The Lysyl Oxidase Inhibitor Β-Aminopropionitrile Reduces Body

RESEARCH ARTICLE The lysyl oxidase inhibitor β-aminopropionitrile reduces body weight gain and improves the metabolic profile in diet-induced obesity in rats Marıá Miana1,2,*, Marıá Galán3,*, Ernesto Martınez-Mart́ ıneź 1,2,4, Saray Varona3, Raquel Jurado-López1,2, Belén Bausa-Miranda1,2, Alfonso Antequera5, Marıá Luaces6, JoséMartınez-Gonzá ́lez3, Cristina Rodrıgueź 3,*,‡ and Victoria Cachofeiro1,2,*,‡

ABSTRACT tissue fibrosis and LOX activity for the clinical management of Extracellular matrix (ECM) remodelling of the adipose tissue this disease. plays a pivotal role in the pathophysiology of obesity. The lysyl KEY WORDS: Lysyl oxidase, Extracellular matrix, Adipose tissue, oxidase (LOX) family of amine oxidases, including LOX and Fibrosis, Obesity, Insulin resistance LOX-like (LOXL) isoenzymes, controls ECM maturation, and upregulation of LOX activity is essential in fibrosis; however, its INTRODUCTION involvement in adipose tissue dysfunction in obesity is unclear. In In recent years, there has been increasing evidence that the this study, we observed that LOX is the main isoenzyme expressed extracellular matrix (ECM) plays a key role in adipose tissue in human adipose tissue and that its expression is strongly development and function, and fibrosis is now recognised as a upregulated in samples from obese individuals that had been crucial player in adipose tissue dysfunction in obesity. In fact, early referred to bariatric surgery. LOX expression was also induced in the deposition of connective tissue occurs in subcutaneous white adipose tissue from male Wistar rats fed a high-fat diet (HFD). adipose tissue after moderate weight gain and further increases with β Interestingly, treatment with -aminopropionitrile (BAPN), a specific obesity (Mutch et al., 2009; Divoux et al., 2010; Henegar et al., and irreversible inhibitor of LOX activity, attenuated the increase in 2008; Alligier et al., 2012). Interestingly, data from animal models body weight and fat mass that was observed in obese animals and of obesity suggest that this abnormal ECM remodelling of white shifted adipocyte size toward smaller adipocytes. BAPN also adipose tissue could contribute to local and systemic metabolic ameliorated the increase in collagen content that was observed in alterations (Halberg et al., 2009; Khan et al., 2009). adipose tissue from obese animals and improved several metabolic Fibrosis arises from the imbalance between ECM synthesis and – parameters it ameliorated glucose and insulin levels, decreased degradation that culminates in an excessive accumulation of ECM homeostasis model assessment (HOMA) index and reduced components. Lysyl oxidase (LOX) activity is crucially involved in plasma triglyceride levels. Furthermore, in white adipose tissue ECM synthesis and processing. To date, five isoenzymes belonging from obese animals, BAPN prevented the downregulation of to the LOX family have been identified, including LOX, LOX-like1 adiponectin and glucose transporter 4 (GLUT4), as well as the (LOXL1), LOXL2, LOXL3 and LOXL4. They are copper- increase in suppressor of cytokine signaling 3 (SOCS3) and dependent amine oxidases that exhibit a high degree of homology dipeptidyl peptidase 4 (DPP4) levels, triggered by the HFD. in the C-terminal domain, which contains the catalytic site. LOX, α Likewise, in the TNF -induced insulin-resistant 3T3-L1 adipocyte the archetypal member of this family and the best characterised, model, BAPN prevented the downregulation of adiponectin and catalyses the covalent cross-linking of collagens and elastin fibres GLUT4 and the increase in SOCS3 levels, and consequently (Rodríguez et al., 2008a,b). This enzyme oxidises lysine and normalised insulin-stimulated glucose uptake. Therefore, our data hydroxylysine residues in these substrates, leading to the synthesis provide evidence that LOX plays a pathologically relevant role in the of highly reactive peptidylsemialdehydes that can spontaneously metabolic dysfunction induced by obesity and emphasise the condense to form the intra- and intermolecular covalent cross- interest of novel pharmacological interventions that target adipose linkages that are responsible for ECM stability. The disturbance of LOX activity induces connective tissue abnormalities related to 1 Departamento de Fisiologıa,́ Facultad de Medicina, Universidad pathological processes, including cardiovascular diseases, as we Complutense, Madrid 28040, Spain. 2InstitutodeInvestigación Sanitaria Gregorio Marañón (IiSGM), Madrid 28007, Spain. 3Centro de Investigación have previously described (Miana et al., 2011; Orriols et al., 2014; Cardiovascular (CSIC-ICCC), IIB-Sant Pau, Barcelona 08025, Spain. Raposo et al., 2004; Rodriguez et al., 2002, 2009; Stefanon et al., 4Cardiovascular Translational Research, NavarraBiomed (FundaciónMiguel Servet), Pamplona 31008, Spain. 5Upper Gastroenterology & Bariatric Surgery 2013). Department, Fuenlabrada University Hospital, Madrid 28942, Spain. Changes in LOX activity could be involved in the disturbance 6Cardiology Department, Cardiovascular Institute, Hospital Clınicó San Carlos, of adipocyte function in obesity. In fact, LOX expression is Madrid 28040, Spain. ob/ob *These authors contributed equally to this work upregulated in the white adipose tissue from mice (Halberg et al., 2009), and it is inhibited during adipocyte differentiation ‡ Authors for correspondence ([email protected]; [email protected]) (Dimaculangan et al., 1994). Furthermore, LOX participates in the

This is an Open Access article distributed under the terms of the Creative Commons Attribution commitment of pluripotent stem cells to the adipocyte lineage License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, (Huang et al., 2009). However, the potential role of LOX activity in distribution and reproduction in any medium provided that the original work is properly attributed. human obesity has been poorly characterised. A transcriptomic

Received 20 January 2015; Accepted 28 March 2015 study revealed an increase in LOX expression in subcutaneous white Disease Models & Mechanisms

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40.2±9.5 (range 30-55) vs 36.0±9.5 (range 26-51) years in TRANSLATIONAL IMPACT the normoweight group. The average BMI was 44.6±3.7 (range 41.5-50.9) in the obese group vs 22.7±3.6 (range 16.4-25.0) in the Clinical issue P Obesity is one of the most serious public health challenges of the 21st control group ( =0.008). No significant differences were observed in century. The risk of a number of major diseases, including type the fasting plasma glucose levels between both groups (114.2±26.0 vs 2 diabetes mellitus, ischemic heart disease, ischemic stroke and 95.8±10.8 mg/dl; P=0.344). However, obese patients showed several common forms of cancers, is dramatically increased in obese higher insulin plasma levels (486.6±173.1 vs 268.4±138.4 pg/dl; individuals. The epidemic proportions achieved by obesity makes P=0.0929), homeostasis model assessment (HOMA) index (3.21± it mandatory to reach a deeper understanding of its underlying 1.49 vs 1.44±0.75; P=0.075) and plasma triglyceride levels (169.4± pathophysiological mechanisms, which could provide novel therapeutic P targets. In recent years, fibrosis has been recognised as a crucial player 44.8 vs 106.6±24.9 mg/dl; =0.0554) than controls, although in adipose tissue dysfunction in obesity. Lysyl oxidase (LOX) activity, neither difference reached statistical significance. As expected, which governs extracellular matrix maturation, is essential for tissue histological analysis of visceral adipose tissue showed an enhanced fibrosis. However, its contribution to adipose tissue dysfunction in adipocyte area (cross-sectional area of single adipocytes) (Fig. 1A; obesity has not been clearly established. P=0.05) and an increase in pericellular collagen (Fig. 1B; P=0.007) in obese patients as compared with control subjects. Among LOX family Results members, LOX exhibited the highest expression level in the adipose This study analyses the role of LOX in adipose tissue remodelling by tissue from control individuals, whereas that of LOXLs was almost using three experimental systems: adipose tissue samples from obese LOX individuals that had been referred for bariatric surgery (weight loss negligible. Interestingly, mRNA levels were significantly surgery), an animal model of diet-induced obesity and cell-based upregulated (about threefold) in the visceral adipose tissue from studies. The authors demonstrate that LOX is the main lysyl oxidase obese patients compared with that of controls. A consequent increase isoenzyme expressed in human adipose tissue and that it is upregulated in LOX protein levels, assessed by western blotting and in samples from both obese individuals and rats fed a high-fat diet. In immunohistochemistry (Fig. 1D,E), was detected in samples from β obese rats, the inhibition of LOX activity through -aminopropionitrile obese subjects. LOX protein levels were correlated with collagen (BAPN, a specific inhibitor of LOX activity) reduces adipose tissue P fibrosis, partially corrects the adipocyte-size distribution pattern (shifting content (r=0.70; =0.0433). As previously described (Kern et al., it toward smaller sizes) and attenuates the increase in body weight and 2003; Sell et al., 2013), visceral adipose tissue from obese patients fat mass. Furthermore, LOX inhibition improves multiple metabolic showed an increase in dipeptidyl peptidase 4 (DPP4) protein levels parameters: normalizing glucose, insulin and triglyceride levels and (220.9±62.6 vs 100.0±10.0 AU; P=0.015; Fig. 1D) and a reduction in reducing the homeostatic model assessment (HOMA) index – a those of adiponectin (22.7±2.8 vs 100.0±52.4 AU; P=0.05; Fig. 1D) measure of insulin resistance. Likewise, in these animals, BAPN compared with normoweight subjects. ameliorates the disturbances in the adipose tissue expression of adiponectin, glucose transporter 4 (GLUT4), suppressor of cytokine signaling 3 (SOCS3) and dipeptidyl peptidase 4, all proteins involved in LOX expression is induced in adipose tissue from HFD-fed the control of insulin sensitivity. Finally, BAPN also normalises the rats insulin-stimulated glucose uptake and protein levels of GLUT4, adiponectin and SOCS3 in the TNFα-induced insulin-resistant 3T3-L1 The expression of LOX in white adipose tissue was investigated in an adipocyte model. animal model of diet-induced obesity that we have previously described (Martínez-Martínez et al., 2014a,b). As shown in Fig. 2A, Implications and future directions LOX mRNA levels were increased approximately 15-fold in the The results reported in this study demonstrate the upregulation of the epididymal adipose tissue of rats fed a high-fat diet (HFD) compared adipose tissue expression of LOX in human obesity and provide with control animals fed normal chow. In agreement, LOX protein evidence that LOX inhibition prevents adipose tissue dysfunction, levels, assessed by western blotting and immunohistochemistry reduces bodyweight gain and improves metabolic disturbances in diet- induced obesity. These findings uncover the pathologically relevant (Fig. 2B,C), were higher in the white adipose tissue from obese contribution of LOX to the metabolic dysfunction induced by obesity and animals. A correlation was observed between protein levels of LOX emphasise the potential of novel pharmacological interventions targeting and collagen content (r=0.8389; P=0.003). The HFD did not affect adipose tissue fibrosis and LOX activity for the clinical management of the expression of bone morphogenetic protein 1 (BMP1), the enzyme obesity. Future studies will help to further clarify the mechanisms that processes pro-LOX to its active form (Fig. 2D). Furthermore, this underlying the beneficial consequences of LOX inhibition in obesity. dietary intervention did not modify the expression of LOX in brown adipose tissue (data not shown). adipose tissue from obese subjects, but its pathophysiological BAPN ameliorates the increase in body weight, adiposity and relevance remains unclear (Henegar et al., 2008). Therefore, the aim adipose tissue fibrosis in HFD-fed rats of this study was to explore the role of LOX activity in adipose To investigate whether LOX contributes to the adipose tissue tissue remodelling and in the metabolic disturbances associated dysfunction associated with obesity, rats subjected to a HFD were with obesity. For this purpose, we have evaluated the impact of treated with BAPN, an irreversible and specific inhibitor of LOX β-aminopropionitrile (BAPN), a specific and irreversible inhibitor activity. As shown in Fig. 3A, the HFD induced a significant increase of LOX activity, in a model of diet-induced obesity. in body weight that reached a significant difference from the third week onwards. After three weeks of treatment, BAPN significantly RESULTS prevented the rise in body weight in HFD rats, but not in animals LOX expression is induced in adipose tissue from morbidly that were fed a standard diet (Fig. 3A). These differences were obese patients sustained until the end of the study (Table 1, Fig. 3A). Similarly, BAPN The expression of LOX and LOXL isoenzymes in visceral adipose reduced the increase in the weight of white adipose tissue (both tissue was investigated in samples from control individuals and epididymal and lumbar) in obese animals (Table 1) and attenuated morbidly obese patients. The mean age of the obese group was their enhanced adiposity (Table 1). It should be noted that the changes Disease Models & Mechanisms

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Fig. 1. Adipocyte size, total collagen content and lysyl oxidase expression in visceral adipose tissue from control subjects and morbidly obese individuals. (A) Quantification of adipocyte area (left) and representative microphotographs of visceral adipose tissue sections from control subjects (CT) and morbidly obese individuals (OB) stained with haematoxylin and eosin and then examined by using light microscopy (magnification 40×; right). (B) Quantification of collagen volume fraction (CVF) normalised for adipocyte number. Right panel shows representative microphotographs of visceral adipose tissue sections from these individuals stained with Picrosirius red that were then examined by using light microscopy (magnification 40×). (C) mRNA levels of lysyl oxidase (LOX) and LOX-like (LOXL) isoenzymes 1, 2, 3 and 4 evaluated using real-time PCR of these samples. Results were normalised to the expression of 18S RNA. (D) LOX, DPP4 and adiponectin (Adipo) protein levels determined by western blotting. β-actin was analysed as loading control. (E) Representative images of the immunohistochemical analysis for LOX are presented (magnification 40×). Histograms represent the mean±s.d. of five subjects. Scale bars: 50 µm. *P<0.05 and **P<0.01 vs control group.

in body weight triggered by BAPN in HFD-fed rats were not related BAPN improves insulin signalling in adipose tissue from to differences in food intake (Table 1). obese animals The changes in adipose tissue mass elicited by BAPN in HFD-fed In order to understand how BAPN improves insulin sensitivity in rats prompted us to determine whether LOX inhibition could obese animals, we analysed the levels of proteins involved in the modulate the adipocyte area. Histological analysis of epididymal control of insulin sensitivity in epididymal adipose tissue. The adipose tissue revealed an increased adipocyte area in the HFD reduction in both glucose transporter 4 (GLUT4) and adiponectin group. A trend towards an attenuation of this parameter was expression observed in the HFD group was normalised through the observed in obese animals that had been treated with BAPN inhibition of LOX activity (Fig. 4A,B). Furthermore, the increase in (Fig. 3B,C). Furthermore, a shift toward smaller adipocytes was the protein levels of DPP4 and suppressor of cytokine signaling 3 detected in BAPN-treated obese animals compared with HFD-fed (SOCS3) triggered by the HFD was completely prevented by BAPN rats (Fig. 3D). Interestingly, LOX inhibition prevented the increase (Fig. 4C,D). in pericellular collagen content observed in obese rats, as analysed by using Picrosirius red staining (Fig. 3E,F). BAPN normalises the expression of GLUT4 and adiponectin, and improves glucose uptake in an in vitro model of insulin BAPN improves the metabolic alterations observed in obese resistance rats To determine whether BAPN directly affects adipocyte function, we Next, we examined whether the inhibition of LOX activity performed in vitro experiments in the TNFα-induced insulin could modify metabolic parameters in obese animals. Treatment resistance model in differentiated 3T3-L1 adipocytes. As shown with BAPN improved fasted glucose and insulin levels and in Fig. 5 and as previously described (Stephens et al., 1997; Sethi consequently reduced HOMA index in the HFD group (Table 1). and Hotamisligil, 1999), TNFα reduced expression of GLUT4 and LOX inhibition also reduced plasma triglycerides in obese animals, adiponectin and increased SOCS3 protein levels in these cells. but no significant differences were observed in the total cholesterol Interestingly, these effects were prevented by BAPN (Fig. 5A-C).

levels (Table 1). Accordingly, BAPN normalised the TNFα-induced decrease in Disease Models & Mechanisms

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Fig. 2. Expression of LOX and BMP1 in epididymal adipose tissue in control and obese rats. (A) Lysyl oxidase (LOX) mRNA levels analysed by using real-time PCR of epididymal adipose tissue from control rats fed a normal chow (CT) and rats fed a high-fat diet (HFD). Results were normalised to cyclophilin expression. (B) LOX protein levels determined by western blotting of the same samples described in A. β-actin was analysed as loading control. (C) Representative images of immunostaining of LOX are presented (magnification 40×). (D) Bone morphogenetic protein-1 (BMP1) mRNA levels were quantified by using real-time PCR analyses of epididymal adipose tissue from control rats fed a normal chow (CT) and rats fed a HFD. Expression was normalised to that of cyclophilin. Graphs represent the mean±s.d. of eight animals. Scale bars: 50 µm. *P<0.05 vs control group.

insulin-stimulated glucose uptake observed in differentiated 3T3-L1 the adipose tissue of rats fed a HFD that was associated with adipocytes (Fig. 5D). enhanced pericellular fibrosis. The strong correlation of LOX expression with pericellular fibrosis in the adipose tissue from both DISCUSSION humans and rats, as well as the ability of BAPN to limit pericellular It is becoming apparent that fibrosis is an essential factor in adipose fibrosis in HFD-fed animals support the notion that LOX makes a tissue dysfunction in obesity (Divoux et al., 2010; Halberg et al., relevant contribution to the fibrotic response in obesity. 2009; Khan et al., 2009). ECM remodelling impacts on the LOX and LOXL isoenzymes differ from other copper amino metabolic function of adipose tissue, and the control of LOX oxidases in their quinone cofactor, the lysine tyrosylquinone activity, a major player in ECM maturation, could be crucial in this (LTQ), which confers particular catalytic properties to these context. The research presented here reveals that LOX is the main enzymes (Rodriguez et al., 2008b). BAPN is a well-known lysyl oxidase isoenzyme expressed in adipose tissue and that its specific and irreversible inhibitor of LOX activity (Tang et al., expression is upregulated in the adipose tissue of morbidly obese 1983; Rodriguez et al., 2008b) that targets the active site of LOX or patients and obese rats. More interestingly, we observed that LOXL isoenzymes. BAPN is a useful tool to study cellular inhibition of LOX activity protects against diet-induced obesity, processes involving LOX and LOXL isoenzymes; however, its thereby limiting weight gain, attenuating the disturbances in adipose pharmacological use as a systemic drug is not indicated. It has been tissue and improving the insulin resistance observed in obese observed that prolonged administration of BAPN evokes a weak animals. neurotoxicity and reduces the mechanical strength of bones (Ahsan Increased expression of ECM components in adipose tissue has et al., 1999; Spencer and Schaumburg, 1983; Turecek et al., 2008). been repeatedly reported in murine models of obesity and in obese Further, lathyrism, a disease characterised by extensive disruption subjects associated with either body mass index (BMI), insulin of connective tissue, is caused by the excessive and long-term sensitivity and/or resistance, and/or inflammation (Abdennour et al., consumption of certain legumes of the genus Lathyrus, which 2014; Divoux et al., 2010; Halberg et al., 2009; Khan et al., 2009; contain high amounts of BAPN and related compounds (Dasler, Tam et al., 2012; Walker et al., 2014). A transcriptomic analysis of 1954). Nevertheless, owing to the lack of viable LOX-deficient adipose tissue from obese individuals revealed the pathological mice (Maki et al., 2002; Hornstra et al., 2003), this inhibitor has relevance of ECM. However, the increase in the expression of LOX been extensively used in short-term in vivo studies to support the and other matrix components observed in these microarray studies involvement of lysyl oxidases in multiple biological processes, was not subsequently validated using other techniques (Henegar including tumour progression and metastasis (Levental et al., 2009; et al., 2008). Accordingly, we found that LOX mRNA levels are Erler et al., 2006), pulmonary arterial hypertension (Nave et al., increased in adipose tissue samples from obese individuals, which 2014) and arterial stiffening (Kothapalli et al., 2012). Similarly, we was confirmed by western blotting and immunohistochemistry. have observed that BAPN prevents the increase in both body weight Furthermore, obese individuals exhibited a marked fibrosis of and fat-pad weight found in obese animals without affecting food adipose tissue, a pathological process that reduces tissue plasticity intake. These changes were accompanied by a shift toward smaller and alters adipocyte function (Khan et al., 2009; Virtue and Vidal- adipocytes. Likewise, in mice of an ob/ob background, the Puig, 2010). In agreement with previous reports in the ob/ob model knockdown of ECM components, such as collagen VI, limited the

(Halberg et al., 2009); we also observed an upregulation of LOX in gain of body weight and reduced the increase in fat mass (Khan Disease Models & Mechanisms

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Fig. 3. Effects of lysyl oxidase inhibition on body weight evolution, adipocyte size and distribution and total collagen content in epididymal adipose tissue from control and obese rats. (A) Body weight evolution along the study. (B) Adipocyte area of epididymal adipose tissue analysed in haematoxylin-eosin- stained sections from control rats fed a normal chow (CT) and rats fed a high-fat diet (HFD) treated with vehicle or with the inhibitor of LOX activity (β-aminopropionitrile, BAPN, 100 mg/kg/day). (C) Representative microphotographs of sections stained with haematoxylin and eosin and then examined by using light microscopy (magnification 40×). (D) Size distribution of adipocytes from epididymal adipose tissue of the animals described in B. (E) Quantification of collagen volume fraction (CVF) normalised for the number of adipocytes analysed in epididymal adipose tissue sections stained with Picrosirius Red. (F) Representative microphotographs of sections stained with Picrosirius Red that were examined by using light microscopy (magnification 40×). Scale bars: † †† 50 µm. Values are mean±s.d. of six to eight animals. *P<0.05, **P<0.01, ***P<0.001 vs control group. P<0.05, P<0.01 vs HFD group. et al., 2009). Therefore, the composition and structure of ECM sign frequently observed in the setting of obesity linked to insulin seems to modulate the metabolism of fat tissue in different animal resistance (Eckel, 2011; Taskinen et al., 2011). models. More interestingly, we have observed that inhibition of LOX Dysfunction of adipose tissue metabolism has a negative impact activity attenuates insulin resistance in obese animals. In fact, on lipid and glucose homeostasis and plays a crucial role in the BAPN reduced insulin and glucose levels and consequently HOMA development of insulin resistance in obesity (Goossens, 2008; index, prevented the upregulation of plasma triglycerides and Guilherme et al., 2008; Trayhurn, 2013). As in obese individuals, ameliorated the changes in protein expression observed in the HFD-fed animals also exhibited a decline in insulin sensitivity, as adipose tissue from HFD rats. Inhibition of LOX activity reversed indicated by an increase in HOMA index. This was associated with the decrease in GLUT4 and adiponectin levels, and the increase in altered expression of several mediators involved in the control of both SOCS3 and DPP4 expression. In view of the low expression metabolic functions in the adipose tissue, including adiponectin, of LOXL isoenzymes in human adipose tissue, these data suggest GLUT4, SOCS3 and DPP4, as previously described in both humans that the upregulation of LOX observed in obesity has multiple and animal models (Emanuelli et al., 2001; Kadowaki et al., 2006; consequences for the physiology of adipose tissue. This functional Kanatani et al., 2007; Kern et al., 2003; Lamers et al., 2011; repercussion of LOX upregulation in systemic and adipose tissue Palanivel et al., 2012; Sell et al., 2013; Shepherd and Kahn, 1999; metabolism has been suggested previously in a transgenic mouse

Wang et al., 2000), and with a significant hypertriglyceridemia, a model that specifically overexpresses HIF-1α in the adipose tissue Disease Models & Mechanisms

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Table 1. Effect of the inhibition of lysyl oxidase activity with β- cellular functions, including the control of epithelial-to-mesenchymal aminopropionitrile (BAPN; 100 mg/kg/day) on general characteristics transition, cell proliferation, migration, adhesion and transformation and metabolic parameters in rats fed a normal chow (CT) and rats fed (Huang et al., 2009). The molecular mechanisms underlying the a high-fat diet (HFD) consequences of LOX inhibition in local and systemic metabolism are CT BAPN HFD HFD+BAPN unknown and, in view of the complex biology of this enzyme, further †† BW (g) 320±24 304±15 374±11*** 332±24 research is warranted. FI (g/day) 20±5 23±2 13±3*** 14±2** In summary, this study demonstrates that LOX could play a † EAT (mg/cm/tibia) 0.09±0.03 0.07±0.03 0.25±0.05** 0.19±0.04**, relevant role in the metabolic dysfunction induced by obesity and † LAT (mg/cm/tibia) 0.11±0.03 0.09±0.03 0.3±0.05*** 0.23±0.05**, † suggests that the inhibition of LOX activity could be a valuable Adiposity (%) 2.3±0.8 2.4±0.8 6.1±1.3** 4.4±1.0*, † strategy to ameliorate obesity-related metabolic disturbances. These Glucose (mg/dl) 97±10 91±9 115±6* 97±8 aspects highlight the benefits as well as the complexity of targeting Insulin (pg/ml) 64±20 50±13 184±91 79±39 † HOMA index 2.1±0.6 1.6±0.5 7.6±3.8* 2.4±1.1 fibrosis as a potential therapeutic intervention in obesity. † TG (mg/dl) 50±11 52±7 71±7* 51±10 TC (mg/dl) 55±7 55±11 61±7 53±6 MATERIALS AND METHODS BW, body weight; EAT, epididymal adipose tissue; FI, food intake; HOMA Subjects index, the homeostasis model assessment; LAT, lumbar adipose tissue; Visceral adipose tissue was obtained from five individuals that had been TG, triglycerides; TC, total cholesterol. Data values represent mean±s.d. of referred for bariatric surgery (all women). Inclusion criteria were: ≥ eight animals. *P<0.05, **P<0.01, ***P<0.001 compared with control group. age 18 years and universally accepted indications: long-term obesity † †† P<0.05, P<0.01 compared with HFD group. (more than 4 years); BMI≥40 despite other weight loss strategies or BMI≥35 in the presence of obesity-related comorbidities (diabetes mellitus, obesity hypoventilation syndrome, obstructive sleep apnoea syndrome and (Halberg et al., 2009). In agreement with our data, the authors of that hypertension). Exclusion criteria were: age>60 years and unacceptable study demonstrated that LOX contributes, at least in part, to the surgical risk. Visceral adipose tissue was also obtained from five metabolic disturbances observed in the model, which were normoweight BMI≤25 individuals that had been referred to non-bariatric improved by the administration of BAPN. Therefore, our data surgery who were included as controls (40% women). The BMI was confirm and extend previous findings on the relevance of LOX in calculated according to the formula: weight (kg)/square of the height adipocyte function. (metres). In accordance with institutional guidelines, all individuals gave The role of LOX in the control of adipocyte function has been informed consent. The study was performed in accordance with the poorly characterised. LOX is downregulated during adipocyte Declaration of Helsinki, and the Ethical Committee of the Fuenlabrada differentiation (Dimaculangan et al., 1994), and our data shows that University Hospital approved the protocol. inhibition of LOX impacts adipocyte homeostasis and improves α Animals insulin resistance in the well-established model of TNF -induced Male Wistar rats of 150 g (Harlan Ibérica, Barcelona, Spain) were fed either insulin resistance. In fact, BAPN normalised the expression of a HFD (33.5% fat; Harlan Teklad no. TD.03307, Haslett, MI, USA; n=16) GLUT4, adiponectin and SOCS3, and improved glucose uptake, or a standard diet (3.5% fat; Harlan Teklad no. TD.2014, Haslett, MI, USA; supporting a direct and more complex role of LOX in adipose tissue n=16) for 6 weeks. Half of the animals of each group received the physiology. Beyond its role in EMC maturation, LOX exhibits other irreversible inhibitor of LOX activity BAPN (100 mg/kg/day; Sigma-

Fig. 4. Effects of lysyl oxidase inhibition on the protein levels of factors involved in the control of insulin sensitivity in epididymal adipose tissue from control and obese rats. Protein levels of (A) glucose transporter type 4 (GLUT4), (B) adiponectin (Adipo), (C) dipeptidylpeptidase 4 (DPP4) and (D) suppressor of cytokine signaling 3 (SOCS3) in epididymal adipose tissue from control rats fed a normal chow (CT) and rats fed a high-fat diet (HFD) that had been treated with vehicle or with the inhibitor of lysyl oxidase (LOX) activity (β-aminopropionitrile, BAPN, 100 mg/ kg/day). Bars on graphs represent the mean±s.d. of six to eight animals, values were normalised to the expression of β- actin. *P<0.05, **P<0.01 vs control † †† group. P<0.05, P<0.01 vs HFD group. ‘C’, control; ‘B’,BAPN;‘H’, HFD; ‘B/H’,HFD+BAPN. Disease Models & Mechanisms

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Fig. 5. LOX inhibition influences glucose uptake and the expression of factors involved in the control of insulin sensitivity in an in vitro model of insulin resistance. Protein levels of (A) glucose transporter type 4 (GLUT4), (B) adiponectin (Adipo) and (C) suppressor of cytokine signaling 3 (SOCS3). 3T3-L1 adipocytes were stimulated with or without TNFα (1.15 and 2.87 nM, equivalent to 20 and 50 ng/ml, respectively) for 72 h in the presence of either vehicle or β-aminopropionitrile, an inhibitor of LOX activity (BAPN, 200 µM). Data that were normalised to the expression of β-actin are expressed as mean±s.d. of four assays in arbitrary units. (D) Insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Bars on graphs represent the mean±s.d. of the counts of [3H]-2- deoxyglucose normalised to the amount of total protein. Values are mean±s.d. of four assays. *P<0.05, **P<0.01 vs unstimulated cells † †† (TNFα=0); P<0.05, P<0.01 vs cells stimulated with the same concentration of TNFα in the absence of BAPN.

Aldrich, St Louis, MO, USA) in the drinking water for the same period, as (www.Biomers.net, Donau, Germany): 5′-GTCCTTCACGACAACAAAC-3′ previously described (Brasselet et al., 2005). The amount of BAPN and 5′-GGGTACTTGTCAGGCCAGTT-3′ (positions 2331 to 2434 effectively taken daily per animal was calculated from the amount of water corresponding to RefSeq: NM_031323.1; amplicon length: 103). 18S rRNA consumed on a daily basis. Animal weight was periodically controlled to (4319413E; RefSeq: X03205.1) was used as an endogenous control for human adjust the target dose of BAPN. Food and water intake were determined samples. Results from rat samples were normalised according to expression of throughout the experimental period. Animals were fasted the day before adenine phosphoribosyltransferase (Aprt) or cyclophilin using SYBR Green euthanasia by anaesthesia with a cocktail of ketamine (Imalgene 1000, and specific oligonucleotides (www.Biomers.net, Donau, Germany) as 70 mg/kg; intraperitoneal, Merial Laboratorios, Barcelona, Spain) and follows: rat Aprt 5′-CGGGCGTGCTGTTCAGGGAT-3′ and 5′-TCAGGT- xilacine (Rompun 2%, 6 mg/kg; Bayer Hispania, Barcelona, Spain). Serum GACCGGCCAGGAGG-3′ (positions 92 to 309 corresponding to RefSeq: and plasma were collected and abdominal adipose tissue was dissected for L04970.1; amplicon length: 217); rat cyclophilin 5′-TCACCAGGGGAGAT- further analysis. Adiposity index was calculated as: sum of fat pads/[(body GGCACAGG-3′ and 5′-CCATGCTCACCCATCCGGGC-3′ (positions 319 weight-fat pad weight)×100]. The Animal Care and Use Committee of to 419 corresponding to RefSeq: NM_022536.2; amplicon length: 101). Universidad Complutense de Madrid approved all experimental procedures Similar results were obtained after normalisation to both housekeeping genes. according to the Spanish Policy for Animal Protection RD53/2013, which The ΔΔCt (or ΔΔCq following MIQE nomenclature) method was used for meets the European Union Directive 2010/63/UE. normalisation. In order to appropriately apply this method, we checked that both the target and the reference genes were amplified with comparable Real-time PCR efficiencies (>95%, calculated on the basis of the slope of calibration curves). Total RNA was isolated using Ultraspec™ (Biotecx, Houston, TX, USA) and Each sample was amplified in duplicate. was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific Inc, Morphological and histological evaluation Waltham, MA, USA). Quantification of mRNA levels was performed by Visceral and epididymal adipose tissue samples were dehydrated, embedded in using real-time PCR and an ABI PRISM 7900HT sequence detection system paraffin and cut into 5-μm-thick sections. Sections were stained with (Applied Biosystems, Thermo Fisher Scientific Inc, Waltham, MA, USA) and Picrosirius Red in order to detect collagen fibres. The area of pericellular TaqMan™ gene expression assays-on-demand (Applied Biosystems, Thermo fibrosis was identified as the ratio of collagen deposition to the total tissue area Fisher Scientific Inc, Waltham, MA, USA) for rat (Rn00566984_m1; RefSeq: after excluding the vessel area from the region of interest. This value was NM_017061.2) and human LOX (Hs00184700_m1; RefSeq:NM_002317.5), normalised by the number of adipocytes. Adipocytes (80-100 per subject or human LOXL1 (Hs00173746_m1; RefSeq: NM_005576.2), human LOXL2 animal) with intact cellular membranes were chosen for determination of the (Hs00158757_m1; RefSeq: NM_002318.2), human LOXL3 (Hs0026 cross-sectional area in hematoxylin-eosin stained sections. For each sample, 1671_m1; RefSeq: NM_032603.2) and human LOXL4 (Hs00260059_m1; 10-15 fields were analysed using a 40× objective (Leica DM 2000; Leica RefSeq: NM_032211.6). Rat BMP1 mRNAwas determined by using real-time Camera AG, Wetzlar, Germany) and quantified (Leica Q550 IWB; Leica PCR with SYBR Green PCR Master mix (Applied Biosystems, Thermo Camera AG, Wetzlar, Germany). A single researcher that was unaware of the

Fisher Scientific Inc, Waltham, MA, USA) and the following primers experimental groups performed the analysis. Disease Models & Mechanisms

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Immunohistological evaluation initiated with 0.9 ml of KRH buffer containing 100 mM insulin for 30 min 3 Sections were deparaffinised in xylene and rehydrated in graded ethanol followed by the addition of 100 µM 2-deoxy-D-glucose and 1 µCi/ml of [ H]- solutions. After blocking, slides were incubated with antibodies against 2-deoxy-D-glucose/ml. After 10 min, cells were washed with an ice-cold LOX (Abcam, Cambridge, UK). Afterwards, samples were incubated with solution of 50 mM D-glucose in PBS three times. Cells were lysed with a buffer a biotinylated secondary antibody (Vector Laboratories Inc, Burlingame, containing 0.5 N NaOH and 0.1% sodium dodecyl sulfate (SDS), and the CA, USA), washed and treated with the Vectastain (ABC) avidin-biotin radioactivity retained bythe cell lysatewas measured using a liquid scintillation peroxidase complex (Vector Laboratories Inc, Burlingame, CA, USA). counter (LS 6500, Beckman Coulter, Fullerton, CA, USA). Measurements Colour was developed using 3,3′-diaminobenzidine (DAB) and sections were made in triplicate and corrected for nonspecific diffusion. Counts of [3H]- were counterstained with haematoxylin. Negative controls, in which the 2-deoxyglucose were normalised to protein levels. primary antibody was omitted, were included to test for non-specific binding (Orriols et al., 2014). Statistical analysis Data are expressed as means and standard deviations. Normality of Cell culture and differentiation distributions and homogeneity of the variance were verified by means of Murine 3T3-L1 preadipocytes were cultured to confluence in Dulbecco’s either the Kolmogorov–Smirnov or Levene’s test. Either Pearson or Spearman modified Eagle’s medium (DMEM; Life Technologies, Thermo Fisher correlation analysis was used to examine the association among different Scientific Inc, Waltham, MA, USA) that was supplemented with 10% (v/v) variables according to whether they were normally distributed or not, calf serum (Biological Industries, Kibbutz Beit-Haemek, Israel). At 2 days respectively. Differences between two groups were analysed by either post-confluence (designated day 0), cells were induced to differentiate with unpaired Student’s t-test test or Mann–Whitney as parametric and non- DMEM containing a standard induction cocktail of 10% (v/v) fetal bovine parametric tests, respectively. Specific differences between more groups were serum (FBS; Biological Industries, Kibbutz Beit-Haemek, Israel), 1 μM analysed using Kruskall–Wallis followed by Dunn’s test for non-normal dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and 100 nM distribution variables. For normal distribution variables, one-way analysis of insulin (all from Sigma-Aldrich, St Louis, MO, USA). After 48 h, this variance followed by Newman–Keuls or Tamahane tests were used whether medium was replaced with DMEM supplemented with 10% FBS and the variance was homogenous or not, respectively. Data analysis was 100 nM of insulin. This medium was changed every 48 h. As a model of performed using the statistical program SPSS version 22.0 (SPSS Inc., insulin resistance, murine TNFα (20 or 50 ng/ml, equivalent to 1.15 and Chicago, IL, USA). The predetermined significance level was α=0.05. 2.87 nM, respectively, Sigma-Aldrich, St Louis, MO, USA) was added to the cell culture medium 7 days after the induction of differentiation, when Acknowledgements We thank Encarna Munoz-Ferrero, Silvia Aguilóand Blanca Martıneź for their more than 95% of the cells had the morphological and biochemical ̃ α technical help, Anthony DeMarco for his help in editing the manuscript and Cristina properties of adipocytes. For treatment with TNF (72 h), fully Fernández for her help in the statistical analysis of the data. differentiated 3T3-L1 adipocytes were treated every 24 h with the cytokine as previously described (Stephens et al., 1997). BAPN (200 µM) Competing interests was added to TNFα-treated cells during the last 24 h of incubation. This The authors declare no competing or financial interests. treatment did not induce cytotoxicity, as analysed by using the XTT-based assay for cell viability (Roche Diagnostics, Indianapolis, IN, USA). Author contributions V.C. and C.R. designed the study, performed experiments and data analysis and wrote the manuscript. M.M. and M.G. performed experiments and data analysis and Preparation of whole cell and tissue extracts helped to write the manuscript. E.M.-M., S.V., R.J.-L. and B.B.-M. performed 3T3-L1 preadipocytes were washed with PBS and cell monolayers were experiments and contributed to the discussion of the manuscript. J.M.-G. harvested in a non-denaturing buffer containing 150 mM NaCl, 10 mM Tris, contributed to the discussion and the writing of the manuscript. A.A. and M.L. were pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, responsible for the clinical study and contributed to discussion of the manuscript. 1mMNa3VO4, 1 µg/ml leupeptin and 1 mM DTT. Samples were extracted V.C. and C.R. had full access to all the data in the study and take responsibility for the for 30 min on ice and centrifuged at 16,000 g at 4°C for 15 min. Supernatants integrity of the data and the accuracy of the data analysis. were analysed for protein content using the BCA kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Total protein from epididymal adipose Funding tissue was obtained by homogenisation in lysis buffer and centrifugation for This work was supported by Plan Estatal I+D+I 2013-2016 [PI12/01729, PI12/ 5 min at 12,700 g (4°C). The tissue extract was then separated from the 01952] and Red de Investigación Cardiovascular [RD12/0042/0033 and RD12/ 0042/0053] of the Instituto de Salud Carlos III (ISCIII), as well as the Ministerio de fat and cellular debris, and analysed for protein content. Economıá y Competitividad (MINECO) [SAF2012-40127]. The study was cofunded by Fondo Europeo de Desarrollo Regional (FEDER), a way to make Europe. M.G. Western blotting was supported by the Sara Borrell Program from ISCIII (CD12/00589). Cell and tissue lysates were separated using SDS-PAGE and transferred to 0.45-μm polyvinylidene difluoride membranes (Immobilon, Merck References Millipore, Darmstadt, Germany). Blots were incubated with antibodies Abdennour, M., Reggio, S., Le Naour, G., Liu, Y., Poitou, C., Aron-Wisnewsky, J., against GLUT4 (Santa Cruz Biotechnology Inc, Heidelberg, Germany), Charlotte, F., Bouillot, J.-L., Torcivia, A., Sasso, M. et al. (2014). Association of adiponectin (Chemicon, Merck Millipore, Darmstadt, Germany), DDP4 adipose tissue and liver fibrosis with tissue stiffness in morbid obesity: links with (Abcam, Cambridge, UK), SOCS3 (Cell Signaling Technology Inc, diabetes and BMI loss after gastric bypass. J. Clin. Endocrinol. Metab. 99, 898-907. Ahsan, T., Lottman, L. M., Harwood, F., Amiel, D. and Sah, R. L. (1999). Integrative Danvers, MA, USA) and LOX (Abcam, Cambridge, UK). Bound cartilage repair: inhibition by beta-aminopropionitrile. J. Orthop. Res. 17, 850-857. antibodies were detected after incubation with a horseradish peroxidase Alligier, M., Meugnier, E., Debard, C., Lambert-Porcheron, S., Chanseaume, E., (HRP)-conjugated IgG and then using the Super Signal West Dura Extended Sothier, M., Loizon, E., Hssain, A. A., Brozek, J., Scoazec, J.-Y. et al. (2012). Duration Substrate (Thermo Fisher Scientific Inc, Waltham, MA, USA). Subcutaneous adipose tissue remodelling during the initial phase of weight gain Equal loading of protein in each lane was verified by Ponceau staining and induced by overfeeding in humans. J. Clin. Endocrinol. Metab. 97, E183-E192. β Brasselet, C., Durand, E., Addad, F., Al Haj Zen, A., Smeets, M. B., Laurent- by western blotting for -actin (Sigma). Maquin, D., Bouthors, S., Bellon, G., de Kleijn, D., Godeau, G. et al. (2005). Collagen and elastin cross-linking: a mechanism of constrictive remodelling after Glucose uptake measurements arterial injury. Am. J. Physiol. Heart Circ. Physiol. 289, H2228-H2233. Fully differentiated 3T3-L1 adipocytes were insulin-deprived and pre-treated Dasler, W. (1954). Isolation of toxic crystals from sweet peas (Lathyrus odoratus). with BAPN in the presence or absence of TNFα for 24 h. Adipocytes were Science 120, 307-308. serum-deprived for 3 h in DMEM supplemented with 2% of fatty-acid-free Dimaculangan, D. D., Chawla, A., Boak, A., Kagan, H. M. and Lazar, M. A. α (1994). Retinoic acid prevents downregulation of ras recision gene/lysyl oxidase bovine serum albumin (BSA) with or without TNF and BAPN. Serum-free early in adipocyte differentiation. Differentiation 58, 47-52. medium was then removed and the cells were washed with 1 ml of Krebs- Divoux, A., Tordjman, J., Lacasa, D., Veyrie, N., Hugol, D., Aissat, A.,

Ringer-HEPES (KRH) buffer pH 7.4 plus 0.2% BSA. Glucose uptake was Basdevant, A., Guerre-Millo, M., Poitou, C., Zucker, J.-D. et al. (2010). Disease Models & Mechanisms

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Fibrosis in human adipose tissue: composition, distribution, and link with lipid Nave, A. H., Mižıková ́, I., Niess, G., Steenbock, H., Reichenberger, F., Talavera, metabolism and fat mass loss. Diabetes 59, 2817-2825. M. L., Veit, F., Herold, S., Mayer, K., Vadász, I. et al. (2014). Lysyl oxidases play Eckel, R. H. (2011). The complex metabolic mechanisms relating obesity to a causal role in vascular remodelling in clinical and experimental pulmonary hypertriglyceridemia. Arterioscler. Thromb. Vasc. Biol. 31, 1946-1948. arterial hypertension. Arterioscler. Thromb. Vasc. Biol. 34, 1446-1458. Emanuelli, B., Peraldi, P., Filloux, C., Chavey, C., Freidinger, K., Hilton, D. J., Orriols, M., Guadall, A., Galán, M., Martı-Pá mies,̀ I., Varona, S., Rodrıguez-́ Hotamisligil, G. S. and Van, O. E. (2001). SOCS-3 inhibits insulin signaling and Calvo, R., Briones, A. M., Navarro, M. A., de Diego, A., Osada, J. et al. (2014). is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of Lysyl oxidase (LOX) in vascular remodelling. Insight from a new animal model. obese mice. J. Biol. Chem. 276, 47944-47949. Thromb. Haemost. 112, 812-824. Erler, J. T., Bennewith, K. L., Nicolau, M., Dornhöfer, N., Kong, C., Le, Q.-T., Chi, Palanivel, R., Fullerton, M. D., Galic, S., Honeyman, J., Hewitt, K. A., Jorgensen, J.-T. A., Jeffrey, S. S. and Giaccia, A. J. (2006). Lysyl oxidase is essential for S. B. and Steinberg, G. R. (2012). Reduced Socs3 expression in adipose tissue hypoxia-induced metastasis. Nature 440, 1222-1226. protects female mice against obesity-induced insulin resistance. Diabetologia 55, Goossens, G. H. (2008). The role of adipose tissue dysfunction in the pathogenesis 3083-3093. of obesity-related insulin resistance. Physiol. Behav. 94, 206-218. Raposo, B., Rodrıguez,́ C., Martınez-Gonzá ́lez, J. and Badimon, L. (2004). High Guilherme, A., Virbasius, J. V., Puri, V. and Czech, M. P. (2008). Adipocyte levels of homocysteine inhibit lysyl oxidase (LOX) and downregulate LOX dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nat. Rev. expression in vascular endothelial cells. Atherosclerosis 177, 1-8. Mol. Cell Biol. 9, 367-377. Rodrıguez,́ C., Raposo, B., Martınez-Gonzá ́lez, J., Casanı,́ L. and Badimon, L. Halberg, N., Khan, T., Trujillo, M. E., Wernstedt-Asterholm, I., Attie, A. D., (2002). Low density lipoproteins downregulate lysyl oxidase in vascular Sherwani, S., Wang, Z. V., Landskroner-Eiger, S., Dineen, S., Magalang, U. J. endothelial cells and the arterial wall. Arterioscler. Thromb. Vasc. Biol. 22, et al. (2009). Hypoxia-inducible factor 1alpha induces fibrosis and insulin 1409-1414. resistance in white adipose tissue. Mol. Cell. Biol. 29, 4467-4483. Rodrıguez,́ C., Martinez-Gonzalez, J., Raposo, B., Alcudia, J. F., Guadall, A. Henegar, C., Tordjman, J., Achard, V., Lacasa, D., Cremer, I., Guerre-Millo, M., and Badimon, L. (2008a). Regulation of lysyl oxidase in vascular cells: lysyl Poitou, C., Basdevant, A., Stich, V., Viguerie, N. et al. (2008). Adipose tissue oxidase as a new player in cardiovascular diseases. Cardiovasc. Res. 79, 7-13. transcriptomic signature highlights the pathological relevance of extracellular Rodrıguez,́ C., Rodrıguez-Sinovas,́ A. and Martınez-Gonzá ́lez, J. (2008b). Lysyl matrix in human obesity. Genome Biol. 9, R14. oxidase as a potential therapeutic target. Drug News Perspect. 21, 218-224. Hornstra, I. K., Birge, S., Starcher, B., Bailey, A. J., Mecham, R. P. and Shapiro, Rodriguez, C., Alcudia, J. F., Martinez-Gonzalez, J., Guadall, A., Raposo, B., S. D. (2003). Lysyl oxidase is required for vascular and diaphragmatic Sanchez-Gomez, S. and Badimon, L. (2009). Statins normalize vascular lysyl development in mice. J Biol Chem. 278, 14387-14393. oxidase down-regulation induced by proatherogenic risk factors. Cardiovasc. Res. Huang, H., Song, T.-J., Li, X., Hu, L., He, Q., Liu, M., Lane, M. D. and Tang, Q.-Q. 83, 595-603. (2009). BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent Sell, H., Bluher, M., Kloting, N., Schlich, R., Willems, M., Ruppe, F., Knoefel, stem cells to the adipocyte lineage. Proc. Natl. Acad. Sci. USA 106, 12670-12675. W. T., Dietrich, A., Fielding, B. A., Arner, P. et al. (2013). Adipose dipeptidyl Kadowaki, T., Yamauchi, T., Kubota, N., Hara, K., Ueki, K. and Tobe, K. (2006). peptidase-4 and obesity: correlation with insulin resistance and depot-specific Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the release from adipose tissue in vivo and in vitro. Diabetes Care. 36, 4083-4090. metabolic syndrome. J. Clin. Invest. 116, 1784-1792. Sethi, J. K. and Hotamisligil, G. S. (1999). The role of TNF alpha in adipocyte Kanatani, Y., Usui, I., Ishizuka, K., Bukhari, A., Fujisaka, S., Urakaze, M., Haruta, metabolism. Semin. Cell Dev. Biol. 10, 19-29. T., Kishimoto, T., Naka, T. and Kobayashi, M. (2007). Effects of pioglitazone on Shepherd, P. R. and Kahn, B. B. (1999). Glucose transporters and insulin action– suppressor of cytokine signaling 3 expression: potential mechanisms for its implications for insulin resistance and diabetes mellitus. N. Engl. J. Med. 341, effects on insulin sensitivity and adiponectin expression. Diabetes 56, 795-803. 248-257. Kern, P. A., Di Gregorio, G. B., Lu, T., Rassouli, N. and Ranganathan, G. (2003). Spencer, P. S. and Schaumburg, H. H. (1983). Lathyrism: a neurotoxic disease. Adiponectin expression from human adipose tissue: relation to obesity, insulin Neurobehav. Toxicol. Teratol. 5, 625-629. resistance, and tumor necrosis factor-alpha expression. Diabetes 52, 1779-1785. Stefanon, I., Valero-Muñoz, M., Fernandes, A. A., Ribeiro, R. F., Jr, Rodrıguez,́ Khan, T., Muise, E. S., Iyengar, P., Wang, Z. V., Chandalia, M., Abate, N., Zhang, C., Miana, M., Martınez-Gonzá ́lez, J., Spalenza, J. S., Lahera, V., Vassallo, B. B., Bonaldo, P., Chua, S. and Scherer, P. E. (2009). Metabolic dysregulation P. F. et al. (2013). Left and right ventricle late remodelling following myocardial and adipose tissue fibrosis: role of collagen VI. Mol. Cell. Biol. 29, 1575-1591. infarction in rats. PLoS ONE 8, e64986. Kothapalli, D., Liu, S.-L., Bae, Y. H., Monslow, J., Xu, T., Hawthorne, E. A., Stephens, J. M., Lee, J. and Pilch, P. F. (1997). Tumor necrosis factor-alpha- Byfield, F. J., Castagnino, P., Rao, S., Rader, D. J. et al. (2012). Cardiovascular protection by ApoE and ApoE-HDL linked to suppression of ECM gene expression induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of and arterial stiffening. Cell Rep. 2, 1259-1271. insulin receptor substrate-1 and GLUT4 expression without a loss of insulin Lamers, D., Famulla, S., Wronkowitz, N., Hartwig, S., Lehr, S., Ouwens, D. M., receptor-mediated signal transduction. J. Biol. Chem. 272, 971-976. ́ Eckardt, K., Kaufman, J. M., Ryden, M., Muller, S. et al. (2011). Dipeptidyl Tam, C. S., Tordjman, J., Divoux, A., Baur, L. A. and Clement, K. (2012). Adipose peptidase 4 is a novel adipokine potentially linking obesity to the metabolic tissue remodelling in children: the link between collagen deposition and age- syndrome. Diabetes 60, 1917-1925. related adipocyte growth. J. Clin. Endocrinol. Metab. 97, 1320-1327. Levental, K. R., Yu, H., Kass, L., Lakins, J. N., Egeblad, M., Erler, J. T., Fong, Tang, S. S., Trackman, P. C. and Kagan, H. M. (1983). Reaction of aortic lysyl S. F. T., Csiszar, K., Giaccia, A., Weninger, W. et al. (2009). Matrix crosslinking oxidase with beta-aminopropionitrile. J. Biol. Chem. 258, 4331-4338. forces tumor progression by enhancing integrin signaling. Cell 139, 891-906. Taskinen, M.-R., Adiels, M., Westerbacka, J., Soderlund, S., Kahri, J., Mäki, J. M., Räsänen, J., Tikkanen, H., Sormunen, R., Mäkikallio, K., Kivirikko, Lundbom, N., Lundbom, J., Hakkarainen, A., Olofsson, S.-O., Orho- K. I. and Soininen, R. (2002). Inactivation of the lysyl oxidase gene Lox leads to Melander, M. et al. (2011). Dual metabolic defects are required to produce aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. hypertriglyceridemia in obese subjects. Arterioscler. Thromb. Vasc. Biol. 31, Circulation 106, 2503-2509. 2144-2150. Martınez-Mart́ ınez,́ E., Jurado-López, R., Valero-Muñoz, M., Bartolomé,M.V., Trayhurn, P. (2013). Hypoxia and adipose tissue function and dysfunction in Ballesteros, S., Luaces, M., Briones, A. M., López-Andrés, N., Miana, M. and obesity. Physiol. Rev. 93, 1-21. Cachofeiro, V. (2014a). Leptin induces cardiac fibrosis through galectin-3, mTOR Turecek, C., Fratzl-Zelman, N., Rumpler, M., Buchinger, B., Spitzer, S., Zoehrer, and oxidative stress: potential role in obesity. J. Hypertens. 32, 1104-1114. R., Durchschlag, E., Klaushofer, K., Paschalis, E. P. and Varga, F. (2008). Martınez-Mart́ ınez,́ E., Miana, M., Jurado-López, R., Bartolomé, M. V., Souza Collagen cross-linking influences osteoblastic differentiation. Calcif. Tissue Int. Neto, F. V., Salaices, M., López-Andrés, N. and Cachofeiro, V. (2014b). The 82, 392-400. potential role of leptin in the vascular remodelling associated with obesity. Virtue, S. and Vidal-Puig, A. (2010). Adipose tissue expandability, lipotoxicity and Int. J. Obes. (Lond.) 38, 1565-1572. the Metabolic Syndrome–an allostatic perspective. Biochim. Biophys. Acta 1801, Miana, M., de Las Heras, N., Rodriguez, C., Sanz-Rosa, D., Martin-Fernandez, 338-349. B., Mezzano, S., Lahera, V., Martinez-Gonzalez, J. and Cachofeiro, V. (2011). Walker, R. W., Allayee, H., Inserra, A., Fruhwirth, R., Alisi, A., Devito, R., Carey, Effect of eplerenone on hypertension-associated renal damage in rats: potential M. E., Sinatra, F., Goran, M. I. and Nobili, V. (2014). Macrophages and fibrosis in role of peroxisome proliferator activated receptor gamma (PPAR-gamma). adipose tissue are linked to liver damage and metabolic risk in obese children. J. Physiol. Pharmacol. 62, 87-94. Obesity(Silver Spring) 22, 1512-1519. Mutch, D. M., Rouault, C., Keophiphath, M., Lacasa, D. and Clément, K. (2009). Wang, Z., Zhou, Y.-T., Kakuma, T., Lee, Y., Kalra, S. P. and Kalra, P. S. (2000). Using gene expression to predict the secretome of differentiating human Leptin resistance of adipocytes in obesity: role of suppressors of cytokine preadipocytes. Int. J. Obes. (Lond.) 33, 354-363. signaling. Biochem. Biophys. Res. Commun. 277, 20-26. Disease Models & Mechanisms

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