Kalypso Karastergiou

Vidya Mohamed-Ali

Mohamed-Ali Adipokines

The autocrine and paracrine roles of adipokines

Keywords: Adipokines, adipogenesis, lipolysis, obesity, adipose tissue, inflammation

Obesity, defined by an excess of adipose tissue, is often associated with the development of various metabolic diseases. The increased and inappropriate deposition of this tissue contributes to hyperglycemia, hyperlipidemia, insulin resistance, endothelial dysfunction and chronic inflammation. Recent evidence suggests that factors expressed and secreted by the adipose tissue, adipokines, may contribute to the development of these abnormalities by mechanisms including inhibition of adipogenesis, adipocyte hypertrophy and death, immune cell infiltration and disruption of tissue metabolism. The presence of adipokine receptors in adipocytes renders these cells available to autocrine and paracrine effects of adipokines. In this review the reported local effects of adipokines on adipose tissue structure, inflammation and regulation of metabolic functions, in the face of over-nutrition and consequent obesity, are outlined. Elucidating the local regulation of white adipocyte development and function could help in the design of effective, tissue-specific therapies for obesity-associated diseases.

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Obesity has been described as a low grade inflammatory condition, with both the cellularity and the secretions of adipose tissue reflecting these changes. Current evidence suggests that over-nutrition leads to adipocyte hypertrophy, followed by cell death, which may act as a stimulus for immune cell infiltration into the tissue [START_REF] Strissel | Adipocyte death, adipose tissue remodeling, and obesity complications[END_REF][START_REF] Murano | Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice[END_REF]. Monocyte infiltration and differentiation in particular has been shown to correlate with adipocyte hypertrophy, as well as body mass [START_REF] Weisberg | Obesity is associated with macrophage accumulation in adipose tissue[END_REF]. These macrophages, in response to the endocrine and metabolic milieu prevalent in the obese adipose tissue, switch their phenotype from one of a non-inflammatory resident macrophage to that of a lipid engorged foam cell, expressing dendritic cell markers, such as CD11c [START_REF] Lumeng | Obesity induces a phenotypic switch in adipose tissue macrophage polarization[END_REF]. The secretions of these macrophages, such as IL-6 and TNFα along with those from the hypertrophied adipocyte, such as MCP-1 and leptin, regulate the pathological changes of obesity, like insulin resistance and endothelial dysfunction (Figure 1). These autocrine/paracrine effects augment the endocrine changes mediated by the molecules in other organs such as liver, heart and skeletal muscle. All the signals released from or expressed by the adipose tissue are referred to as adipokines here, even though these include cytokines, chemokines and products of tissue enzyme activity (Table 1).

A c c e p t e d M a n u s c r i p t 3 i) Adipokines, adipose tissue structure and regional distribution. Two histologically and functionally distinct types of adipose tissue have been identified: white (WAT) and brown adipose tissue (BAT). In mice, BAT exists throughout adulthood, while in humans it was thought to exist mainly in the neonatal period, having largely disappeared within the first years after birth. However, recent evidence suggests that adults retain metabolically active BAT depots that can be cold-induced and respond to sympathetic nervous system activation [START_REF] Frühbeck | BAT: a new target for human obesity?[END_REF].

While BAT is specialized in the production of heat and lipid oxidation, WAT functions as insulation, an exemplary store of excess energy as triacylglycerols, mediator of glucose homeostasis and an important endocrine/immune organ that secretes adipokines, that include inflammatory cytokines, chemokines, acute phase proteins and complementlike factors (Table 1 lists some of these). These adipokines may act as endocrine factors that regulate many central and peripheral processes, including appetite, energy metabolism, glucose and lipid metabolism, inflammatory responses, angiogenesis, blood pressure and reproductive function. The endocrine effects of adipokines have been extensively and expertly reviewed elsewhere [START_REF] Halberg | The adipocyte as an endocrine cell[END_REF].

WAT is composed of many cell types, with about 50% of it being adipocytes, 10% resident macrophages and the rest comprising preadipocytes, endothelial and epithelial cells. Both the adipocytes and the non-adipocyte cells secrete adipokines. Adiponectin, and to a lesser extent, leptin, are largely adipocyte products, with signals such as MCP-1 and IL-6 being produced by the hypertrophied fat cell and the non-fat cells producing many of the other inflammatory signals. In obesity, there is macrophage infiltration into WAT, with the number of these macrophages being directly correlated to adipocyte size (Cousin et al., 1999). In addition the phenotype of the macrophage is also altered to reflect a more inflammatory state (M1; [START_REF] Lumeng | Obesity induces a phenotypic switch in adipose tissue macrophage polarization[END_REF]). It appears that these alterations in the cellularity of the tissue may lead to changes in local adipokines secretion and can potentially cause dysregulation of WAT-mediated disposal of dietary glucose and lipids, and in turn, contribute to hyperglycemia, hyperlipidemia, insulin resistance and chronic inflammation [START_REF] Trayhurn | Endocrine and signalling role of adipose tissue: new perspectives on fat[END_REF]. Depot specific differences in adipose tissue secretory function are also dependent on the anatomical location of this tissue, which can accumulate in different compartments within the body. It is deposited peripherally in subcutaneous depots, such as mammary and gluteofemoral regions, and internally in visceral depots, as in the omental and epicardial regions. The expansion of these depots may occur both through adipocyte hypertrophy and hyperplasia. Accumulation of excess fat in the visceral compartment, even if subcutaneous fat mass is normal, carries the greater metabolic risk [START_REF] Weyer | Enlarged subcutaneous abdominal adipocyte size, but not obesity, predicts type II diabetes independent of insulin resistance[END_REF][START_REF] Wajchenberg | Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome[END_REF][START_REF] Smith | Contributions of total body fat, abdominal subcutaneous adipose tissue compartments, and visceral adipose tissue to the metabolic complications of obesity[END_REF]. Insulin resistance, the precursor to type 2 diabetes, is closely associated to adipocyte hypertrophy in visceral, as opposed to subcutaneous, adipose tissue. It has been proposed that inhibition of adipogenesis, the process of conversion of pre-adipocytes to lipid-laden adipocytes, in subcutaneous depots, in the face of a continuing ability to differentiate visceral adipocytes normally, leads to type 2 diabetes [START_REF] Danforth | Failure of adipocyte differentiation causes type II diabetes mellitus?[END_REF]. This may be because the excess lipid that would have been stored in the subcutaneous depots is shunted to liver and skeletal muscle A c c e p t e d M a n u s c r i p t 4 and results in impaired insulin action within these tissues and the development of peripheral insulin resistance [START_REF] Smith | Contributions of total body fat, abdominal subcutaneous adipose tissue compartments, and visceral adipose tissue to the metabolic complications of obesity[END_REF]. Visceral depots also secrete more of the proinflammatory cytokines, such as IL-6, visfatin and MCP-1 (Greenberg andObin, 2006, Madani, et al., 2009), which may be responsible for the insulin resistance. Conversely, in comparison with visceral adipose tissue, the subcutaneous depots appear less metabolically active and secrete more leptin and less free fatty acids [START_REF] Wajchenberg | Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome[END_REF]. Therefore, excessive accumulation of subcutaneous adipose tissue is more likely to be associated with hyperleptinemia and may lead to central leptin resistance and greater susceptibility to further weight gain [START_REF] Wajchenberg | Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome[END_REF][START_REF] Smith | Contributions of total body fat, abdominal subcutaneous adipose tissue compartments, and visceral adipose tissue to the metabolic complications of obesity[END_REF]. Thus it is apparent that differential regional deposition of adipose tissue can impact on disease outcome, perhaps mediated by their altered adipokine repertoire.

ii) Adipokine receptors and signal transduction. The presence of several of the adipokine receptors in adipocytes from both subcutaneous and visceral depots potentially renders these cells available to autocrine/paracrine regulation by adipokines. It has been demonstrated that human adipocytes express leptin receptors, both the long form (huOb-R), as well as the short forms B219.1 to B219.3 [START_REF] Kielar | Leptin receptor isoforms expressed in human adipose tissue[END_REF][START_REF] Apran-Husmann | Effects of leptin on the differentiation and metabolism of human adipocytes[END_REF]. The IL-6 receptor, together with the signal-transducing gp130 unit, is also present in isolated mature human adipocytes, as well as in those differentiated in vitro [START_REF] Päth | Human breast adipocytes express interleukin-6 (IL-6) and its receptor system: increased IL-6 production by beta-adrenergic activation and effects of IL-6 on adipocyte function[END_REF]. The two TNFα receptors, (TNFR1 and TNFR2) are ubiquitously expressed, and interestingly, the expression of TNFR2 in human adipose tissue is positively correlated with BMI [START_REF] Hotamisligil | Differential regulation of the p80 tumor necrosis factor receptor in human obesity and insulin resistance[END_REF]. Similarly, both adiponectin receptors are expressed (mRNA) in human adipose tissue and in isolated adipocytes from both subcutaneous and omental depots. Their levels correlate negatively with indices of obesity and metabolic dysfunction and are increased by physical exercise [START_REF] Rasmussen | Adiponectin receptors in human adipose tissue: effects of obesity, weight loss, and fat depots[END_REF][START_REF] Nannipieri | Pattern of expression of adiponectin receptors in human adipose tissue depots and its relation to the metabolic state[END_REF][START_REF] Blüher | Gene expression of adiponectin receptors in human visceral and subcutaneous adipose tissue is related to insulin resistance and metabolic parameters and is altered in response to physical training[END_REF]. However, expression of the receptors is not proof of locally acting adipokines. Most of the evidence comes from in vitro studies that have used supra-physiological or pharmacological doses of these molecules to elicit a response. In the absence of tissue specific receptor knockout models or pharmacological inhibitors of the receptors local effects remains speculative.

In addition to the specific adipokine receptors, all toll-like receptors (TLRs 1-10, apart from 5 and 7) are expressed both in the stromavascular fraction and in mature adipocytes, from human subcutaneous and visceral adipose tissue [START_REF] Kopp | Innate immunity and adipocyte function: ligand-specific activation of multiple Toll-like receptors modulates cytokine, adipokine, and chemokine secretion in adipocytes[END_REF]. These TLRs are essential innate immune receptors that classically alert the immune system to the presence of pathogens [START_REF] Zhang | Mammalian toll-like receptors: from endogenous ligands to tissue regeneration[END_REF]. They recognize a large number of ligands, which includes LPS, zymosan and glycosylphosphotidylinositol lipid, and are capable of inducing the expression of a variety of inflammatory genes, like cytokines and chemokines. They can thus attract and activate T-cells in response to chemokines, such as RANTES. Their role in obesity or in response to excess energy intake is currently being investigated.

A c c e p t e d M a n u s c r i p t 5

The signalling pathways that are apparent in the adipocytes and can be activated by the adipokines include those most commonly implicated in inflammation such as the NFκB and JAK/STAT pathways (Table 2).

iii) Adipokine regulation of adipose mass and blood flow.

During adipogenesis, fibroblast-like preadipocytes differentiate into lipid-laden and insulin-responsive adipocytes. This process occurs in several stages and involves a cascade of transcription factors, among which peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPs) are considered the crucial determinants of adipocyte fate. The involvement of additional factors, including Krupel-like factors (KLFs), Wingless and INT-1 proteins (Wnts), and cell-cycle proteins has also been reported [START_REF] Lefterova | New developments in adipogenesis[END_REF]. Differentiation of fibroblast-like preadipocytes to mature adipocytes is a continuous process in adipose tissue. Although the number of total adipocytes in an individual is determined during childhood and adolescence, there is constant turnover and about 10% of all adipocytes are renewed per year at all adult ages and throughout the spectrum of BMI [START_REF] Spalding | Dynamics of fat cell turnover in humans[END_REF]. Several of the proinflammatory adipokines, such as TNFα, act as potent inhibitors of adipogenesis, possibly via inhibition of PPARγ-dependent phenomena, as shown by the fact that thiazolidinediones prevent TNFα-induced anti-adipogenesis [START_REF] Cawthorn | TNFα and adipocyte biology[END_REF]. Furthermore, both in 3T3L1 [START_REF] Cawthorn | Tumour necrosis factor-alpha inhibits adipogenesis via a beta-catenin/TCF4(TCF7L2)-dependent pathway[END_REF] and in human preadipocytes [START_REF] Isakson | Impaired Preadipocyte Differentiation in Human Abdominal Obesity[END_REF], this TNFα-effect is dependent on Wnt/β-catenin/T-cell factor 4 pathway.

IL-6 (20 ng/ml) also inhibits differentiation and lipid accumulation of 3T3L1 fibroblasts. The main adipogenic transcription factor affected by IL-6 is C/EBPα, whereas earlier activation of C/EBPδ, C/EBPβ and PPARγ remain intact [START_REF] Gustafson | Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes[END_REF]. Chemerin, acting via the relevant receptor in 3T3L1 and human preadipocytes also impairs PPARγ expression and cellular differentiation (Gorlaski et al., 2007[START_REF] Roh | Chemerin -A new adipokine that modulates adipogenesis via its own receptor[END_REF]. Interestingly, with all these adipokines, not only is adipogenesis is inhibited, but in addition, the cells acquire a more macrophage-like phenotype [START_REF] Isakson | Impaired Preadipocyte Differentiation in Human Abdominal Obesity[END_REF] and express a number of inflammatory genes, like IL-6, MCP1 and PAI-1 [START_REF] Gustafson | Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes[END_REF], Gorlaski et al., 2007). Resistin had no clear effect on the in vitro differentiation of 3T3-L1 adipocytes [START_REF] Kim | A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation[END_REF][START_REF] Gong | Resistin promotes 3T3-L1 preadipocyte differentiation[END_REF], however, in vivo data from a transgenic mouse model support an inhibitory role of resistin in adipogenesis [START_REF] Kim | Dominant inhibitory adipocyte-specific secretory factor (ADSF)/resistin enhances adipogenesis and improves insulin sensitivity[END_REF]. The same appears to apply to PAI-1, as primary adipocytes from PAI-1 deficient mice show accelerated differentiation and overexpression of PAI-1 in 3T3L1 adipocytes suppressed CEBPα and PPARγ expression, and, subsequently differentiation [START_REF] Liang | Plasminogen activator inhibitor-1 modulates adipocyte differentiation[END_REF].

Data on adipokines that may act as promoters of differentiation are scarce. In this context the effects of adiponectin on adipogenesis appear to be diverse. In brown pre-adipocytes derived from human bone marrow, adiponectin suppressed differentiation, via activation of COX-2 and increased PGE2 synthesis [START_REF] Yokota | Paracrine regulation of fat cell formation in bone marrow cultures via adiponectin and prostaglandins[END_REF]. On the other hand, in 3T3L1 adipocytes adiponectin overexpression promoted preadipocyte proliferation and accelerated expression of adipogenic transcription factors and adipogenesis [START_REF] Fu | Adiponectin promotes adipocyte differentiation, insulin sensitivity, and lipid accumulation[END_REF]. There are also some data that macrophage migration inhibitory factor (MIF), A c c e p t e d M a n u s c r i p t 6 which is induced and released by adipocytes after treatment with TNFα [START_REF] Hirokawa | Identification of macrophage migration inhibitory factor in adipose tissue and its induction by tumor necrosis factor-alpha[END_REF], might facilitate adipogenesis. Knocking down of MIF in 3T3L1 preadipocytes restricted their proliferation and suppressed expression of C/EBPδ; as a result expression and activation of C/EBPα and PPARγ and cell differentiation were also inhibited [START_REF] Ikeda | Knockdown of Macrophage Migration Inhibitory Factor Disrupts Adipogenesis in 3T3-L1 Cells[END_REF].

The reninangiotensin system (RAS) and the relevant receptors, AT 1 and AT 2 , are expressed in adipose tissue [START_REF] Engeli | Physiology and pathophysiology of the adipose tissue renin-angiotensin system[END_REF]. The effects of the locally produced angiotensin II appear to be dependent on the balance between the expression of the two receptors. Thus, initial in vitro experiments suggested that angiotensin II promotes preadipocyte differentiation via the AT 2 receptor [START_REF] Engeli | Physiology and pathophysiology of the adipose tissue renin-angiotensin system[END_REF]. Subsequently, however, it was shown that the in vivo effects of angiotensin II are mediated primarily via AT 1 and in this case the end result is expansion of adipose tissue, primarily because of adipocyte hypertrophy, and thus metabolic dysfunction [START_REF] Massiéra | Adipose angiotensinogen is involved in adipose tissue growth and blood pressure regulation[END_REF][START_REF] Tomono | Blockade of AT1 receptor improves adipocyte differentiation in atherosclerotic and diabetic models[END_REF].

While not truly adipokines, products of enzyme activity within the adipose tissue such as NEFA and the prostaglandins, also have documented, potent autocrine/paracrine effects on adipose tissue differentiation and metabolism [START_REF] Frayn | Non-esterified fatty acid metabolism and postprandial lipaemia[END_REF][START_REF] Richelsen | Release and effects of prostaglandins in adipose tissue[END_REF][START_REF] Négrel | Prostacyclin as a critical prostanoid in adipogenesis[END_REF]. Prostacyclin especially has been shown to be an inducer of adipogenesis by activating both C/EBP and PPAR [START_REF] Vassaux | Differential response of preadipocytes and adipocytes to prostacyclin and prostaglandin E2: physiological implications[END_REF].

The nitric oxide (NO) pathway has also come under some scrutiny as endothelial function, determined largely by bioavailability of NO, and insulin sensitivity, are both modulated by adiposity [START_REF] Caballero | Endothelial dysfunction in obesity and insulin resistance: a road to diabetes and heart disease[END_REF][START_REF] Ritchie | The role of insulin and the adipocytokines in regulation of vascular endothelial function[END_REF]. Although it has been postulated that adipokines, such as leptin, TNFα and IL-6, act on the endothelium to produce detrimental effects [START_REF] Clarke | Insulin Resistance: Insulin Action and its Disturbances in Disease[END_REF], to date none has clearly been identified. NO is generated in adipose tissue and adipocytes [START_REF] Ryden | Increased expression of eNOS protein in omental versus subcutaneous adipose tissue in obese human subjects[END_REF]. While both eNOS, and to a lesser extent iNOS, are expressed in adipose tissue and adipocytes, only eNOS expression increases with obesity [START_REF] Ryden | Increased expression of eNOS protein in omental versus subcutaneous adipose tissue in obese human subjects[END_REF]. Asymmetric dimethylarginine (ADMA) is a novel risk factor for cardiovascular disease that inhibits nitric oxide (NO) bioavailability and causes endothelial dysfunction [START_REF] Vallance | Blocking NO synthesis: how, where and why?[END_REF]. ADMA is cleared by catabolism through the activity of dimethylarginine dimethylaminohydrolase (DDAH1 and 2; [START_REF] Vallance | Blocking NO synthesis: how, where and why?[END_REF]. In obesity the ADMA generated may act as an autocrine and/or paracrine inhibitor of eNOS, thus potentially affecting several NO-mediated functions of the adipose tissue, such as local blood flow [START_REF] Ardilouze | Nitric oxide and beta-adrenergic stimulation are major regulators of preprandial and postprandial subcutaneous adipose tissue blood flow in humans[END_REF], lipolysis [START_REF] Gaudiot | Modulation of WATlipolysis by nitric oxide[END_REF], glucose metabolism [START_REF] Tanaka | Nitric oxide stimulates glucose transport through insulinindependent GLUT4 translocation in 3T3-L1 adipocytes[END_REF] and mitochondrial biogenesis [START_REF] Nisoli | Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals[END_REF].

The role of NO in energy expenditure, food intake and body weight gain has been explored recently in eNOS -/-mice [START_REF] Nisoli | Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide[END_REF]. In the lean state these animals show no significant differences to wild-type littermates. However, on a weight-gaining diet the eNOS -/-mice were significantly heavier, with this weight gain attributed to greater feed efficiency (described as weight gain per gram of food intake). Adipose tissue from these eNOS -/-animals also had less mitochondrial DNA and mitochondrial enzymes, COXIV and Cytc, suggesting lower mitochondrial biogenesis (Nisoli et al., Recent studies have looked at ADMA in human obesity and shown a close relationship between BMI and plasma levels of ADMA and the L-arg/ADMA ratio, independent of other metabolic risk factors [START_REF] Eid | Relationship Between Obesity, Smoking, and the Endogenous Nitric Oxide Synthase Inhibitor, Asymmetric Dimethylarginine[END_REF]. Overweight and obese subjects had significantly elevated ADMA levels and lower L-arg/ADMA ratio compared to lean or normal individuals. Enzymes responsible for the biosynthesis and degradation of ADMA are expressed in the human adipose tissue [START_REF] Spoto | The enzymatic machinery for ADMA synthesis and degradation is fully expressed in human adipocytes[END_REF]. This study showed ADMA release from adipose tissue from two healthy patients and reported higher DDAH2 in visceral adipose tissue and higher DDAH1 in the subcutaneous depot [START_REF] Spoto | The enzymatic machinery for ADMA synthesis and degradation is fully expressed in human adipocytes[END_REF]. Interestingly adiponectin has been shown to reduce ADMA accumulation in human cultured endothelial cells [START_REF] Eid | Insulin and adiponectin inhibit the TNFalpha-induced ADMA accumulation in human endothelial cells: the role of DDAH[END_REF]. Modulation of this pathway in the adipose tissue has not, as yet, been investigated, especially with reference to the adipokines.

iv) Role of adipokines in inflammation.

The secretory function of adipocytes can also be modulated by adipokines, as shown mainly in in vitro models. TNFα is known to suppress adiponectin production by adipocytes, while it induces a number of pro-inflammatory mediators like IL-6, MCP-1 and PAI-1 [START_REF] Cawthorn | TNFα and adipocyte biology[END_REF], primarily via activation of Foxo1 [START_REF] Ito | Foxo1 increases pro-inflammatory gene expression by inducing C/EBPb in TNF-a-treated adipocytes[END_REF]. Treatment of differentiated human adipocytes with TNF also significantly decreased the expression of angiotensinogen and haptoglobin (Wang et al., 2005). With regards to leptin, TNFα is thought to stimulate release of a preformed pool of leptin by 3T3L1 adipocytes and by murine adipose tissue in vivo [START_REF] Kirchgessner | Tumor necrosis factor-alpha contributes to obesity-related hyperleptinemia by regulating leptin release from adipocytes[END_REF]. On the other hand, in human adipose tissue explants, TNFα has a stimulatory effect on leptin expression and release only in the presence of dexamethasone [START_REF] Trujillo | Tumor necrosis factor alpha and glucocorticoid synergistically increase leptin production in human adipose tissue: role for p38 mitogen-activated protein kinase[END_REF]. Finally, TNFα upregulates expression of apelin in 3T3F442A adipocytes, in murine and human adipose tissue and increases circulating levels of apelin in mice in vivo [START_REF] Daviaud | TNFalpha upregulates apelin expression in human and mouse adipose tissue[END_REF]. Therefore, TNFα might mediate the increased apelin levels noted in obesity [START_REF] Boucher | Apelin, a newly identified adipokine up-regulated by insulin and obesity[END_REF].

IL-6 has similar effects to TNFα on adipokine regulation. Given that it suppresses expression of PPARγ in rat adipocytes [START_REF] Tanaka | Down regulation of peroxisome proliferator-activated receptorgamma expression by inflammatory cytokines and its reversal by thiazolidinediones[END_REF], 3T3L1 and 3T3F442A adipocytes [START_REF] Lagathu | Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion and induced insulin resistance in adipocyte: prevention by rosiglitazone[END_REF] and in human subcutaneous adipose tissue explants [START_REF] Sopasakis | High local concentrations and effects on differentiation implicate interleukin-6 as a paracrine regulator[END_REF], it is not surprising that IL-6 inhibits adiponectin expression and/or release (Fasshauer et al., 2003a[START_REF] Sopasakis | High local concentrations and effects on differentiation implicate interleukin-6 as a paracrine regulator[END_REF][START_REF] Simons | Pro-inflammatory delipidizing cytokines reduce adiponectin secretion from human adipocytes without affecting adiponectin oligomerization[END_REF]. Furthermore, IL-6 can induce PAI-1 [START_REF] Rega | Inflammatory cytokines interleukin-6 and oncostatin m induce plasminogen activator inhibitor-1 in human adipose tissue[END_REF], MCP-1 [START_REF] Fasshauer | Monocyte chemoattractant protein 1 expression is stimulated by growth hormone and interleukin-6 in 3T3-L1 adipocytes[END_REF], as well as its own expression and release (Fasshauer et al., 2003b[START_REF] Lagathu | Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion and induced insulin resistance in adipocyte: prevention by rosiglitazone[END_REF]. Finally, IL-6 acts as a negative regulator of visfatin expression in 3T3L1 adipocytes [START_REF] Kralisch | Interleukin-6 is a negative regulator of visfatin gene expression in 3T3-L1 adipocytes[END_REF] and does not alter leptin release [START_REF] Bruun | Effects of proinflammatory cytokines and chemokines on leptin production in human adipose tissue in vitro[END_REF]. Release of pro-inflammatory cytokines, TNFα and IL-6, is also induced by resistin in isolated human subcutaneous adipocytes, an effect which is possibly mediated by the NFĸB and JNK pathways (Kusminski et al., 2007). It has been proposed that leptin, at least in part, regulates its own release by adipose tissue, based on findings in mice deficient for the leptin receptor, which present with high circulating leptin levels [START_REF] Zhang | The Leptin Receptor Mediates Apparent Autocrine Regulation of Leptin Gene Expression[END_REF]. Furthermore, leptin stimulates adiponectin and visfatin expression and release by A c c e p t e d M a n u s c r i p t 8 adipocytes and murine adipose tissue, both ex vivo and in vivo [START_REF] Delporte | Leptin treatment markedly increased plasma adiponectin but barely decreased plasma resistin of ob/ob mice[END_REF], Tan et al., 2009).

Adiponectin exerts anti-inflammatory effects on adipocytes, as in other cellular populations, primarily via inhibition of the NFĸB pathway [START_REF] Ajuwon | Adiponectin inhibits LPS-induced NF-ĸB activation and IL-6 production and increases PPARγ2 expression in adipocytes[END_REF]. As a result, release of a number of pro-inflammatory cytokines by adipocytes is significantly suppressed (IL-6, IL-8, GROα, MCP-1) [START_REF] Dietze-Schroeder | Autocrine action of adiponectin on human fat cells prevents the release of insulin resistance-inducing factors[END_REF][START_REF] Ajuwon | Adiponectin inhibits LPS-induced NF-ĸB activation and IL-6 production and increases PPARγ2 expression in adipocytes[END_REF][START_REF] Sell | Cytokine secretion by human adipocytes is differentially regulated by adiponectin, AICAR, and troglitazone[END_REF]. These changes might be of particular pathophysiological significance, given that they are adequate to inhibit adipocyte-induced skeletal muscle insulin resistance [START_REF] Dietze-Schroeder | Autocrine action of adiponectin on human fat cells prevents the release of insulin resistance-inducing factors[END_REF]. Similar anti-inflammatory actions might be exerted by vaspin, which was able to induce expression of adiponectin, while inhibiting expression of leptin, resistin and TNFα in murine WAT [START_REF] Hida | Visceral adipose tissuederived serine protease inhibitor: A unique insulin-sensitizing adipocytokine in obesity[END_REF]. Finally, apelin administration resulted in increased circulating adiponectin and reduced leptin concentrations without changes in mRNA expression of these two adipokines in adipose tissue. It seems likely however, that these effects might be secondary to wholebody changes in adiposity and energy expenditure, rather than the primary apelin effect [START_REF] Higuchi | Apelin, an APJ receptor ligand, regulates body adiposity and A c c e p t e d M a n u s c r i p t 18 favors the messenger ribonucleic acid expression of uncoupling proteins in mice[END_REF].

From these in vitro data it is reasonable to speculate that in obesity the inflammatory signals are amplified in the adipose tissue and perpetuate both the cellular and the secretory changes reported. However no concrete evidence is currently available to support this hypothesis.

In view of adipose tissue expansion in obesity, the ability to form new vessels to prevent adipocyte hypoxia, or the failure to do so, might be of particular importance [START_REF] Trayhurn | Hypoxia in adipose tissue: a basis for the dysregulation of tissue function in obesity?[END_REF]. Among the adipokines, leptin is known to promote angiogenesis in several experimental models [START_REF] Sierra-Honigmann | Biological action of leptin as an angiogenic factor[END_REF][START_REF] Bouloumié | Leptin, the product of Ob gene, promotes angiogenesis[END_REF]. In adipose tissue it also induces angiostatic signals (angiopoietin-2) and tissue de-vascularization [START_REF] Cohen | Leptin induces angiopoietin-2 expression in adipose tissues[END_REF]. Adiponectin, by activating AMP kinase in endothelial cells, is also able to induce angiogenesis [START_REF] Ouchi | Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells[END_REF], as shown in adiponectin-deficient mice, which have impaired responses to ischemia [START_REF] Shibata | Adiponectin stimulates angiogenesis in response to tissue ischemia through stimulation of ampactivated protein kinase signaling[END_REF]. IL-6 also has the potential to act as an angiogenic factor, by inducing release of VEGF, as has been reported in both human subcutaneous and visceral adipose tissue explants and in isolated adipocytes and preadipocytes [START_REF] Rega | Vascular endothelial growth factor is induced by the inflammatory cytokines interleukin-6 and oncostatin m in human adipose tissue in vitro and in murine adipose tissue in vivo[END_REF]. Adipose derived VEGF may also directly induce angiogenesis in the tissue [START_REF] Vona-Davis | Angiogenesis, adipokines and breast cancer[END_REF]. Apelin and HGF have also been shown to induce angiogenesis, both in human umbilical vein endothelial cells in vitro, and in adipose tissue in transplantation model in vivo. Interestingly, given that hypoxia induces its expression in 3T3F442A adipocytes, they may represent a compensatory mechanism of adipocytes against oxygen deprivation in obesity and other disease states (Kunduzova et al., 2008;[START_REF] Bell | A central role for hepatocyte growth factor in adipose tissue angiogenesis[END_REF]. v) Autocrine/paracrine regulation of glucose and lipid metabolism. Deposition of energy in adipose tissue occurs primarily via hydrolysis of circulating triglycerides by lipoprotein lipase (LPL), uptake of fatty acids and re-esterification to glycerol within the cell or via de novo lipogenesis. Interestingly, ASP is a potent stimulator of triglyceride synthesis in adipocytes in vitro [START_REF] Baldo | The adipsin-acylation stimulating protein system and regulation of intracellular triglyceride synthesis[END_REF] and is A c c e p t e d M a n u s c r i p t 9 released by adipose tissue in vivo in the postprandial period, when net fatty acid uptake by the tissue occurs [START_REF] Saleh | Coordinated release of acylation stimulating protein (ASP) and triacylglycerol clearance by human adipose tissue in vivo in the postprandial period[END_REF]. TNFα has opposing effects on free fatty acid uptake by adipocytes, probably by interfering with the expression and activity of LPL and fatty acid binding and transport proteins [START_REF] Cawthorn | TNFα and adipocyte biology[END_REF]. It has also been shown that IL-6 inhibits LPL activity in 3T3L1 adipocytes and in murine adipose tissue in vivo [START_REF] Greenberg | Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3-L1 adipocytes: a possible role for interleukin 6 in cancer cachexia[END_REF], whereas resistin increases LPL activity [START_REF] Kim | Resistin is a key mediator of glucosedependent insulinotropic polypeptide (GIP) stimulation of lipoprotein lipase (LPL) activity in adipocytes[END_REF]. In view of some of the opposing effects of these adipokines on fatty acid uptake and the lack of in vivo data it is difficult to speculate on their precise role in obesity.

In the face of negative energy balance, adipose tissue releases free fatty acids after intracellular hydrolysis of triglycerides by adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL). TNFα is a potent stimulator of lipolysis. At least two distinct mechanisms have been implicated: downregulation of perilipin, leading to exposure of the lipid droplet to the lipases, and suppression of the antilipolytic GTPbinding membrane proteins Ga i [START_REF] Rydén | Tumour necrosis factor-α in human adipose tissuefrom signalling mechanisms to clinical implications[END_REF]. Leptin also induces lipolysis in mouse or rat adipocytes [START_REF] Frühbeck | In vitro lipolytic effect of leptin on mouse adipocytes: evidence for a possible autocrine/paracrine role of leptin[END_REF]. In human adipocytes, however, from either lean or obese healthy subjects, leptin treatment failed to modulate lipolysis (basal or isoprotenerol-stimulated or insulin-mediated anti-lipolysis), [START_REF] Apran-Husmann | Effects of leptin on the differentiation and metabolism of human adipocytes[END_REF][START_REF] Elimam | In vitro effects of leptin on human adipocyte metabolism[END_REF].

It has been reported that IL-6 increases basal, as well as isoprenaline-stimulated, lipolysis in 3T3L1 [START_REF] Petersen | Acute IL-6 treatment increases fatty acid turnover in elderly humans in vivo and in tissue culture in vitro[END_REF] and in human mammary adipocytes [START_REF] Päth | Human breast adipocytes express interleukin-6 (IL-6) and its receptor system: increased IL-6 production by beta-adrenergic activation and effects of IL-6 on adipocyte function[END_REF]; however, very high IL-6 concentrations (≥100 ng/ml) were used in these experiments. Under physiological conditions, adipocytes are chronically exposed to IL-6 at the lower ng/ml range, which however exceed circulating concentrations by orders of magnitude [START_REF] Sopasakis | High local concentrations and effects on differentiation implicate interleukin-6 as a paracrine regulator[END_REF]. Finally, recent data indicate that chemerin acts as an inhibitor of both basal and stimulated lipolysis in 3T3L1 adipocytes, possibly via a reduction in intracellular cAMP levels (Gorlaski et al., 2007).

Insulin plays a central role in the regulation of adipocyte metabolism, by acting as a potent inhibitor of lipolysis, while stimulating glucose uptake and fatty acid reesterification. TNFα antagonises the effects of insulin on adipocytes in multiple levels (Cawthorn andSethi, 2008, Rydén M and[START_REF] Rydén | Tumour necrosis factor-α in human adipose tissuefrom signalling mechanisms to clinical implications[END_REF]. Firstly, it reduces protein levels of GLUT-4, insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) in adipocytes, possibly by suppressing PPARγ expression and activity. Secondly, it disrupts downstream insulin signaling by inhibiting tyrosine phosphorylation of IR and IRS-1. Finally, it increases the intracellular concentrations of diacylglycerols and ceramides, which then in turn, compromise insulin signaling further. Leptin also interferes with insulin signaling [START_REF] Pérez | Leptin impairs insulin signaling in rat adipocytes[END_REF][START_REF] Kraus | Leptin secretion and negative autocrine crosstalk with insulin in brown adipocytes[END_REF] and insulin-mediated glucose uptake [START_REF] Kraus | Leptin secretion and negative autocrine crosstalk with insulin in brown adipocytes[END_REF] in mouse or rat adipocytes. As was noted for lipolysis, these findings are not reflected in human adipocytes: in this case, leptin does not appear to affect basal glucose uptake, and there is controversy as to whether or not it inhibits insulin-induced glucose uptake [START_REF] Zhang | Intrinsic Site-Specific Differences in the Expression of Leptin in Human Adipocytes and Its Autocrine Effects on Glucose Uptake[END_REF][START_REF] Apran-Husmann | Effects of leptin on the differentiation and metabolism of human adipocytes[END_REF] and insulin-induced lipogenesis [START_REF] Elimam | In vitro effects of leptin on human adipocyte metabolism[END_REF]. The initial reports regarding IL-6 effects on glucose uptake by 3T3L1 adipocytes were contradictory [START_REF] Stouthard | Interleukin-6 enhances glucose transport in 3T3-L1 adipocytes[END_REF][START_REF] Huppertz | Comparison of the effects of insulin, PDGF, interleukin-6, and interferon-gamma on glucose transport in 3T3-L1 cells: lack of cross-talk between tyrosine kinase receptors and JAK/STAT pathways[END_REF]. Subsequently, high concentrations of IL-6 (≥20 ng/ml) were A c c e p t e d M a n u s c r i p t 10 shown to suppress expression, both at mRNA and protein levels, of GLUT-4 and IRS-1 and thus to reduce glucose uptake. In contrast to TNFα, IL-6 does not interfere with the phosphorylation of IR and IRS-1 [START_REF] Rotter | Interleukin-6 (IL-6) induces insulin resistance in 3T3-L1 adipocytes and is, like IL-8 and tumor necrosis factor-alpha, overexpressed in human fat cells from insulin-resistant subjects[END_REF][START_REF] Lagathu | Chronic interleukin-6 (IL-6) treatment increased IL-6 secretion and induced insulin resistance in adipocyte: prevention by rosiglitazone[END_REF]. Resistin seems to act as another inhibitor of glucose uptake in differentiated human subcutaneous preadipocytes and adipocytes, although its effects appear modest [START_REF] Mcternan | Resistin and type 2 diabetes: regulation of resistin expression by insulin and rosiglitazone and the effects of recombinant resistin on lipid and glucose metabolism in human differentiated adipocytes[END_REF]. Insulin-sensitizing effects of other adipokines have also been reported. Overexpression of adiponectin in 3T3L1 adipocytes resulted in increased expression of GLUT4 and enhanced insulin-mediated glucose uptake [START_REF] Fu | Adiponectin promotes adipocyte differentiation, insulin sensitivity, and lipid accumulation[END_REF]; on the other hand, treatment of pig adipocytes with adiponectin (full-length and globular at 30 μg/ml) inhibited insulin-mediated lipogenesis [START_REF] Jacobi | Cloning and expression of porcine adiponectin, and its relationship to adiposity, lipogenesis and the acute phase response[END_REF]. Vaspin, a product of murine visceral adipose tissue, induced expression of GLUT-4 in WAT after in vivo administration and improved whole-body insulin sensitivity and glucose tolerance [START_REF] Hida | Visceral adipose tissuederived serine protease inhibitor: A unique insulin-sensitizing adipocytokine in obesity[END_REF]. Omentin, which is also preferentially expressed and release by visceral adipose tissue, appears to increase adipocyte insulin sensitivity and promote insulin-stimulated glucose uptake, via stimulation of Akt phosphorylation [START_REF] Yang | Identification of omentin as a novel depotspecific adipokine in human adipose tissue: possible role in modulating insulin action[END_REF]. Finally, chemerin promotes insulin-mediated IRS-1 phosphorylation and concomittantly enhances glucose uptake in 3T3L1 adipocytes [START_REF] Takahashi | A c c e p t e d M a n u s c r i p t 26 Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism[END_REF].

In addition to studies on adipocytes, whole-body animal models can offer further insight in the function of adipokines. Thus, it has been suggested that leptin in vivo inhibits fat accumulation in adipose tissue. It is known that high circulating leptin levels in obesity are not effective in body weight regulation due to the accompanying leptin resistance. However, induction of hyperleptinemia in leptin-sensitive lean rats results in rapid depletion of fat stores. In addition, constitutive overexpression of the leptin receptor in adipose tissue, which overcomes leptin resistance, prevents high fat diet-induced obesity. At a molecular level, this is accompanied by induction of leptin signaling pathways (STAT-3), activation of AMPK and PCG-1α, and upregulation of UCP-1 and UCP-2 in WAT (Wang et al., 2005). Because of the failure of adipose tissue as an energy store, these mice show ectopic fat deposition and metabolic dysregulation [START_REF] Wang | Adipogenic capacity and the susceptibility to type 2 diabetes and metabolic syndrome[END_REF]. Data from a transgenic model suggest that resistin impairs fatty acid re-esterification in the adipose tissue and thus leads to increased free fatty acid release and systemic insulin resistance [START_REF] Pravenec | Fat-specific transgenic expression of resistin in the spontaneously hypertensive rat impairs fatty acid re-esterification[END_REF].

Conclusion.

There is accumulating evidence that in obesity, adipose tissue expands, through adipocyte hypertrophy that is facilitated by inhibition of adipogenesis and adipocyte death. These areas of cell death form focal points for macrophage infiltration into the tissue. The infiltrating macrophage switch from a non-inflammatory phenotype to form foam cells. Continued accumulation of body fat is accompanied by adipocyte hyperplasia and hypertrophy, along with further increases in macrophage numbers. All stages of this process are accompanied by changes in, and are mediated by, autocrine and paracrine effects of adipokines on adipogenesis, angiogenesis, inflammation and adipocyte metabolism. Many of these effects may impact on the development of endothelial dysfunction and insulin resistance, important precursors of various metabolic diseases, such as atherosclerosis and type 2 diabetes. Understanding the putative role of these molecules in shifting the adipocytes from the large, insulin-resistant to the small, insulin-responsive phenotypes, may constitute an attractive strategy for treating metabolic

  induction of specific adipokines may facilitate such a shift in adipose tissue cellularity as well as in adipocyte morphology and function. Thus, elucidating the mechanisms underlying WAT secretory function and the ability to modulate this may, in future, be important in treating the pathological consequences of obesity. ; TNF: tumour necrosis factor; MIF: macrophage migration inhibitory factor; MCP: macrophage chemotactic protein; RANTES: regulated upon activation, normally T cell expressed and secreted; MIP: macrophage inhibitory protein; PAI: plasminogen activator inhibitor; HGF: hepatocyte growth factor; NGF: nerve growth factor; VEGF: vascular endothelial growth factor; VCAM: vascular cell adhesion molecule; ICAM-1: intracellular adhesion molecule; COX: cyclo-oxygenase; NO: nitric oxide; ADMA: asymmetric dimethylarginine; RAS: renin angiotensin system; MMP: matrix metalloproteinase; TIMP: tissue inhibitor of metalloproteinases; RBP: retinol binding protein.

  Figure legends

Figure

  Figure

Acknowledgements.

Aspects of this work were funded by grants from Heart Research UK (RG 2580/09/11), European Commission (EXGENESIS: LSHM-CT-2004-005272) and The Wellcome Trust (Grant number GR078055MA).

A c c e p t e d M a n u s c r i p t 20 Kunduzova, O., Alet, N., Delesque-Touchard, N., Millet, L., Castan-Laurell, I., Muller, C., Dray, C., Schaeffer, P., Herault, J.P., Savi, P., Bono, F., Valet, P., 2008. Apelin/APJ signaling system: a potential link between adipose tissue and endothelial angiogenic processes. FASEB J. 3T3L1 [START_REF] Gustafson | Cytokines promote Wnt signaling and inflammation and impair the normal differentiation and lipid accumulation in 3T3-L1 preadipocytes[END_REF] Porcine adipocytes [START_REF] Yang | Interleukin-6 stimulates lipolysis in porcine adipocytes[END_REF] 3T3L1 (Fasshauer et al., 2003[START_REF] Kralisch | Interleukin-6 is a negative regulator of visfatin gene expression in 3T3-L1 adipocytes[END_REF] 3T3L1 [START_REF] Fasshauer | Monocyte chemoattractant protein 1 expression is stimulated by growth hormone and interleukin-6 in 3T3-L1 adipocytes[END_REF] (↓) CEBPα


Keywords: breast, metaplastic carcinoma, fibromatosis-like

Keloid-type of fibromatosis-like metaplastic carcinoma of the breast with transformation into biphasic tumor

Sir, metaplastic carcinoma of the breast with fibromatosis-like pattern of growth is a recently described type of breast carcinoma 1 . It was originally thought that such tumors exhibited a high risk of local recurrence but showed no tendency to distal spread. In the second published series following the original report, it has been demonstrated that these tumors may occasionally metastasize distantly but not, as also shown in the first study, to the axillary lymph nodes 2 . Morphologically, fibromatosis-like pattern of growth was the only observed pattern but in some cases a minor (less than 5 %) glandular or squamoid component was also present [1][2] . Histological features of recurrent tumors were similar to the initial one 1 . Here, we report a case of fibromatosis-like metaplastic carcinoma with unusual morphology that progressively changed in several recurrences and metastasized to axillary lymph nodes and also distantly. The patient was a 48-year-old woman with a lump in the left breast of recent onset. On aspiration cytology, the tumor was suspicious for malignancy. It was excised and diagnosed as spindle cell tumor, possibly infiltrating myoepithelioma. Excisional margins were free of tumor. No immunohistochemistry was performed since it was not available at that time. Two years later, tumor locally recurred, was excised, only to recur one year later. The patient refused mastectomy, so the tumor was only excised and recurred again 10 months later; simple mastectomy was performed. One and a half year later, a recurrent tumor appeared in a postmastectomy scar. It was excised together with the three enlarged axillary lymph nodes. At the same time, pulmonary metastases were demonstrated on X-ray. Thoracic region was postoperatively irradiated and chemotherapy was introduced. Despite such treatment, the disease ran a downhill course, pleural effusion and skleletal metastases appeared and the patient died in respiratory failure, 6 years following initial surgery. Grossly, the initial tumor was well delineated, white-gray, firm, and measured 1 cm in diameter. Histologically, it was mostly paucicellular with abundant keloid-like hyalinized collagen matrix with some entrapped preexistent breast ducts surrounded by numerous plasma cells. Keloid-like collagen fibres only focally showed some fascicular arrangement, they were largely distributed haphazardly (Figure 1 A). Tumor cells were ovoid to spindle, with minimal pleomorphism (Figure 1 B), only focally denser and slightly pleomorphic. Mitoses were extremely rare. No epithelial structures or epithelioid cell nests were found; tumor was embedded in its entirety. The first recurrent tumor was morphologically similar to the primary, while in the second recurrence focal neoplastic tubular/ductal structures appeared in otherwise predominantly spindle cell tumor with keloid like matrix (Figure 1 C). In the mastectomy specimen, such epithelial formations of low to intermediate grade malignancy were more numerous, focally cribriform, while spindle cell component was generally more cellular, less collagenized, with focal pseudovascular retiform structures; tumor infiltrated skin and underlying skeletal muscle. In 2 of 3 lymph nodes excised together with postmastectomy scar recurrence metastases were seen, showing predominantly epithelial component with some foci of condensed ovoid to spindle tumor cells without or minimal keloid-like collagen (Fig 1 D). Immunohistochemical reactions performed on a later date on primary and recurrences, showed strong diffuse cytokeratin 5, 14, and MNF116 reactivity of spindle cells in all tumors (Fig 2 A-B), while luminal cytokeratin 18 was only positive in ductal epithelial structures. In addition, neoplastic spindle cells also showed positive reaction, although less extensively, for smooth muscle actin, and for p63. Estrogen and progesterone receptors and HER-2 were negative in spindle cell component but estrogen receptors were positive in ductal epithelial component of recurrent tumors. Although morphological spectrum of fibromatosis-like metaplastic carcinoma of the breast is well recognized and its clinical behaviour mostly agreed upon 1-4 , our case deserves recognition for the following reasons: 1. initial tumor presented as a pure spindle cell lesion with unusual keloid-like matrix what is also rarely seen in fibromatosis proper 5 . 2. well to moderately differentiated neoplastic ducts and tubules that were not present in the primary appeared in recurrent tumors producing a biphasic morphologic appearance. (subsequently becoming more numerous; spindle cell tumor appearently " differentiated" into histologically obvious carcinoma)-deleted 3. contrary to previous claims, lymph node metastases in fibromatosis-like metaplastic carcinoma are possible, if only after repeated recurrences, with less obvious spindle cell component.

  Keloid type of fibromatosis-like metaplastic carcinoma of the breast with transformation into biphasic tumor in recurrences and lymph node metastases.

  Figure 1. A, Low power view of the initial fibromatosis-like carcinoma with abundant keloidlike matrix and some entrapped preexistent small ducts. B, Higher magnification to show oval to spindle tumor cells of deceptively benign morphology. C, Ductal neoplastic elements admixed with fibromatosis-like component in the second recurrence. D, Lymph node metastasis with ductal component (right-hand side) and condensed ovoid to spindle cell component (left-hand side).

Figure 2 .

 2 Figure 2. A, Strong positivity of spindle cells for MNF116. B, CK5 positive reaction of spindle cells and lack of it in ductal cells.

Published on behalf of the British Division of the International Academy of Pathology Histopathology


Thomas Bräuniger
email: t.braeuniger@fkf.mpg.de

S Ghedia

M Jansen

Covalent bonds in α-SnF 2 monitored by J-couplings in solid-state NMR spectra

Keywords: 19F-NMR, 119Sn-NMR, J-couplings, heteronuclear spin decoupling, alpha-SnF2 F-NMR, 119 Sn-NMR, J-couplings, heteronuclear spin decoupling, α-SnF 2 2

The stable ambient-temperature phase of tin(II) fluoride, α-SnF 2 , has been studied by 19 F and 119 Sn magic-angle spinning (MAS) NMR spectroscopy.

A large chemical shift anisotropy (CSA) is observed in the spectra of both nuclides, which is indicative of the presence of stereochemically active lone electron pair on Sn. Symmetric satellite lines with low intensity have been detected in the 119 Sn MAS spectra acquired under 19 F decoupling, and are assigned to indirect J-couplings between the magnetically active Sn isotopes. The detection of J-couplings is evidence for the existence of genuine covalent bonds in α-SnF 2 , a fact which has been deduced before from X-ray crystallography, which revealed the presence of Sn-F distances smaller than the sum of the respective ionic radii.

Introduction

Tin(II) fluoride, SnF 2 , is known to crystallise as monoclinic phase (α-SnF 2 ) at ambient conditions [1,2]. At elevated temperatures an orthorhombic phase (β-SnF 2 ) and a tetragonal phase (γ-SnF 2 ) also occur [3], which convert back to α-SnF 2 at standard conditions. The structure of α-SnF 2 is pictured in Fig. 1. It is constructed from an asymmetric unit that contains two tin and four fluorines [1]. Both these elements may be observed by nuclear magnetic Fig. 1 resonance (NMR) spectroscopy (see Table 1), with 119 Sn being the preferred tin isotope as comparatively, it possesses the highest sensitivity. Solid-state NMR has indeed been applied to characterise structure and dynamics of α-SnF 2 [3][4][5]. However, mostly due to apparent inavailability of modern Table 1 magic-angle spinning (MAS) equipment, the NMR data published so far neither achieve satisfactory site resolution for 19 F [3,4] nor for 119 Sn [5].

Here we demonstrate that a reliable NMR characterization of tin(II) fluoride is feasible by using fast MAS and efficient 19 F spin decoupling. The MAS spectra recorded under these conditions possess sufficient resolution to reveal the effects of indirect J-coupling. Unlike dipolar couplings, which act through space, J-couplings are mediated by covalent bonds, and are difficult to observe in solid-state NMR, because they are usually much smaller than dipolar couplings [6][START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF][START_REF] Massiot | [END_REF]. For 119 Sn however, J-couplings tend to be comparatively large (in the range of kHz), and have been reported before in the literature for a variety of compounds [9][10][11][12][13][14][15][16][17][18][19][20][21]. The existence of J-couplings in α-SnF 2 shown here is further evidence for the existence of covalent Sn-F bonds in tin(II) fluoride.

Experimental

α-SnF 2 (99%) was purchased from Sigma Aldrich and used without further treatment. Phase purity was checked by powder XRD. Furthermore, the sample was inspected for elements other than Sn and F by EDX (energydispersive X-ray spectroscopy), but no evidence for such elements was found.

Magic-angle spinning (MAS)

Results and Discussion

In comparison to NMR spectra recorded from solutions, static solid-state NMR spectra exhibit very broad, featureless resonances. For nuclei with spin I = 1/2, this is usually due to two predominant effects: (a) direct dipolar coupling (both homo-and heteronuclear), and (b) chemical shift anisotropy (CSA). Since both these effects have the same spatial dependence, they may be reduced or removed by application of magic-angle spinning (MAS) [6,[START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF].

The extent to which the broadening caused by dipolar couplings and CSA is removed from the spectrum depends on the relative magnitude of these interactions compared to the MAS frequency.

The magnitude of the dipolar interaction is determined by the angle θ jk between the vector connecting the two spins j and k, and the direction of the external magnetic field, the gyromagnetic ratios γ i of the involved nuclei, and the distance r jk between the two spins [START_REF] Levitt | Spin Dynamics: Basics of Nuclear Magnetic Resonance[END_REF]. The largest secular dipolar coupling, which is observed for θ jk = 0 (i.e. with the internuclear vector parallel to B 0 ) is usually called the dipolar coupling constant, b jk . In units of Hertz, b jk is given by Eq. 1.

b jk = - µ 0 8π 2 γ j γ k h r 3 jk (1) 4
The shortest distances for heteronuclear and homonuclear pairs of nuclei reported for the X-ray structure [1] are 2.03 Å, namely between Sn(2) and F(1), and 2.69 Å, between F(2) and F(4). With Eq. 1 and the γ i values listed in Table 1, we calculate maximum dipolar coupling constants of ≈ 5.0 kHz for heteronuclear 119 Sn-19 F couplings, and ≈ 5.5 kHz for homonuclear 19 F-19 F couplings. It has been estimated that MAS frequencies in excess of five times the relevant dipolar interactions are necessary to completely remove the dipolar broadening [6,26]. The second relevant interaction, the chemical shift anisotropy (CSA), is also comparatively large for both 19 F and 119 Sn. In the case of fluorine, CSA's of several hundred ppm's have been reported [27], whereas for 119 Sn, CSA's up to a thousand ppm have been found [11]. For a magnet with a field of 9.4 T, this translates into interactions between 40 kHz ( 19 F), and 150 kHz ( 119 Sn). Therefore, it is clear that very fast MAS is needed to reduce both dipolar and CSA interactions sufficiently to be able to resolve spectral lines belonging to different crystallographic sites in α-SnF 2 .

As a third contribution to line broadening, J-couplings have to be taken into account for 119 Sn, since it has been documented previously [9][10][11][12][13][14][15][16][17][18][19][20][21] that they may also be of the order of kHz. In fact, the J-couplings for 119 Sn can be so large that it has been possible to determine the anisotropy of this interaction in some organo-tin compounds [14] by using off-magic angle spinning. Under exact magic-angle spinning conditions, only the isotropic part of the indirect J-coupling remains, but because of its comparatively large magnitude, we expect J-couplings -if they are present -to affect the NMR spectra of α-SnF 2 even at high MAS speeds.

119 Sn MAS-NMR

In Fig. 2, the 119 Sn NMR spectra of α-SnF 2 at 25 kHz MAS are shown, acquired with and without application of spin-decoupling on 19 F, under otherwise identical conditions. It is obvious from Fig. 2b that residual dipolar Fig. 2 couplings (in combination with indirect J-couplings, see below) strongly broaden the spectral lines, so application of efficient 19 F spin-decoupling is essential. The sideband pattern observed in the 119 Sn NMR spectrum acquired with decoupling (Fig. 2a) spans almost 1000 ppm, which is caused by a large anisotropy of the chemical shift. Two isotropic bands (which do not 5 alter position when the MAS speed is changed) are located at -949.4 and -1024.7 ppm in the 119 Sn spectrum, which correspond to the two independent Sn positions in the crystal structure [1]. A better quality spectrum of 119 Sn with 19 F spin-decoupling is shown in Fig. 3a. In this spectrum it can Fig. 3 be seen that the main lines of the sideband pattern are flanked by doublets of satellite lines, with the spacing of these doublets being unaffected by the spinning speed. These satellites should therefore be attributed to indirect J-coupling between the magnetically active tin isotopes in α-SnF 2 . Because of the very low natural abundance of 115 Sn, the presence of the satellite lines is due mainly to coupling between the observed 119 Sn and neighbouring 117,119 Sn isotopes. Since the natural abundance of both 117 Sn and 119 Sn is below 9% (cf. Table 1), most of the observed 119 Sn nuclei are unaffected, and only a small percentage contributes to the J-coupling satellites. To determine the magnitude of the J-couplings, the high-intensity first-order spinning sidebands were deconvoluted using the Dmfit program [START_REF] Massiot | [END_REF], as shown in Fig. Of course, such a lone pair is absent in SnO 2 , which is reflected in a comparatively small CSA of about 140 ppm [11]. Also, the tin spectra of SnO 2 do not exhibit any J-coupling effects, since bonding is largely of ionic nature (rutile type). By contrast, the significant degree of covalent character in SnO (PbO type) and α-SnF 2 , permits indirect spin coupling that is observed in the MAS spectrum of each compound, with a J value of about 8 kHz in SnO [11]. Thus, the presence of J-couplings in α-SnF 2 is additional evidence for the existence of genuine covalent bonds, which has been deduced before from X-ray crystallography by comparing the observed Sn-F distances to the sum of the respective ionic radii [1].

3.2 119 Sn J -couplings and crystal structure This level of connectivity is used in the structural representation shown in Fig. 1. The co-ordination of this arrangement, as also described in Ref. [2],

is such that the Sn( as a bond into the model. As may be seen from Table 2, Sn( 1) is now connected to four Sn(2) and two Sn(1) via various fluorine bridges. For Table 2 Sn(2), the situation is identical in that it is connected to four Sn(1) and two Sn (2). With this model, we may explain the satellite pattern seen in the 119 Sn spetrum in the following way. The J-coupling via the shortest fluorine bridges (top two rows for each tin species in Table 2) is strongest, causing the satellites with about 5.4 kHz spacing. The remaining four fluorine bridges, involving at least one significantly longer tin-fluorine bond, all give rise to J-couplings of the order of 1.4 kHz, causing the inner satellites. As these are only visible as shoulders to the main line, fitting the exact positions is prone to some error. Since each tin species may experience the smaller J-coupling via four fluorine bridges, there is a reasonable probability that two magnetically active isotopes may be simultaneously present. This would result in a triplet structure caused by the J-coupling, and could explain the satellites with a spacing of about 3.3 kHz, if we accept that there is an error of about 0.2 kHz in the determination of the inner satellites. The 3.3 kHz satellites would thus represent the outer triplet lines (with corresponding low intensities), whereas the centre line of the triplet is hidden within the majority resonance of uncoupled nuclei.

With this model, we are able to qualitatively describe the appearance of the 119 Sn spectrum, however, the actual J-coupling pattern may be even more intricate. In particular, a roof effect could be expected for the 119 Sn-F-119 Sn couplings, as the chemical shift difference between the two tin res-8 onances is only about 11 kHz at 9.4 T. Within the quality of the available spectra, we do not observe such an effect, and other, unobserved subtleties may be similarly present. Obviously, the resolution of the 119 Sn spectrum would greatly benefit from faster MAS, but the 1.3 mm probe used for the acquisition of the fast 19 F spectra at 60 kHz MAS does not reach the necessary high resonance frequency for 119 Sn.

It should also be mentioned that the strongest J-coupling should exist for the 19 F-119 Sn pair, which is connected by a direct bond. Because 19 F decoupling is applied, these couplings will be suppressed in the 119 Sn spectrum shown in Fig. 3. However, some evidence of tin-fluorine coupling can be deduced from the 19 F MAS spectrum, as discussed below.

19 F MAS-NMR

The 19 F NMR spectra of α-SnF 2 at 25 kHz and 60 kHz MAS are shown in Fig. 4. The spectrum at 25 kHz MAS (Fig. 4b) exhibits an extensive Fig. 4 sideband pattern, caused by a combination of residual dipolar couplings and chemical shift anisotropy. At 60 kHz MAS (Fig. 4a), however, we can expect the dipolar couplings to be completely averaged, so that the small spinning sidebands still visible are caused by CSA only. Even at this high spinning frequency, the two main 19 F bands remain fairly broad, with a full-width-at-half-height (FWHH) of approximately 4 kHz. This broadening must be attributed to a range of indirect J-couplings present in the bond network of α-SnF 2 . Similar to the 119 Sn spectra discussed above, some satellite lines can also be discerned in the 19 F spectra (indicated by arrows in Fig. 4a). These satellites appear at a distance of approximately 8 and 4.5 kHz from the main band, are independent of the spinning speed, and do not belong to 'probe background' (the latter was checked by measuring other fluorinated compounds). The satellite line at 8 kHz (corresponding to a J-coupling of 16 kHz) could tentatively be attributed to 19 F directly bonded to 119 Sn, because of the strong coupling and the low intensity of the satellite line. However, since the pairing lines of the satellite doublets are hidden under the second main band, the analysis can not be conducted in the same quantitative manner as for the 119 Sn spectra.

The considerable line broadening present in the 19 F NMR spectra of α-SnF 2 , even at 60 kHz MAS, results in the observation of only two isotropic 9 bands, which are centered around -42 and -50.5 ppm. Thus, the four fluorine sites reported in the crystal structure [1] cannot be fully resolved.

In addition to the main bands, a low-intensity line is observed in the 19 F spectrum at around 113 ppm (marked by an asterisk in Fig. 4a), which remains narrow and stays at the same position also for lower spinning speeds.

We attribute this line to some unknown minor impurity, which, judging from the small line width, appears to be very mobile. A chemically plausible candidate, which is also expected to show high mobility, would be hydrogen fluoride. Since the 19 F chemical shift of HF is known to depend very strongly on the environment [29], this is however difficult to verify.

Conclusions

Magic-angle spinning (MAS) NMR spectra of 19 F and 119 Sn have been recorded and analysed for the stable room-temperature phase of tin(II) fluoride, α-SnF 2 . The spinning sideband pattern observed for both nuclides indicate the existence of a large chemical shift anisotropy (CSA), which is consistent with the presence of a stereochemically active Sn lone electron pair. In the 119 Sn spectra, two isotropic bands are observed, belonging to the two independent Sn sites in the crystal structure [1]. Also, symmetric satellite lines with low intensity have been detected in the 119 Sn spectra, which are attributed to indirect J-couplings between the magnetically active tin isotopes. By spectral deconvolution of the satellite doublets, the J-couplings could be quantified to be 5.4, 3.3 and 1.4 kHz. By considering tin-fluorine distances of up to 2.675 Å to be covalent bonds, a structural model of α-SnF 2 could be found that qualitatively explains the observed Jcoupling pattern. Indications for the presence of J-couplings have also been found in the 19 F spectrum of α-SnF 2 . Quite uncommonly, J-coupling thus appears to be the main source of the considerable 19 F line broadening still present in the 60 kHz MAS spectrum. This residual line broadening does not permit sufficient resolution to observe all four fluorine sites reported in the crystal structure [1]. The detection of J-couplings is in itself evidence for the existence of genuine covalent bonds in α-SnF 2 , a fact which has been deduced before from X-ray crystallography by identifying Sn-F distances smaller than the sum of the respective ionic radii [1].

6 Page

 6 3b. First, to ensure that the satellite doublet remains symmetric around the central line, the spinning sideband tool of Dmfit was used to determine the positions of the satellites. These positions were then fixed, and the intensity of the satellites was subsequently fitted with Lorentzian lines, resulting in the deconvolution depicted in Fig.3b. (The native Jcoupling tool in Dmfit could not be used because it expects both satellites to have identical intensity, which our 119 Sn spectra do not always exhibit because of the relatively poor signal-to-noise ratio.) The 119 Sn NMR spectrum of α-SnF 2 can be fitted with a minimum of 3 doublets, resulting in J-couplings of approximately 5.4, 3.3 and 1.4 kHz. We have also recorded a Hahn-echo spectrum (not shown), where the observed satellite lines can be caused by coupling of 119 Sn to 117 Sn only. Deconvolution of this echo spectrum resulted in values very similar to those shown in Fig.3b. However, according to the connectivities that can be derived from the crystal structure of α-SnF 2[1,2], more than just the 3 satellites used for fitting here are expected to show up in the spectrum. This will be discussed in more detail below.In terms of chemical bond character, it is instructive to compare the findings presented here with a previous NMR study of SnO and SnO 2[11].Similar to α-SnF 2 , the 119 Sn spectrum of SnO displays a CSA-broadened pattern spanning about 1000 ppm. This large anisotropy in the shielding can be attributed to the presence of a stereochemically active Sn lone electron pair in both compounds.

  Since J-couplings are mediated by covalent bonds, the satellite pattern observed in the 119 Sn spectrum of α-SnF 2 (Fig.3) should reflect the connectivities of the crystal structure, where the tin atoms are connected via fluorine bridges. In general, a 119 Sn NMR spectrum acquired with a single pulse should show J-couplings for both 117 Sn-F-119 Sn and 119 Sn-F-119 Sn pairs, whenever crystallographically distinct tin species are connected via Sn(1)-F-Sn(2) bridges. For bridges among identical tin species, i.e. Sn(1)-F-Sn(1) and Sn(2)-F-Sn(2), only 117 Sn-F-119 Sn couplings are relevant because of the magnetic equivalence of crystallographically identical pairs. It also follows that bridges among identical species add to the J-coupling satellites with only about half the intensity, because the 119 Sn isotope does not contribute.However, the actual number of tin-fluorine-tin bridges existing in the structure depends on which tin-fluorine distances are thought to represent covalent bonds. When considering only distances up to the sum of the ionic radii (2.27 Å according to Ref.[1]), the resulting structure consists of an arrangement of Sn 4 F 8 rings[1,2]. In this model, the two crystallographically distinct tin species, designated Sn(1) and Sn(2), are connected via F(2) and F(4) forming puckered rings. These rings possess comparatively short bonds, from 2.068 to 2.273 Å. Clearly, the J-coupling observed in the 119 Sn 7 spectrum is not consistent with having bonds only within these rings. If longer distances are also considered, additional bonds connect the Sn 4 F 8 among themselves via fluorine bridges involving bond distances > 2.4 Å.

Figure 1 :

 1 Figure 1: The structure of the monoclinic phase of tin(II) fluoride, α-SnF 2 , based on the co-ordinates published in Ref. [1]. Puckered rings with comparatively short bonds are formed by tin (Sn(1) and Sn(2), dark spheres) and fluorine (F(2) and F(4), light spheres), which are emphasized in the drawing. These rings are connected via the F(1) species along the a-axis direction, and via F(3) along the c-axis direction [1, 2], involving longer bond distances (> 2.4 Å).

Figure 2 :

 2 Figure 2: 119 Sn MAS NMR spectra at 25 kHz spinning speed (2k accumulations, 25 s recycle delay): (a) with, and (b) without 19 F spin decoupling. The isotropic bands (marked by arrows) are located at -949.4 and -1024.7 ppm, respectively.

Figure 3 :

 3 Figure 3: 119 Sn MAS NMR spectrum at 25 kHz spinning speed (5k accumulations, 60 s recycle delay): (a) full MAS spectrum, (b) deconvolution ofsatellites caused by J-coupling, using the high-intensity first-order spinning sidebands, which are marked by arrows in (a). The spectrum can be fitted with a minimum of 3 doublets, with J-couplings of approximately 5.4, 3.3 and 1.4 kHz. From structural data[1,2], however, an entire range of J-couplings is expected, which are not resolved here.

Figure 4 : 16 Page

 416 Figure 4: 19 F MAS NMR spectra: (a) 60 kHz spinning speed (32 accumulations, 5 s recycle delay); (b) 25 kHz spinning speed (128 accumulations, 10 s recycle delay). The isotropic bands are centered around -42 and -50.5 ppm, respectively. The arrows in (a) indicate satellite lines attributed to J-couplings, whereas the asterisk marks a line due to an impurity (see text for details).

  19 F and 119 Sn NMR spectra were recorded on Bruker MAS double-resonance probe with 1.3 mm rotors. Neither probe showed any substantial 19 F background signal.Recycle delays and number of transients are listed in the respective figure captions. For 19 F spin decoupling, the recently suggested SW f -TPPM sequence[22] was used. The sequence consisted of 11 pulse pairs with a phase angle of ±12.5 o , employing a linear sweep profile[23] with a sweep window from 0.69 -1.46. These parameters were optimised on a fluorinated model compound. More details on the application of SW f -TPPM for19 F

	with a 14.1 T magnet, and a
	a Bruker DPX 400 spectrometer with a 9.4 T magnet, using a Bruker
	MAS double-resonance probe with 2.5 mm rotors. The 19 F spectrum at
	3

decoupling will be published elsewhere

[24]

. The 19 F spectra were referenced against CFCl 3 by using solid CaF 2 as a secondary standard, with the 19 F resonance of CaF 2 assigned to -107.7 ppm

[6]

. Similarly, the 119 Sn NMR spectra were referenced by measuring SnO 2 as a secondary standard, setting this 119 Sn resonance at -604.3 ppm

[25]

.

  1) species are tetrahedrally surrounded by three fluorines and a lone pair, whereas Sn(2) is surrounded by five fluorine and a lone pair in octahedral fashion. Again, this structural model does not fit our 119 Sn NMR data, as it would result in different coupling patterns for the 119 Sn

resonances belonging to Sn(1) and Sn(2), whereas we see a very similar satellite pattern for both. We may however find the desired equivalent coordination for both tin species if we include one further distance (2.675 Å)

10 Table 1 .

 101 Nuclear properties of the NMR-observable nuclei in SnF 2 (from

	Ref. [30]).
	Isotope Spin I	Natural	Magnetogyric	Relative	Frequency
			abundance	ratio, γ a	receptivity b	ratio c
	19 F	1 2	100%	25.181	0.834	94.094 MHz
	115 Sn	1 2	0.34%	-8.801	1.21 × 10 -4 32.718 MHz
	117 Sn	1 2	7.68%	-9.588	3.54 × 10 -3 35.632 MHz
	119 Sn	1 2	8.59%	-10.032	4.53 × 10 -3 37.290 MHz

a in 10 7 rad s -1 T -1 b Receptivity relative to 1 H. c With 1 H at 100 MHz.

14

Acknowledgments

We would like to thank C. Vinod Chandran and Ivan Halasz (MPI Stuttgart) for skillful help in preparing the figures for this article. Sharon Ashbrook, John Griffin and Phil Wormald (St. Andrews, UK) are acknowledged for their instrumental assistance to acquire the 19 F-MAS spectrum at 60 kHz.