The matricellular protein SPARC/osteonectin as a newly identified factor up-regulated in obesity

Alterations in the expression level of genes may contribute to the development and pathophysiology of obesity. To find genes differentially expressed in adipose tissue during obesity, we performed suppression subtractive hybridization on epididymal fat mRNA from goldthioglucose (GTG) obese mice and from their lean littermates. We identified the secreted protein acidic and rich in cysteine (SPARC), a protein that mediates cell-matrix interactions and plays a role in modulation of cell adhesion, differentiation, and angiogenesis. SPARC mRNA expression in adipose tissue was markedly increased (between 3- and 6-fold) in three different models of obesity, i.e. GTG mice, ob/ob mice, and AKR mice, after 6 weeks of a high fat diet. Immunoblotting of adipocyte extracts revealed a similar increase in protein level. Using a SPARC-specific ELISA, we demonstrated that SPARC is secreted by isolated adipocytes. We found that insulin administration to mice increased SPARC mRNA in the adipose tissue. Food deprivation had no effect on SPARC expression, but after high fat refeeding SPARC mRNA levels were significantly increased. Our results reveal both hormonal and nutritional regulation of SPARC expression in the adipocyte, and importantly, its alteration in obesity. Finally, we show that purified SPARC increased mRNA levels of plasminogen activator inhibitor 1 (PAI-1) in cultured rat adipose tissue suggesting that elevated adipocyte expression of SPARC might contribute to the abnormal expression of PAI-1 observed in obesity. We propose that SPARC is a newly identified autocrine/paracrine factor that could affect key functions in adipose tissue physiology and pathology.     

Effect of angiotensin peptides on PAI-1 expression and production in human adipocytes.

Angiotensin (Ang) II is the active component of the renin-angiotensin-system (RAS), but its degradation products have also been shown to exhibit biological activity. This system, which mainly controls blood pressure and electrolyte homeostasis, was recently found to be completely expressed in human adipose tissue. The major determinant in the fibrinolytic system is the plasminogen activator inhibitor-1 (PAI-1). Both PAI-1 and components of the RAS are over-expressed in the obese state. We have recently shown that Ang II is able to induce PAI-1 expression and release via the AT1-receptor in human fat cells in primary culture, and have provided the first evidence that two metabolites, Ang III and Ang IV, may have a similar stimulatory effect on PAI-1 release. We have now performed additional experiments to further characterize the role of the angiotensin peptides in the production of PAI-1. Ang III and Ang IV showed a time- and dose-dependent stimulation of PAI-1 protein release. Concomitantly, mRNA-levels were markedly elevated. Using specific receptor blockers, all angiotensin peptides seem to induce PAI-1 expression via the angiotensin receptor subtype 1. However, components of the renin-angiotensin-system seem to play an important role in the control of fibrinolysis in adipose tissue. We conclude that PAI-1 production by adipose tissue may contribute to the elevated thromboembolic risk in obesity.     

Protection from high fat diet-induced increase in ceramide in mice lacking plasminogen activator inhibitor 1

Obesity increases the risk for metabolic and cardiovascular disease, and adipose tissue plays a central role in this process. Ceramide, the key intermediate of sphingolipid metabolism, also contributes to obesity-related disorders. We show that a high fat diet increased ceramide levels in the adipose tissues and plasma in C57BL/6J mice via a mechanism that involves an increase in gene expression of enzymes mediating ceramide generation through the de novo pathway (e.g. serine palmitoyltransferase) and via the hydrolysis of sphingomyelin (acid sphingomyelinase and neutral sphingomyelinase). Although the induction of total ceramide in response to the high fat diet was modest, dramatic increases were observed for C16, C18, and C18:1 ceramides. Next, we investigated the relationship of ceramide to plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of plasminogen activation and another key player in obesity. PAI-1 is consistently elevated in obesity and thought to contribute to increased artherothrombotic events and more recently to obesity-mediated insulin resistance. Interestingly, the changes in ceramide were attenuated in mice lacking PAI-1. Mechanistically, mice lacking PAI-1 were protected from diet-induced increase in serine palmitoyltransferase, acid sphingomyelinase, and neutral sphingomyelinase mRNA, providing a mechanistic link for decreased ceramide in PAI-1-/- mice. The decreases in plasma free fatty acids and adipose tumor necrosis factor-alpha in PAI-1-/- mice may have additionally contributed indirectly to improvements in ceramide profile in these mice. This study has identified a novel link between sphingolipid metabolism and PAI-1 and also suggests that ceramide may be an intermediary molecule linking elevated PAI-1 to insulin resistance.     

Regulation of plasminogen activitor inhibitor-1 in human adipose tissue: interaction between cytokines, cortisol and estrogen.

To investigate further the role of plasminogen activator inhibitor-1 (PAI-1) in human adipose tissue, the regulation of cytokines, cortisol (dexamethasone) as well as estrogen on PAI-1 were determined in human adipose tissue fragments. PAI-1 activity was increased in human adipose tissue fragments incubated for 48 h with interleukin-1beta (IL-1beta) (2.6-fold, p < 0.01) and tumor necrosis factor-alpha (2.3-fold, p < 0.01). Incubation with interleukin-6 revealed a non-significant decrease in PAI-1 activity. Parallel findings were obtained when studying the PAI-1 mRNA expression. Dexamethesone increased PAI-1 activity after incubation for 8 h (p < 0.05) and enhanced the stimulation of IL-1beta after 8 h incubation. However, after 24 and 48 h, dexamethasone significantly reduced the IL-1beta induced increase in PAI-1 activity by 24-52% (p < 0.05), accordingly, PAI-1 mRNA expression was reduced 60%. Finally, the induction of PAI-1 activity and PAI-1 mRNA expression by IL-1beta was attenuated by estrogen (17.8+/-4.9%, p < 0.05 and 20.9+/-5.8%, p < 0.05, respectively). These results indicate that multiple cytokines, estrogen and dexamethasone may be involved in the regulation of PAI-1 biosynthesis in human adipose tissue, and suggest that there are interactions between cytokines and these steroid hormones. The interplay between these hormones may be of importance for the levels of PAI-1 observed in obesity and associated states.     

Fat cell function and fibrinolysis.

Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of plasminogen activators and may be the principal regulator of plasminogen activation in vivo. PAI-1 levels are elevated in insulin-resistant subjects and are associated with an increased risk of atherothrombosis. After adjustment for metabolic parameters, increased PAI-1 levels were no longer considered as a cardiovascular risk factor. The mechanisms underlying the strong association between PAI-1 levels and the metabolic disturbances found in insulin resistance are still not understood. Several studies have suggested that visceral adipose tissue may be a major source of PAl-1. Accordingly, adipose tissue PAI-1 production particularly that from visceral fat, was found to be elevated in obese human subjects. Within human adipose tissue, stromal cells appear to be the main cells involved in PAI-1 synthesis. This review discusses the potential mechanisms linking adipose tissue to plasma PAI-1 levels such as the intervention of cytokines (TNFalpha and TGFbeta), free fatty acids and hormones (insulin and glucocorticoids). Moreover alteration of adipose tissue cellular composition induced by the modulation of PAI-1 expression opens a novel field of interest.     

Bone marrow plasminogen activator inhibitor-1 influences the development of obesity

Plasma levels of plasminogen activator inhibitor-1 (PAI-1) are elevated in obesity and correlate with body mass index. The increase in PAI-1 associated with obesity likely contributes to increased cardiovascular risk and may predict the development of type 2 diabetes mellitus. Although adipocytes are capable of synthesizing PAI-1, the bulk of evidence indicates that cells residing in the stromal fraction of visceral fat are the primary source of PAI-1. We hypothesized that bone marrow-derived PAI-1, e.g. derived from macrophages located in visceral fat, contributes to the development of diet-induced obesity. To test this hypothesis, male C57BL/6 wild-type mice and C57BL/6 PAI-1 deficient mice were transplanted with either PAI-1(-/-), PAI-1(+/-), or PAI-1(+/+) bone marrow. The transplanted animals were subsequently fed a high fat diet for 24 weeks. Our findings show that only the complete absence of PAI-1 protects from the development of diet-induced obesity, whereas the absence of bone marrow-derived PAI-1 protects against expansion of the visceral fat mass. Remarkably, there is a link between the PAI-1 levels, the degree of inflammation in adipose tissue, and the development of obesity. Based on these findings we suggest that bone marrow-derived PAI-1 has an effect on the development of obesity through its effect on inflammation.     

Visceral fat is a determinant of PAI-1 activity in diabetic and non-diabetic overweight and obese women.

Plasminogen activator inhibitor type 1 (PAI-1), an inhibitor of fibrinolysis and an important and independent cardiovascular risk factor, has been shown to be elevated in obesity and type 2 diabetes. Recent study results have suggested that adipose tissue--visceral fat in particular--could play an important role in the fibrinolytic process.In order to assess the specific role of this fat distribution, we measured PAI-1 activity (AU/ml) and visceral fat (CT-scan at level L4-L5) in 2 groups of 30 overweight and obese diabetic and overweight and obese non-diabetic women. Subjects were matched for age, weight, body mass index, fat mass and total abdominal fat. Visceral adipose tissue and PAI-1 were significantly higher in diabetic women (p = 0.022 and p = 0.004 respectively) than in non-diabetic patients. Visceral fat correlated significantly with PAI-1 activity, even after correction for insulin and triglycerides (r = 0.28, p = 0.034). Stepwise regression analysis showed visceral fat as the most important determinant factor for PAI-1 in the whole group and in the non-diabetic group. In the diabetic group, fasting insulin was the most important determinant. These results show that visceral fat is more important than BMI or total body fat in the determination of PAI-1 levels. Furthermore, the increased amount of visceral fat in type 2 diabetics may contribute to the increase of PAI-1 activity levels and the subsequent increased risk for thrombovascular disease, regardless of BMI and total fatness.     

Glucocorticoid-regulated adipose tissue secretion of PAI-1, but not IL-6, TNFalpha or leptin in vivo.

OBJECTIVE: Glucocorticoids are well-known regulators of adipose tissue metabolism and endocrine function. The aim of this study was to examine glucocorticoid effect on plasminogen activator inhibitor 1 (PAI-1), tumour necrosis factor alpha (TNFalpha), leptin and interleukin-6 (IL-6) adipose tissue secretion. MATERIAL AND METHODS: Twelve healthy postmenopausal women with mean BMI of 28.9 kg/m(2) (+/- 0.8 SEM) received 25 mg prednisolone daily for 7 days. Before and after glucocorticoid treatment adipose tissue secretion of PAI-1, leptin, IL-6 and TNFalpha were measured, and adipocyte PAI-1 mRNA as well as anthropometrical and bio-chemical data were obtained. RESULTS: Anthropometric measurements remained unaffected. Analyses of venous blood-samples showed a borderline increase of insulin levels (p = 0.062). PAI-1 secretion from adipose tissue increased (1.9 +/- 0.2 vs. 3.5 +/- 0.5 ng/g triglycerides, p = 0.012), but PAI-1 mRNA levels did not (0.19 +/- 0.02 vs. 0.21 +/- 0.04 arbitrary units after normalised to beta-actin, p = 0.51). There were no apparent differences in IL-6, TNFalpha or leptin secretion after glucocorticoid exposure. CONCLUSION: This study shows an increased secretion of PAI-1, but not IL-6, TNFalpha or leptin, from abdominal adipose tissue after in vivo glucocorticoid treatment, which may be a finding of pathophysiological importance given the well-known effect of glucocorticoid excess on metabolic aberrations and cardiovascular morbidity.     

Monocyte chemotactic protein-1 and its role in insulin resistance

PURPOSE OF REVIEW: In obesity, there is a strong link between increased adipose tissue mass and development of insulin resistance in tissues such as liver and muscle. Under these conditions, adipose tissue synthesizes various pro-inflammatory chemokines such as monocyte chemotactic protein-1. This review provides a summary of recent knowledge on the role of monocyte chemotactic protein-1 in adipose tissue inflammation and insulin resistance. RECENT FINDINGS: Monocyte chemotactic protein-1 is a proinflammatory adipokine that is believed to play a role in the pathogenesis of obesity and diabetes. New in-vitro data demonstrate that monocyte chemotactic protein-1 has the ability to induce insulin resistance in adipocytes and skeletal muscle cells. By using mice that either overexpress monocyte chemotactic protein-1 or are deficient in monocyte chemotactic protein-1 or its receptor, exciting new insights have been obtained into the role of monocyte chemotactic protein-1 in adipose tissue inflammation and insulin resistance. SUMMARY: Monocyte chemotactic protein-1 is an adipokine with insulin-resistance-inducing capacity that is related to increased adipose tissue mass in obesity and insulin resistance. It plays an important role in adipose tissue inflammation by recruiting macrophages into fat. Monocyte chemotactic protein-1 is thus a therapeutic target, and may represent an important factor linking adipose tissue inflammation, obesity and type 2 diabetes.     

Dicarbonyl stress in clinical obesity

The glyoxalase system in the cytoplasm of cells provides the primary defence against glycation by methylglyoxal catalysing its metabolism to D-lactate. Methylglyoxal is the precursor of the major quantitative advanced glycation endproducts in physiological systems - arginine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. Glyoxalase 1 of the glyoxalase system was linked to anthropometric measurements of obesity in human subjects and to body weight in strains of mice. Recent conference reports described increased weight gain on high fat diet-fed mouse with lifelong deficiency of glyoxalase 1 deficiency, compared to wild-type controls, and decreased weight gain in glyoxalase 1-overexpressing transgenic mice, suggesting a functional role of glyoxalase 1 and dicarbonyl stress in obesity. Increased methylglyoxal, dicarbonyl stress, in white adipose tissue and liver may be a mediator of obesity and insulin resistance and thereby a risk factor for development of type 2 diabetes and non-alcoholic fatty liver disease. Increased methylglyoxal formation from glyceroneogenesis on adipose tissue and liver and decreased glyoxalase 1 activity in obesity likely drives dicarbonyl stress in white adipose tissue increasing the dicarbonyl proteome and related dysfunction. The clinical significance will likely emerge from on-going clinical evaluation of inducers of glyoxalase 1 expression in overweight and obese subjects. Increased transcapillary escape rate of albumin and increased total body interstitial fluid volume in obesity likely makes levels of glycation of plasma protein unreliable indicators of glycation status in obesity as there is a shift of albumin dwell time from plasma to interstitial fluid, which decreases overall glycation for a given glycemic exposure.     

The double role of epicardial adipose tissue as pro- and anti-inflammatory organ.

Obesity is associated with low grade inflammation. Whether this is just an adaptive response to excess adiposity to maintain a normal oxygen supply or a chronic activation of the innate immune system is still unknown. Recent research has focused on the origin of the inflammatory markers in obesity and the extent to which adipose tissue has a direct effect. The production of adipokines by visceral adipose tissue is of particular interest since their local secretion by visceral fat depots may provide a novel mechanistic link between obesity and the associated vascular complications. Growing evidences suggest that the epicardial adipose tissue, the visceral fat depot located around the heart, may locally interact with myocardium and coronary arteries. Epicardial fat is a source of adiponectin and adrenomedullin, adipokines with anti-inflammatory properties, and several proinflammatory cytokines as well as Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin 1 (IL1), IL-1 h, Interleukin (IL6), Monocyte Chemoattractive Protein-1 (MCP-1), Nerve Growth Factor (NGF), resistin, Plasminogen Activator Inhibitor-1 (PAI-1), and free fatty acids. Epicardial adipose tissue could locally modulate the heart and vasculature, through paracrine secretion of pro- and anti-inflammatory cytokines, thereby playing a possible role in the adiposity-related inflammation and atherosclerosis. On the other hand, epicardial fat could exert a protective effect through adiponectin and adrenomedullin secretion as response to local or systemic metabolic or mechanical insults. Future studies will continue to provide new and fascinating insights into the double role of epicardial adipose tissue in the development of cardiovascular pathology and/or in protecting the heart and arteries.     

Expression of aggrecan(ases) during murine preadipocyte differentiation and adipose tissue development

The expression and potential functional role of aggrecan in adipogenesis and adipose tissue development was investigated in murine models of obesity. Aggrecan, as well as the two aggrecanases ADAMTS-4 and ADAMTS-5 (A Disintegrin And Metalloproteinase with Thrombospondin motif) mRNAs, are expressed in subcutaneous (SC) and gonadal (GON) adipose tissues of mice. Their presence was confirmed by western blotting using adipose tissue extracts. In mice with nutritionally induced obesity (high fat diet) as well as in lean controls, aggrecan mRNA expression was downregulated whereas ADAMTS-4 and ADAMTS-5 were upregulated with time. In mice with genetically determined obesity (ob/ob), ADAMTS-5 mRNA was upregulated in both SC and GON adipose tissues, as compared to wild-type (WT) mice (p<0.001). Enhanced aggrecanase expression levels in these tissues were associated with significantly elevated levels of G1-NITEGE, a degradation product of aggrecan. Thus, aggrecan levels were high at the early stages of adipose tissue development in mice, whereas its production decreased and its degradation increased during development of obesity. A functional role of aggrecan in promoting early stages of adipogenesis is supported by the findings that it stimulated the in vitro differentiation of 3T3-F442A preadipocytes and the de novo in vivo accumulation of fat in Matrigel plaques injected into WT mice. Proteoglycans in the extracellular matrix of adipose tissue, such as aggrecan, may contribute to the regulation of lipid uptake and obesity in mice.     

Differential expression of plasminogen activator inhibitor-1, tumor necrosis factor-alpha,TNF-alpha converting enzyme and ADAMTS family members in murine fat territories

Our objective was to investigate expression of A disintegrin and metalloproteinase (ADAM) and ADAM proteins with a thrombospondin (TS) motif (ADAMTS) family members in adipose tissue of lean and obese mice. Five-week-old male mice were kept on standard chow (SFD) or on high fat diet (HFD) for 15 weeks, and subcutaneous (SC) and gonadal (GON) adipose tissue, as well as mature adipocytes and stromal-vascular (S-V) cells were harvested. mRNA levels of plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNF-alpha), ADAM-17 (TACE or TNF-alpha converting enzyme), ADAMTS-1 and ADAMTS-8 were quantified in isolated adipose tissues and cell fractions, and during differentiation of murine preadipocytes. The HFD resulted in a significantly enhanced weight of isolated SC and GON fat pads, and in enhanced blood levels of glucose, cholesterol and PAI-1. ADAM-17, TNF-alpha, PAI-1, ADAMTS-1 and ADAMTS-8 mRNA were detected in both SC and GON adipose tissue of lean mice (SFD). In SC adipose tissue of obese mice (HFD), the expression of ADAM-17 and PAI-1 was enhanced and that of ADAMTS-1 reduced, whereas in GON adipose tissue expression of TNF-alpha was enhanced and that of ADAMTS-8 reduced. In lean and obese mice, expression of ADAM-17, ADAMTS-1 and ADAMTS-8 was higher in the S-V cell fraction than in mature adipocytes. During differentiation of murine 3T3-F442A preadipocytes, expression of ADAM-17 and ADAMTS-1 remained virtually unaltered, whereas that of ADAMTS-8 decreased as adipocytes matured. Several ADAM and ADAMTS family members are expressed in adipose tissue and during differentiation of preadipocytes. Modulation of their expression upon development of obesity is adipose tissue-dependent.     

Differences in plasminogen activator inhibitor 1 in subcutaneous versus omental adipose tissue in non-obese and obese subjects.

Human adipose tissue can produce plasminogen activator inhibitor-1 (PAI-1). It has been suggested that high levels of PAI-1 are of importance in enhanced cardiovascular disease observed among obese subjects, especially abdominally obese individuals. In the present study, we investigated the level of mRNA and production of PAI-1 in adipose tissue from two adipose tissue depots (omental vs. subcutaneous). Adipose tissue from both depots was obtained from obese (mean BMI, 46.9 kg/m 2) and non-obese (mean BMI, 23.9 kg/m 2) women. PAI-1 mRNA was measured both in fresh adipose tissue obtained immediately after surgery and after the adipose tissue (fragments) had been incubated for up to 72 h. In immediately frozen adipose tissue, PAI-1 mRNA expression was similar in omental and subcutaneous adipose tissue. No differences between obese and non-obese women were found. However, when adipose tissue fragments were cultured, PAI-1 mRNA and PAI-1 production were significantly higher in omental than in subcutaneous adipose tissue (p < 0.05). In the culture system, the production of PAI-1 in obese subjects was higher than in non-obese subjects in both subcutaneous (p < 0.05) and in omental adipose tissue (p = 0.19). In order to test whether these regional differences observed after incubation of the adipose tissue were due to differences in local accumulation of cytokines that may stimulate PAI-1 by a paracrine or autocrine manner, we investigated the expression of transforming growth factor beta1 (TGF-beta1) mRNA and tumor necrosis factor alpha (TNF-alpha) mRNA and protein. No differences between the two fat depots were found. In conclusion, no differences in PAI-1 expression between omental and subcutaneous adipose tissue were observed in biopsies frozen immediately after removal, but after incubation of adipose tissue (which somehow stimulates PAI-1 production), higher levels of PAI-1 were found in omental adipose tissue than in subcutaneous adipose tissue. Finally, PAI-1 production in adipose tissue from obese women was higher in non-obese women after incubation for 72 h.     

Plasminogen activator inhibitor-1 in the pathogenesis of asthma

Plasminogen activator inhibitor (PAI)-1 is the main inhibitor of the fibrinolytic system and is known to play an essential role in tissue remodeling. Recent evidence indicates that chronic asthma may lead to tissue remodeling such as subepithelial fibrosis and extracellular matrix (ECM) deposition in the airways. However, the role of PAI-1 in asthma is unknown. Recently the mast cell (MC), which plays a major role in asthma, was found as a novel source of PAI-1, and a large number of MCs expressing PAI-1 are infiltrated in the airways of patients with severe asthma. Furthermore, PAI-1-deficient mice show reduced ECM deposition in the airways of a murine model of chronic asthma by inhibiting MMP-9 activity and fibrinolysis. In a human study, the 4G allele frequency was significantly higher in the asthmatic patients than in the control group. In view of the findings that the 4G allele is associated with elevated plasma PAI-1 level, elevated PAI-1 level in the lung may contribute to the development of asthma. In summary, PAI-1 may play an important role in the pathogenesis of asthma and further studies evaluating the mechanisms of PAI-1 action may lead to the development of a novel therapeutic target for the treatment and prevention of asthma.