Norikazu Maeda

Uric Acid Secretion from Adipose Tissue and Its Increase 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. 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.

Positive Feedback Regulation Between Adiponectin and T-Cadherin Impacts Adiponectin Levels in Tissue and Plasma of Male Mice

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.

Adiponectin Protein Exists in Aortic Endothelial Cells

Aims Inflammation is closely associated with the development of atherosclerosis and metabolic syndrome. Adiponectin, an adipose-derived secretory protein, possesses an anti-atherosclerotic property. The present study was undertaken to elucidate the presence and significance of adiponectin in vasculature. Methods and Results Immunofluorescence staining was performed in aorta of wild-type (WT) mice and demonstrated that adiponectin was co-stained with CD31. Thoracic aorta was cut through and then aortic intima was carefully shaved from aorta. Western blotting showed the existence of adiponectin protein in aortic intima, while there was no adiponectin mRNA expression. Adiponectin knockout (Adipo-KO) and WT mice were administered with a low-dose and short-term lipopolysaccharide (LPS) (1 mg/kg of LPS for 4 hours). The endothelium vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were highly increased in Adipo-KO mice compared to WT mice after LPS administration. Conclusions Adiponectin protein exists in aortic endothelium under steady state and may protect vasculature from the initiation of atherosclerosis.

Visualized macrophage dynamics and significance of S100A8 in obese fat

Significance Infiltration of immune cells into adipose tissue has been observed in obesity, but, until now, these data were based on immunohistological microscopic analysis. Here, to our knowledge for the first time, we applied an intravital multiphoton imaging technique to adipose tissue with lysozyme M-EGFP transgenic (LysMEGFP) mice whose EGFP was expressed in the myelomonocytic lineage. Mobility of LysMEGFP-positive macrophages was activated just 5 d after high-fat and high-sucrose (HF/HS) diet before the development of obesity. Furthermore, a significant increase of S100A8, one of the alarmins, was detected in fat tissue just 5 d after HF/HS diet. S100A8 stimulated chemotactic migration, and neutralization of S100A8 suppressed the HF/HS diet-induced activation of LysMEGFP-positive cells. Time-lapse intravital imaging first identified the very early event exhibiting increased mobility of adipose macrophages. 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 (LysMEGFP) mice whose EGFP was specifically expressed in the myelomonocytic lineage. Mobility of LysMEGFP-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 LysMEGFP-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.

Ultrastructural Localization of Adiponectin protein in Vasculature of Normal and Atherosclerotic mice

Adiponectin, adipose-specific secretory protein, abundantly circulates in bloodstream and its concentration is around 1000-fold higher than that of other cytokines and hormones. Hypoadiponectinemia is a risk factor for atherosclerosis. There is little or no information on ultrastructural localization of adiponectin in the vasculature. Herein we investigated the localization of vascular adiponectin in the aorta using the immunoelectron microscopic technique. In wild-type (WT) mice, adiponectin was mainly detected on the luminal surface membrane of endothelial cells (ECs) and also found intracellularly in the endocytic vesicles of ECs. In the atherosclerotic lesions of apolipoprotein E-knockout (ApoE-KO) mice, adiponectin was detected in ECs, on the cell surface membrane of synthetic smooth muscle cells, and on the surface of monocytes adherent to ECs. Changes in adiponectin localization within the wall of the aorta may provide novel insight into the pathogenesis of atherosclerosis.

Vascular complications and changes in body mass index in Japanese type 2 diabetic patients with abdominal obesity

BackgroundAlthough many Asian type 2 diabetic patients have been considered to be not obese and have low capacity of insulin secretion, the proportion of obese patients with visceral fat accumulation has increased in recent years. We found previously considerable number of Japanese non-obese subjects (body mass index (BMI) <u200925xa0kg/m2) with visceral fat accumulation and multiple cardiovascular risk factors. The aim of the study was to investigate the difference in clinical features of type 2 diabetic patients with and without visceral fat accumulation, focusing on vascular complications and changes in BMI.MethodsWe enrolled 88 Japanese hospitalized type 2 diabetic patients. Abdominal obesity represented waist circumference (WC) of ≥85xa0cm for males and ≥90xa0cm for females (corresponding to visceral fat area of 100xa0cm2). Subjects were divided into two groups; with or without abdominal obesity.ResultsHypertension, dyslipidemia and cardiovascular diseases were significantly more in the patients with abdominal obesity. The prevalence of cardiovascular disease in the non-obese patients (BMI <u200925xa0kg/m2) with abdominal obesity were similar in obese patients (BMI ≥25xa0kg/m2). The mean BMI of the patients with abdominal obesity was <u200925xa0kg/m2 at 20xa0years of age, but reached maximum to more than 30xa0kg/m2 in the course. Furthermore, substantial portion of the type 2 diabetic patients (52% in males and 43% in females) were not obese at 20xa0year-old (BMIu2009<u200925xa0kg/m2), but developed abdominal obesity by the time of admission.ConclusionThese results emphasize the need to control multiple risk factors and prevent atherosclerotic disease in patients with abdominal obesity. The significant weight gain after 20xa0years of age in patients with abdominal obesity stresses the importance of lifestyle modification in younger generation, to prevent potential development of type 2 diabetes and future atherosclerotic cardiovascular disease.

Systemic arteriosclerosis and eating behavior in Japanese type 2 diabetic patients with visceral fat accumulation.

BackgroundVisceral fat accumulation is a major etiological factor in the progression of type 2 diabetes mellitus and atherosclerosis. We described previously visceral fat accumulation and multiple cardiovascular risk factors in a considerable number of Japanese non-obese subjects (BMI <25 kg/m2). Here, we investigated differences in systemic arteriosclerosis, serum adiponectin concentration, and eating behavior in type 2 diabetic patients with and without visceral fat accumulation.MethodsThe study subjects were 75 Japanese type 2 diabetes mellitus (age: 64.8u2009±u200911.5 years, meanu2009±u2009SD). Visceral fat accumulation represented an estimated visceral fat area of 100 cm2 using the bioelectrical impedance analysis method. Subjects were divided into two groups; with (nu2009=u200953) and without (nu2009=u200922) visceral fat accumulation. Systemic arteriosclerosis was scored for four arteries by ultrasonography. Eating behavior was assessed based on The Guideline for Obesity questionnaire issued by the Japan Society for the Study of Obesity.ResultsThe visceral fat accumulation (+) group showed significantly higher systemic vascular scores and significantly lower serum adiponectin levels than the visceral fat accumulation (−) group. With respect to the eating behavior questionnaire items, (+) patients showed higher values for the total score and many of the major sub-scores than (−) patients.ConclusionsType 2 diabetic patients with visceral fat accumulation showed 1) progression of systemic arteriosclerosis, 2) low serum adiponectin levels, and 3) differences in eating behavior, compared to those without visceral fat accumulation. Taken together, the findings highlight the importance of evaluating visceral fat area in type 2 diabetic patients. Furthermore, those with visceral fat accumulation might need to undergo more intensive screening for systemic arteriosclerosis and consider modifying their eating behaviors.

Effect of adiponectin on cardiac β-catenin signaling pathway under angiotensin II infusion.

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.

A Novel Role for Adipose Ephrin-B1 in Inflammatory Response

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.

Adipose hypothermia in obesity and its association with period homolog 1, insulin sensitivity, and inflammation in fat.

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.