Visceral WAT (vWAT)

It is well recognized that development of this fat depot is responsible for a detrimental effect on metabolism and therefore represents a risk factor for the development of cardiovascular diseases, as well as dyslipidemia, insulin resistance and hypertension [90]. The metabolic syndrome is characterised by several criteria, one of them being the measurement of the waist circumference [91], which assesses the amount of vWAT, thereby demonstrating the importance of this tissue in metabolic diseases.

Visceral WAT is made of the retro- and the intraperitoneal fat depots, the latter consisting in the omental, mesenteric and, in rodents, in the epididymal WAT (eWAT) fat pad. The amount of vWAT positively correlates with glucose intolerance, plasma LDL cholesterol, triglyceride

31 (TG) and cholesterol levels, as well as with hypertension and dyslipidaemia [92, 93]. Moreover, analysis of the insulin signalling pathway in both human scWAT and vWAT shows that vWAT expresses higher levels of various proteins involved in this pathway, as well as a better insulin sensitivity [94]. Visceral WAT is also more metabolically active, having a higher lipolytic activity and producing more of some adipokines [95].

In rodents, vWAT is located in the thorax and in the abdomen cavities. It is intraperitoneal or retroperitonal (rpWAT). The rpWAT is mainly composed of mesenteric (mWAT), gonadal (gWAT) and perirenal (prWAT) WAT. Gonadal WAT is well developed in rodents, whereas mWAT is limited [79].

Figure 3: Fat mass repartition in mice. Taken from The adipose organ, Cinti, Prostaglandins Leukot Essent Fatty Acids. 2005 Jul;73(1):9-15 Repartition of adipose depots in mice. A: deep cervical, B: anterior subcutaneous, C: visceral mediastinic, D: visceral mesenteric, E: visceral retroperitoneal, F: visceral perirenal, periovarian, parametrial and perivesical, G: posterior subcutaneous.

32 2.3 Development

In many species, WAT formation starts before birth. WAT expansion occurs immediately after birth and is due to an increase in both the size and number of adipocytes, known as hypertrophy and hyperplasia, respectively. Moreover, many studies suggest that WAT expansion in adult does not only result in an increase in adipocyte size, but also by an increase in cell number [96, 97]. Adipocyte hyperplasia was indeed observed in many rodent models [98, 99]. In humans, even if hyperplasia stays controversial, adult human preadipocytes were still shown to be able to differentiate into mature adipocytes in vitro [100], indicating the capacity to generate new mature adipocytes even at adulthood.

Moreover, the SVF fraction can differentiate in vitro into many different cell types, such as adipocytes, myocytes and osteoblasts [101], confirming the plasticity of WAT. It was recently shown that vWAT in humans arises from WT1 expressing cells, which is not the case for scWAT and BAT. The authors also demonstrated that vWAT has heterogeneous progenitors in the mesoderm [102].

This suggests the presence of multipotent precursors in human WAT, in addition to preadipocytes. However, molecular mechanisms involved in cell determination in adipocyte lineage remain poorly unravelled. The mechanisms involved in the differentiation of a fibroblast into a mature adipocyte is, however, well described and is called adipogenesis.

2.4 Differentiation

During the adipocyte differentiation process, acquisition of an adipocyte phenotype is characterised by chronologic changes in the expression of various genes. This is reflected by the appearance of early, intermediate and late markers and by the accumulation of TG. These

33 changes are mainly seen at the translational level. Changes in gene expression during the early and late phases were mainly characterised in an adipocyte mouse cell line, the 3T3-L1 cells [103].

The first step at the beginning of differentiation consists in stopping the proliferation process. In fact, after an exponential phase of growth (regulated by mitogenic factors), adipoblasts, that look like fibroblasts, reach a confluence step that will stop the proliferation.

When cells are stopped at the G0/G1 phase of mitosis, they express early pre-adipocyte markers, like lipoprotein lipase (LPL) and α2 chain collagen type IV. Expression of preadipocyte factor 1 (pref-1) stops at this moment, as it has an inhibitory effect on adipocyte differentiation [104]. Preadipocytes can continue differentiation only if they pass through a clonal expansion phase, which includes several cycles of postconfluent mitosis [105].

However, primary adipocytes from human WAT do not need cell division to enter into the differentiation process [106].

At least two families of transcriptional factors are induced during the early phase of adipocyte differentiation: C/EBP and PPAR. PPARγ is expressed only in adipocytes, its expression is low but detectable in preadipocytes and increases during the differentiation process to maintain a high level in mature adipocytes. A transitory induction of C/EBP-β and C/EBP-δ precedes the induction of PPARγ; It decreases before the late phase, and is accompanied by an increase in C/EBP-α expression just before induction of specific adipocyte differentiation genes [107, 108]. During differentiation, morphology of the cell is completely remodelled: they pass from a fibroblastic morphology to a spherical form. Cytoskeleton extracellular matrix elements are drastically changed with a decrease of 90 to 95% of actin and tubulin synthesis during the early phase of differentiation in order to allow for a correct differentiation [109]. In 3T3-L1 cells, a

34 decrease in procollagen type I and III and an increase in type IV collagen and chondroitin was measured [110, 111]. Different specific markers are expressed at the cell surface during these phases, as summarized by Figure 4.

Figure 4: Main markers identified during white adipocyte development. Taken from Weighing in on adipocyte precursors, Berry R, Cell Metab. 2014 Jan 7;19(1):8-20. Identification of different markers during white adipocyte differentiation.

During the last phase of differentiation, the adipocytes start to be able to synthesise FAs from glucose (de novo lipogenesis), and acquire their insulin sensitivity. Expression and activity of various proteins involved in TG metabolism, such as ATP citrate lyase, malic enzyme, acetyl Co-A carboxylase, stearoyl-CoA desaturase, glycerol-3- phosphate acyltransferase, glycerol-3-phosphate dehydrogenase, fatty acid synthase, and GLUT4 are increased between 10 and 100 times. The expression of genes encoding for other proteins indirectly involved in

35 lipid metabolism also increases. This is the case for proteins such as aP2, CD36, perilipin, adipsin, leptin, PEPCK [103, 112, 113].

2.5 WAT as an endocrine organ

WAT has long been thought to be a mere storage tissue, only involved in FA release depending on the metabolic needs. But WAT is also a real endocrine organ able to receive hormonal signalling coming from the whole body and to react by secreting hormones named adipokines. These hormones have an impact on many target tissues, such as the liver or skeletal muscles and participate in the general control of energy balance. Some of these adipokines, like leptin or adiponectin activate neural circuits in the hypothalamus, mainly aimed at regulating EE and lipid catabolism [114]. Moreover, under stress, WAT secretes pro- or anti-inflammatory cytokines regulated by the WAT mass and the physiological state of the organism [115].

Table 1: Adipokines secreted by WAT and their impact on the tissue

name expression Main functions Leptin adipocytes Reflects fat mass

Satiety hormone, direct action on the hypothalamus

Stimulates lipolysis, inhibits lipogenesis, increases FA oxidation Adiponectin adipocytes Increases insulin sensitivity

Increases FA oxidation Anti-inflammatory action Il-6 adipocytes Pro-inflammatory cytokine

Inhibits insulin and leptin pathways TNFα macrophages Pro-inflammatory cytokine

Induces insulin resistance Increases lipolysis in adipocytes Expression increased in obesity Pref-1 preadipocytes Inhibits adipogenesis

Its overexpression in WAT impairs insulin sensitivity [116]

36 TGFβ adipocytes/

macrophages

Growth factor

Anti-inflammatory cytokine

Induces proliferation, differentiation and apoptosis [117]

Expression increased in obesity MCP1 adipocytes/

macrophages

Anti-inflammatory chemokine

Recruits macrophages on inflammatory sites Increases lipolysis and leptin secretion 2.6 Lipid homeostasis

One of the main functions of WAT is to store excess energy as TG, which can be degraded into FAs to be used by other tissues in response to metabolic needs, such as during food restriction periods. Synthesis of TG from FAs is named lipogenesis. Adipocytes are able to store large amounts of TG in their lipid droplet surrounded by a protein named perilipin, without causing any lipotoxicity to the cell [118].

2.6.1 Lipogenesis

TG stored in adipocytes are synthetized from fatty acids (FA) and glycerol, after being transformed, respectively into acyl-CoA and glycerol-3-phosphate (G3P). Most FAs come from circulating plasma lipids, whereas G3P has two main origins: glycolysis and glyceroneogenesis.

Plasma circulating FA levels are mainly non esterified FA (NEFA) linked to albumin, or TG incorporated within lipoproteins, mainly very low density lipoproteins (VLDL) or chylomicrons.

TG are first hydrolysed by lipoprotein lipase (LPL), an enzyme linked to capillaries of WAT and skeletal muscles, in order to release FAs [119]. Its expression and activity is increased in WAT during the postprandial period, mainly under diet enriched in carbohydrates, due to a

37 stimulatory effect of insulin. In contrast, LPL expression and activity are decreased during a starvation period or in response to a high fat diet (HFD) [120].

Long chain fatty acids (LCFA) need specific transporters to cross the plasma membrane [121]. White adipocytes express different FA transporters, such as "cluster of differentiation 36" (CD36), "fatty acid transport protein" (FATP) and "fatty acid binding protein" (FABP). CD36 is responsible for the majority of FA uptake [122]. Insulin improves this transport by stimulating the expression of these transporters, as well as their presence at the plasma membrane level [123].

FA are soluble in the cytosol only. To avoid cytotoxic effects, they are linked to a cytosolic protein, the FABP, which transports FAs to the action site of acyl-CoA synthase.

Human white adipocytes express two type of FABP: "adipocyte protein2" (AP2) (product of the FABP4 gene) and the "keratinocyte lipid-binding protein" (KLBP). AP2 is exclusively expressed in adipocytes and is the main molecule involved in FA transport, whereas KLPB is also expressed in macrophages [124]. The first step in FA metabolism after being linked to FABP is the activation of LCFA to LCFA-CoA through the acyl-CoA synthase. LCFA-CoA are then oxidized or orientated to the synthesis of more complex lipids, such as TG. Oxidation of LCFA-CoA occurs in the mitochondria after their transport inside this organelle by carnitine-palmitoyl transferase I (CPT1).

Another source of FA is through de novo lipogenesis, which is the synthesis of new FA molecules from non-lipid substrates, mainly carbohydrates in mammals. Expression and activity of enzymes involved in lipogenesis and glycolysis are closely related in the liver and WAT, which are lipogenic tissues. De novo lipogenesis is less active in humans than in rodents and it contributes to a small amount of TG production in adipocytes [125, 126].

38 Synthesis of G3P is needed for the esterification of FA into TG. G3P is produced from glucose (via glycolysis) or from glyceroneogenesis [127]. The limiting step of this latter process is the cytosolic form of phosphoenol pyruvate carboxykinase (PEPCK). Relative contribution of glycolysis and glyceroneogenesis to produce G3P depends on nutritional and pharmacological factors. Global availability of G3P controls FA esterification coming from de novo lipogenesis or circulating lipids, as well as partial re-esterification of FA released by hydrolysis of TG.

Figure 6: Lipogenesis pathways in white adipocytes. These pathways enable energy storage as lipids. FA are transported as lipoproteins (chylomicrons and VLDL), FA and glucose are transformed into acylCoA and G3P, respectively, which are both needed for triacylglycerol synthesis (TAG on the Figure). DAG: diacylglycerol, MAG: monoacyglycerol, LPA:

lysophosphatidic acid, PA: phosphatidic acid.

39 2.6.2 Lipolysis

In WAT, TG are hydrolysed when energy needs are not completely covered by food intake. TG in adipocytes are successively hydrolysed into diacylglycerols (DAG) and monoacylglycerols (MAG) to ultimately produce three molecules of FA and one molecule of glycerol. Glycerol produced by lipolysis (TG degradation into FAs and glycerol) is released into the bloodstream to be transformed to G3P by glycerol kinase in the liver or to be used by other tissues. This glycerol release depends in part on aquaporine, a canal protein in the plasma membrane. FAs produced by lipolysis are also released in the circulation or re-esterified directly as TG, inside the adipocytes. The intracellular FA cycling depends on G3P availability and on the re-esterification of FAs thanks to the triglyceride synthase. In the basal state, this cycling is negligible, but it increases during starvation periods, stress [128] or in pathological situations, such as hyperthyroidism [129]. The extent of FA re-esterification plays a role in the regulation of plasma FA levels.

Hormone sensitive lipase (HSL) plays a key role in hydrolysis of TG. Lipolysis is activated by hormones or mediators activating the adenylate cyclase system (glucagon, adrenaline, noradrenaline), inducing an intracellular increase in cyclic adenosine mono phosphate (cAMP) that phosphorylates protein kinase A, which will then phosphorylate HSL and perilipin.

Inhibition of HSL expression in mice induces an accumulation of DAG instead of TG. HSL hydrolyses DAG and then a MAG lipase releases the last FA and one glycerol. MAG lipase is the limiting enzyme in the release of glycerol and free fatty acids (FAs).

40 Adipose triglyceride lipase (ATGL) is another lipase present in WAT. Inhibition of both ATGL and HSL induces an inhibition of 90% of TG hydrolysis [130]. In fact, TG hydrolysis depends mainly on ATGL activity and less on HSL activity.

Figure 7: Lipolysis pathways in white adipocytes. TG (TAG on the Figure) are degraded between meals when body needs energy. The FAs produced will be then be used by the mitochondria (β oxidation) to produce ATP or they will be exported to the tissues which are able to use FAs (mainly skeletal muscles, heart, liver, adipose tissue).

3) BAT

BAT is characteristic of mammals, more precisely of hibernating animals because this tissue is responsible for heat production by consuming lipids [131]. The association between BAT and hibernation was described for the first time by Gessner in 1551 in marmots.

41 In contrast to white adipose tissue, brown adipose tissue (BAT), which is darker due to the presence of cytochrome c in the mitochondria and a higher degree of vascularization, is specialised in adaptive thermogenesis [132]. Even if the role of BAT has been well studied in rodents and human new-borns, its persistence and importance in adult humans are currently actively studied and its functions remain to be precisely determined [133-135].

3.1 Brown adipocyte

BAT is composed of brown adipocytes mainly. In contrast to white adipocytes that contain a unique and big lipid droplet, brown adipocytes are multilocular, have a central nucleus and a large cytoplasm that contains a lot of mitochondria. This tissue is highly vascularized and innervated.

Figure 8: Brown adipocyte. Histology of brown adipose tissue stained with hematoxylin eosin (HE). As schematized on the right panel, the cell has a central nucleus, a large cytoplasm full of active mitochondria and lot a small lipid droplets.

Brown adipocytes have a common origin with skeletal muscles, which is not the case for white adipocytes [136]. Thus, it was demonstrated by cell tracing that both brown adipocytes and myocytes express Myf5, strongly suggesting that they share a common precursor [137].

42 3.2 UCP1 in mitochondria

In eukaryotes, mitochondria are responsible for oxidative metabolism. It contains enzymes that catalyse the citric acid cycle and FA oxidation, ultimately producing ATP as an energy source for the cell. This organelle has a smooth outer and an inner membrane. Porine is a constituent of the outer membrane, which allows free circulation of proteins with a molecular weight lower than 10kDa. The inner membrane contains a lot of invaginations named cristae. The number of cristae (increasing the membrane surface) reflects the respiratory activity of the cell. The proteins that achieve the electron transport, linked with oxidative phosphorylation, lie within the inner membrane. This membrane contains 75% of proteins and is richer in this constituent than the outer membrane. It is only permeable to O2, CO2 and water. It also contains many proteins involved in the transport of metabolites, such as ATP, ADP, pyruvate, Ca²⁺ and phosphate. Internal compartment is constituted of a gel (50%

of water) named matrix, containing high concentrations of soluble enzymes involved in oxidative metabolism (such as citric acid cycle enzymes). Overall, the various systems present in mitochondrial membranes allow for the maintenance of ionic gradients through the membranes.

Major implication of the mitochondria in energy metabolism is due to the occurrence of many metabolic pathways within this organelle, as well as its ability to convert energy into ATP. Pr Peter D. Mitchell received a Nobel Prize in 1978 for his chimio-osmotic theory [138]. According to this theory, electron transfer coming from NADH or FADH2 to oxygen, via different complexes of the respiratory chain, is associated with protons’ expulsion from the mitochondrial matrix into the intermembrane space. This proton flux creates an

43 electrochemical gradient on either side of the inner membrane of the mitochondria.

Dissipation of this gradient by ATP synthase in the presence of ADP allows for ATP synthesis.

Thus, ATP production depends, via the protons’ gradient, on the function of the respiratory chain. It is named coupled respiration [139].

Figure 9: Mitochondrial coupled respiration. Taken from: Mitochondrial uncoupling proteins in the CNS: in support of function and survival. Horvath et al, Nature Reviews Neuroscience 6, 829-840, November 2005. Glucose or FA oxidation produces NADH and FADH2, releasing electrons that will be used by the electron transport chain or complexes one to four. This releases protons into the intermembrane space. Proton’s reuptake by ATP synthase produce ATP from ADP.

This mechanism implies that the inner membrane is impermeable to protons. It is the case for the majority of cells, but one exception exists in animals, in the mitochondria of brown adipocytes responsible for thermogenesis. This process is possible thanks to a high permeability for protons through the inner membrane. Indeed, it allows dissipating the electrochemical gradient independently from ATP synthase. The consequence is that the

44 respiratory chain and ATP production are dissociated and energy is released as heat. The whole process is called uncoupled respiration. A few years after its initial description by Nicholls [138], a protein, which was a member of the mitochondrial anionic transporters, was discovered and its gene cloned [140]. As this protein, inserted in the inner membrane, acted as a proton channel and was responsible for an increased inner membrane permeability to protons, it was called the “uncoupling protein 1” or UCP1. Thus, UCP1 decreases ATP production by using protons from the respiratory chain [141].

Figure 10: Uncoupled respiration. Adapted from: Mitochondrial uncoupling proteins in the CNS: in support of function and survival. Horvath et al, Nature Reviews Neuroscience 6, 829-840, November 2005. UCP1 dissipates the protons’ gradient resulting from the electron transport chain to generate heat, decreasing ATP production by ATP synthase.

Importance of UCP1 in the metabolic regulation was highlighted by several transgenic models in mice. Surprisingly, Ucp1 KO mice have the same phenotype as wild-type mice at 22°C. At this temperature, mice develop other pathways to induce thermogenesis, such as shivering thermogenesis, which probably explains why they can maintain a normal body weight gain and fat content. However, at thermoneutrality (30°C), Ucp1 KO mice become

UCP1

45 obese, probably due to the fact that BAT, via UCP1, is the unique way to dissipate energy excess when animals do not need to maintain their body temperature [142]. It was also shown that under a HF diet, Ucp1 KO mice become more obese than wild-type mice, demonstrating that UCP1 activity is indispensable for diet-induced thermogenesis [143].

3.3 Beige adipocyte

Occurrence of brown-like adipocytes in rodents in WAT was shown to contribute to non-shivering thermogenesis [144]. These recruitable adipocytes were named BRITE for

“BRown to whITE” or beige adipocytes. Their morphology is very close to that of brown adipocytes. They have a central nucleus and various multilocular lipid droplets. However, in contrast to brown adipocytes, they are not coming from a Myf5+ cell lineage [137]. Moreover, differences between mice strains were observed in the development of UCP1-positive cells in

“BRown to whITE” or beige adipocytes. Their morphology is very close to that of brown adipocytes. They have a central nucleus and various multilocular lipid droplets. However, in contrast to brown adipocytes, they are not coming from a Myf5+ cell lineage [137]. Moreover, differences between mice strains were observed in the development of UCP1-positive cells in