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Objective Visceral fat obesity is located upstream of metabolic syndrome and atherosclerotic diseases. Accumulating evidences indicate that several immunocytes including macrophages infiltrate into adipose tissue and induce chronic low-grade inflammation. We recently analyzed the association between visceral fat adiposity and the gene expression profile in peripheral blood cells in human subjects and demonstrated the close relationship of visceral fat adiposity and disturbance of circadian rhythm in peripheral blood cells. In a series of studies, we herein investigated the association of visceral fat adiposity and mRNA levels relating to inflammatory genes in peripheral blood cells. Approach and Results Microarray analysis was performed in peripheral blood cells from 28 obese subjects. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted by using blood cells from 57 obese subjects. Obesity was defined as body mass index (BMI) greater than 25 kg/m2 according to the Japanese criteria. Gene expression profile analysis was carried out with Agilent whole human genome 4 × 44 K oligo-DNA microarray. Gene ontology (GO) analysis showed that 14 genes were significantly associated with visceral fat adiposity among 239 genes relating to inflammation. Among 14 genes, RT-PCR demonstrated that S100A8, S100A9, and S100A12 positively correlated with visceral fat adiposity in 57 subjects. Stepwise multiple regression analysis showed that S100A8 and S100A12 mRNA levels were closely associated with HOMA-IR and S100A9 mRNA was significantly related to adiponectin and CRP. Conclusions Peripheral blood mRNA levels of S100 family were closely associated with insulin resistance and inflammation.

Adiponectin, an adipocyte-derived protein abundant in the circulation, is thought to be protective against atherosclerosis. However, it is not fully understood how the association of adiponectin with vascular cells and its antiatherogenic effect are connected. In this study, T-cadherin was essential for accumulation of adiponectin in the neointima and atherosclerotic plaque lesions, and the adiponectin-T-cadherin association protected against vascular injury. In the apolipoprotein E-knockout (ApoE-KO) mice, adiponectin and T-cadherin colocalized on endothelial cells and synthetic smooth muscle cells in the aortic intima. Notably, aortic adiponectin protein disappeared in T-cadherin/ApoE double-knockout (Tcad/ApoE-DKO) mice with significant elevation of blood adiponectin concentration. Furthermore, in Tcad/ApoE-DKO mice, carotid artery ligation resulted in a significant increase of neointimal thickness compared with ApoE-KO mice. Finally, on a high-cholesterol diet, Tcad/ApoE-DKO mice increased atherosclerotic plaque formation, despite a 5-fold increase in plasma adiponectin level compared with that in ApoE-KO mice. In vitro, knockdown of T-cadherin from human aortic smooth muscle cells (HASMCs) with synthetic phenotype significantly reduced adiponectin accumulation on HASMCs and negated the inhibitory effect of adiponectin on proinflammatory change. Collective evidence showed that adiponectin accumulates in the vasculature via T-cadherin, and the adiponectin-T-cadherin association plays a protective role against neointimal and atherosclerotic plaque formations.-Fujishima, Y., Maeda, N., Matsuda, K., Masuda, S., Mori, T., Fukuda, S., Sekimoto, R., Yamaoka, M., Obata, Y., Kita, S., Nishizawa, H., Funahashi, T., Ranscht, B., Shimomura, I. Adiponectin association with T-cadherin protects against neointima proliferation and atherosclerosis.

Chronic low-grade inflammation of adipose tissue plays a crucial role in the pathophysiology of obesity. Immunohistological microscopic analysis in obese fat tissue has demonstrated the infiltration of several immune cells such as macrophages, but dynamics of immune cells have not been fully elucidated and clarified. Here, by using intravital multiphoton imaging technique, to our knowledge for the first time, we analyzed and visualized the inflammatory processes in adipose tissue under high-fat and high-sucrose (HF/HS) diet with lysozyme M-EGFP transgenic (LysM(EGFP)) mice whose EGFP was specifically expressed in the myelomonocytic lineage. Mobility of LysM(EGFP)-positive macrophages was shown to be activated just 5 d after HF/HS diet, when the distinct hypertrophy of adipocytes and the accumulation of macrophages still have not become prominent. Significant increase of S100A8 was detected in mature adipocyte fraction just 5 d after HF/HS diet. Recombinant S100A8 protein stimulated chemotactic migration in vitro and in vivo, as well as induced proinflammatory molecules, both macrophages and adipocytes, such as TNF-α and chemokine (C-C motif) ligand 2. Finally, an antibody against S100A8 efficiently suppressed the HF/HS diet-induced initial inflammatory change, i.e., increased mobilization of adipose LysM(EGFP)-positive macrophages, and ameliorated HF/HS diet-induced insulin resistance. In conclusion, time-lapse intravital multiphoton imaging of adipose tissues identified the very early event exhibiting increased mobility of macrophages, which may be triggered by increased expression of adipose S100A8 and results in progression of chronic inflammation in situ.

Adiponectin (Adipo), a multimeric adipocyte-secreted protein abundant in the circulation, is implicated in cardiovascular protective functions. Recent work documented that Adipo locally associates with responsive tissues through interactions with T-cadherin (Tcad), an atypical, glycosylphosphatidylinositol (GPI)-anchored cadherin cell surface glycoprotein. Mice deficient for Tcad lack tissue-associated Adipo, accumulate Adipo in the circulation, and mimic the Adipo knockout (KO) cardiovascular phenotype. In reverse, Tcad protein is visibly reduced from cardiac tissue in Adipo-KO mice, suggesting interdependent regulation of the 2 proteins. Here, we evaluate the effect of Adipo on Tcad protein expression. Adipo and Tcad proteins were colocalized in aorta, heart, and skeletal muscle. Adipo positively regulated levels of Tcad protein in vivo and in endothelial cell (EC) cultures. In Tcad-KO mice, binding of endogenous and exogenously administered Adipo to cardiovascular tissues was dramatically reduced. Consistently, knockdown of Tcad in cultured murine vascular ECs significantly diminished Adipo binding. In search for a possible mechanism, we found that enzymatic cleavage of Tcad with phosphatidylinositol-specific phospholipase C increases plasma Adipo while decreasing tissue-bound levels. Similarly, pretreatment of cultured ECs with serum containing Adipo attenuated phosphatidylinositol-specific phospholipase C-mediated Tcad cleavage. In vivo administration of adenovirus producing Adipo suppressed plasma levels of GPI phospholipase D, the endogenous cleavage enzyme for GPI-anchored proteins. In conclusion, our data show that both circulating and tissue-bound Adipo levels are dependent on Tcad and, in reverse, regulate tissue Tcad levels through a positive feedback loop that operates by suppressing phospholipase-mediated Tcad release from the cell surface.

Obesity is an epidemic matter increasing risk for cardiovascular diseases and metabolic disorders such as type 2 diabetes. We recently examined the association between visceral fat adiposity and gene expression profile of peripheral blood cells in human subjects. In a series of studies, Opa (Neisseria gonorrhoeae opacity-associated)-interacting protein 5 (OIP5) was nominated as a molecule of unknown function in adipocytes and thus the present study was performed to investigate the role of OIP5 in obesity. Adenovirus overexpressing Oip5 (Ad-Oip5) was generated and infected to 3T3-L1 cells stably expressing Coxsackie-Adenovirus Receptor (CAR-3T3-L1) and to mouse subcutaneous fat. For a knockdown experiment, siRNA against Oip5 (Oip5-siRNA) was introduced into 3T3-L1 cells. Proliferation of adipose cells was measured by BrdU uptake, EdU-staining, and cell count. Significant increase of Oip5 mRNA level was observed in obese white adipose tissues and such increase was detected in both mature adipocytes fraction and stromal vascular cell fraction. Ad-Oip5-infected CAR-3T3-L1 preadipocytes and adipocytes proliferated rapidly, while a significant reduction of proliferation was observed in Oip5-siRNA-introduced 3T3-L1 preadipocytes. Fat weight and number of adipocytes were significantly increased in Ad-Oip5-administered fat tissues. Oip5 promotes proliferation of pre- and mature-adipocytes and contributes adipose hyperplasia. Increase of Oip5 may associate with development of obesity.

Obesity is associated with heart failure and cardiac hypertrophy. Adiponectin has been shown to play a protective role for cardiovascular diseases. The β-catenin signaling pathway is deeply involved in cardiac hypertrophy. However, the effect of adiponectin on β-catenin signaling has not been investigated in cardiac hypertrophy. Present study aimed to clarify the involvement of adiponectin and β-catenin signaling pathway in the mouse model of angiotensin II (AngII)-induced cardiac hypertrophy. In hearts of Wild type (WT) mice, AngII dose-dependently augmented cytosolic β-catenin protein level. WT and adiponectin knockout (Adipo-KO) mice were administered with AngII at 2.4 mg/kg/day for 14 days and were also injected with adenovirus expressing the adiponectin (Ad-Adipo) or the β-galactosidase (Ad-βgal). Cardiac mRNA levels relating to hypertrophy and β-catenin signaling were increased in Adipo-KO mice and these changes were reversed by Ad-Adipo. Phosphorylation of Akt was increased in Adipo-KO mice and such increases were reversed by Ad-Adipo. Furthermore, the phosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser(9) and cytosolic β-catenin level were increased in Adipo-KO mice and they were significantly reduced by Ad-Adipo treatment. Phosphorylation of mammalian target of rapamycin (mTOR) was reduced by Ad-Adipo-mediated adiponectin supplementation in WT and Adipo-KO mice. The current study suggests that adiponectin attenuates AngII-induced cardiac hypertrophic signals partly through Akt/GSK3β/β-catenin and Akt/mTOR pathways.

Objective Adipose tissue inflammation plays an important role in the pathogenesis of obesity-associated complications, such as atherosclerosis. Adiponectin secreted from adipocytes has various beneficial effects including anti-inflammatory effect. Obesity often presents with hypoadiponectinemia. However, the mechanism and adiponectin movement in obesity remain uncharacterized. Here we investigated tissue distribution of adiponectin protein in lean and obese mice. Methods Adiponectin protein levels were evaluated by enzyme-linked immunosorbent assay and western blotting. Adipose tissues were fractionated into mature adipocyte fraction (MAF) and stromal vascular fraction (SVF). Results Adiponectin protein was detected not only in MAF but also in SVF, which lacks adiponectin mRNA expression, of adipose tissue remarkably. SVF adiponectin protein level was higher in obese mice than in lean mice. The mechanism of adiponectin accumulation was investigated in adiponectin-deficient (APN-KO) mice after injection of plasma from wild-type mice. These mice showed accumulation of exogenous adiponectin, which derived from wild type mice, in adipose tissues, and the adiponectin was more observed in SVF of diet induced obese APN-KO mice than lean APN-KO mice. Among the adiponectin binding proteins, T-cadherin mRNA and protein levels in SVF of obese mice were remarkably higher than in lean mice. Oxidative stress levels were also significantly higher in SVF of obese mice than lean mice. Mechanistically, H2O2 up-regulated T-cadherin mRNA level in murine macrophages. Conclusions The results demonstrated adiponectin targets to adipose SVF of obese mice. These findings should shed a new light on the pathology of adipose tissue inflammation and hypoadiponectinemia of obesity.

Obesity is often accompanied by hyperuricemia. However, purine metabolism in various tissues, especially regarding uric acid production, has not been fully elucidated. Here we report, using mouse models, that adipose tissue could produce and secrete uric acid through xanthine oxidoreductase (XOR) and that the production was enhanced in obesity. Plasma uric acid was elevated in obese mice and attenuated by administration of the XOR inhibitor febuxostat. Adipose tissue was one of major organs that had abundant expression and activities of XOR, and adipose tissues in obese mice had higher XOR activities than those in control mice. 3T3-L1 and mouse primary mature adipocytes produced and secreted uric acid into culture medium. The secretion was inhibited by febuxostat in a dose-dependent manner or by gene knockdown of XOR. Surgical ischemia in adipose tissue increased local uric acid production and secretion via XOR, with a subsequent increase in circulating uric acid levels. Uric acid secretion from whole adipose tissue was increased in obese mice, and uric acid secretion from 3T3-L1 adipocytes was increased under hypoxia. Our results suggest that purine catabolism in adipose tissue could be enhanced in obesity. Background: Purine metabolism in adipose tissue is largely unknown. Results: Adipose tissue has abundant xanthine oxidoreductase activity. Uric acid is secreted from adipose tissues and cells, and the secretion is augmented in obese mice. Conclusion: Adipose tissue can secrete uric acid in mice. Significance: Dysfunction of obese adipose tissue could be related to overproduction of uric acid.

S100A8/A9 complex, calprotectin, which serves as an endogenous ligand for immune pathways, is associated with atherosclerosis. These proteins are reported to have several functions such as activating NADPH oxidase, binding toll-like receptor 4 and associated with the receptor for advanced glycation end-products. We recently reported S100A8 mRNA was highly expressed in mouse white adipose tissues and differentiated 3T3-L1 adipocytes. However, regulation of S100A9 expression in murine adipose tissue remains to be elucidated. The results of our studies in male Japanese, obese and control mice and cultured cells showed: (1) serum levels of S100A8/A9 complex, calprotectin, correlated with visceral fat area, body mass index, subcutaneous fat area, and leukocyte count in 500 Japanese men, and (2) higher mRNA expression levels of S100A8 in mature adipocyte fraction and S100A9 in stromal vascular cell fraction of obese mice, compared with those of lean mice. Overexpression of S100A8 and S100A9 in obese adipose tissue may be involved, at least partly, in not only high circulating levels of S100A8/A9 complex in abdominal obesity but also adipose and systemic tissue inflammation.

Visceral fat adiposity plays an important role in the development of metabolic syndrome. We reported previously the impact of human visceral fat adiposity on gene expression profile of peripheral blood cells. Genes related to circadian rhythm were highly associated with visceral fat area and period homolog 1 (PER1) showed the most significant negative correlation with visceral fat area. However, regulation of adipose Per1 remains poorly understood. The present study was designed to understand the regulation of Per1 in adipose tissues. Adipose Per1 mRNA levels of ob/ob mice were markedly low at 25 and 35 weeks of age. The levels of other core clock genes of white adipose tissues were also low in ob/ob mice at 25 and 35 weeks of age. Per1 mRNA was mainly expressed in the mature adipocyte fraction (MAF) and it was significantly low in MAF of ob/ob mice. To examine the possible mechanisms, 3T3-L1 adipocytes were treated with H2O2, tumor necrosis factor-α (TNF-α), S100A8, and lipopolysaccharide (LPS). However, no significant changes in Per1 mRNA level were observed by these agents. Exposure of cultured 3T3-L1 adipocytes to low temperature (33°C) decreased Per1 and catalase, and increased monocyte chemoattractant protein-1 (Mcp-1) mRNA levels. Hypothermia also worsened insulin-mediated Akt phosphorylation in 3T3-L1 adipocytes. Finally, telemetric analysis showed low temperature of adipose tissues in ob/ob mice. In obesity, adipose hypothermia seems to accelerate adipocyte dysfunction.

Aims Ephrin-B1 (EfnB1) was selected among genes of unknown function in adipocytes or adipose tissue and subjected to thorough analysis to understand its role in the development of obesity. Methods and Results EfnB1 mRNA and protein levels were significantly decreased in adipose tissues of obese mice and such reduction was mainly observed in mature adipocytes. Exposure of 3T3-L1 adipocytes to tumor necrosis factor-α (TNF-α) and their culture with RAW264.7 cells reduced EFNB1 levels. Knockdown of adipose EFNB1 increased monocyte chemoattractant protein-1 (Mcp-1) mRNA level and augmented the TNF-α-mediated THP-1 monocyte adhesion to adipocytes. Adenovirus-mediated adipose EFNB1-overexpression significantly reduced the increase in Mcp-1 mRNA level induced by coculture of 3T3-L1 adipocytes with RAW264.7 cells. Monocyte adherent assay showed that adipose EfnB1-overexpression significantly decreased the increase of monocyte adhesion by coculture with RAW264.7 cells. TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was reduced by EFNB1-overexpression. Conclusions EFNB1 contributes to the suppression of adipose inflammatory response. In obesity, reduction of adipose EFNB1 may accelerate the vicious cycle involved in adipose tissue inflammation.