-Hp (but not of free Hb or Hp) from the plasma, the degradation to free globin and heme, and the final recovery of the erythrocyte iron. So Hp participates to the maintenance of iron homeostasis by conveying Hb to the liver, the spleen and not to the kidney.
The organ most exposed to hemolytic damage is definitely the kidney: in the absence of Hp, free Hb can damage in fact the glomeruli of the kidney accumulating as ferric intracellular stores (Lim SK et al., 1998).
Hp has also antioxidant properties, since it protects tissues against the oxidative damage caused by free Hb and by its metabolite heme: Hb is a powerful oxidizing agent and can lead to the formation of reactive oxygen species (ROS). Thus the binding to Hp results in the formation of a stable complex that enhances the peroxidase activity of Hb, hampers the intracellular accumulation of free radicals and the formation of NO.
Hp also has an inhibitory action on the biosynthesis of prostaglandins in vitro and in vivo, by acting both on the pathway of cyclooxygenase (COX) and lipoxygenase (LOX); it also promotes angiogenesis, a process that is involved in physiological and pathological inflammatory conditions (embryonic development, tumor growth, systemic vasculitis, myocardial infarction).
The inflammatory process, in fact, due to occlusion or stenosis of the lumen, often results in ischemia or infarction. This suggests an important role of this molecule in the process of tissue
15 repair and, in the case of ischemia, in the formation of new collateral vessels as a compensatory mechanism.
We have also recently demonstrated an important Hp chemotactic activity for monocytes in vitro (Maffei M et al., 2009). This novel property is mediated by the interaction with the chemokine receptor CCR2 and our data indicate that pharmacological inhibition of CCR2 abolishes monocytes migration towards Hp. In this regard Hp, which is upregulated in the WAT of the obese, could participate to the massive infiltration of monocytes observed in obesity, thus confirming its role as intersection between obesity and inflammation.
1.9 Aim
Given the inflammatory / chemoattractant nature of Hp and its specific over-expression and release by the WAT of the obese subject (rodent and human), we wanted to further elucidate the role and the biological significance of Hp in the adipose tissue and in metabolism.
The aim of my thesis project was to investigate whether and how changes in Hp expression might interfere with the onset of obesity-associated complications, often attributed to the altered inflammatory profile of the obese subject and if changes in Hp expression might be relevant to metabolism and WAT inflammatory profile. Based on the Hp increase observed in the WAT of obese subjects, a specific focus of this study is to elucidate how Hp deficiency impacts on WAT in terms of adipocyte size, gene expression and adipogenic potential. These issues were primarily investigated in vivo by employing the rodent Hp -/- model (Hp−/− mouse), for which no metabolic characterization had been so far carried out (Lim SK et al., 1998) and in vitro by using:
murine 3T3-L1 pre-adipocytes, primary human subcutaneous (SC) adipocytes and mouse embryonic fibroblasts (MEFs).
