A pilot investigation of visceral fat adiposity and gene expression profile in peripheral blood cells.

Evidence suggests that visceral fat accumulation plays a central role in the development of metabolic syndrome. Excess visceral fat causes local chronic low-grade inflammation and dysregulation of adipocytokines, which contribute in the pathogenesis of the metabolic syndrome. These changes may affect the gene expression in peripheral blood cells. This study for the first time examined the association between visceral fat adiposity and gene expression profile in peripheral blood cells. The gene expression profile was analyzed in peripheral blood cells from 28 obese subjects by microarray analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using peripheral blood cells from 57 obese subjects. Obesity was defined as body mass index (BMI) greater than 25 kg/m(2) according to the Japanese criteria, and the estimated visceral fat area (eVFA) was measured by abdominal bioelectrical impedance. Analysis of gene expression profile was carried out with Agilent whole human genome 4 × 44 K oligo-DNA microarray. The expression of several genes related to circadian rhythm, inflammation, and oxidative stress correlated significantly with visceral fat accumulation. Period homolog 1 (PER1) mRNA level in blood cells correlated negatively with visceral fat adiposity. Stepwise multiple regression analysis identified eVFA as a significant determinant of PER1 expression. In conclusion, visceral fat adiposity correlated with the expression of genes related to circadian rhythm and inflammation in peripheral blood cells.

Reduction of visceral fat correlates with the decrease in the number of obesity-related cardiovascular risk factors in Japanese with Abdominal Obesity (VACATION-J Study).

AIM: Visceral fat accumulation is associated with obesity-related cardiovascular risk factor accumulation and atherosclerosis. The present study investigated whether one-year reduction of the visceral fat area (VFA) correlates with a decrease in the number of such factors in Japanese with or without visceral fat accumulation. METHODS: The study subjects comprised 5,347 Japanese, who underwent health check-ups in 2007 and 2008, including measurements of VFA and subcutaneous fat area (SFA) by computed tomography at 9 centers in Japan. Subjects with one or more such factor(s) were categorized into tertiles based on the one-year change in VFA. We investigated the multivariate age, sex, and one-year change in SFA-adjusted odds ratios (ORs) and 95% confidence intervals (CI) for reductions in the number of risk factors in each of the three categories based on the one-year change in VFA, in subjects with one or more such factors (n= 3,648). RESULTS: In the entire group (n=3,648), the OR and 95%CI for reductions in the number of risk factors in the first tertile were 0.804 (0.673-0.962, p=0.0172), compared with the second tertile set at 1.0. Subjects with VFA <100cm(2) showed no reduction in the number of risk factors. In subjects with VFA≥100 cm(2), OR in the first tertile was 0.788 (0.639-0.972, p=0.0257) relative to the second tertile set at 1.0. CONCLUSIONS: In subjects with multiple cardiovascular risk factors, visceral fat reduction correlated with a decrease in the number of such factors in subjects with VFA≥100cm(2), but not in those with VFA<100cm(2).

Coexistence of visceral fat and multiple risk factor accumulations is strongly associated with coronary artery disease in Japanese (the VACATION-J study).

AIM: Multiple risk factor syndrome is a target for the prevention of coronary artery disease (CAD). A cluster of multiple risk factors, such as hypertension, glucose intolerance, and/or dyslipidemia, is encountered in Japanese without and with excess visceral fat. The present study investigated the relationship between multiple risk factor accumulation and CAD in Japanese without and with visceral fat accumulation. METHODS: The study subjects comprised 257 Japanese with suspected CAD (males/females= 153/ 104), who underwent 64-row multislice computed tomography (CT) coronary angiography and visceral fat area (VFA) measurement by CT. Based on the Japanese criteria for visceral fat accumulation, they were divided into those with VFA <100 and ≥10 cm(2). RESULTS: In subjects with VFA <100 cm(2), the age- and sex-adjusted odds ratios (ORs) for 2 and 3 risk factors were 5.33 (95% confidence intervals; 1.04-27.38, p=0.0449) and 4.07 (0.72-23.15, p=0.1138), respectively, compared with VFA <100 cm(2) and 0 risk factor set at 1.0 (p=0.0569 for trend). In contrast, the respective ORs for subjects with VFA ≥100 cm(2) were much higher [6.46 (1.25-33.44, p=0.0261) and 20.42 (3.60-115.73, p=0.0007)] (p<0.0001 for trend). The multivariate adjusted model demonstrated a significant relative excess CAD risk of 1.08 (p=0.0484) and 5.01 (p<0.0001) for the interactions of 2 risk factors and VFA ≥100 cm(2), and 3 risk factors and VFA ≥100 cm(2), whereas multiple risk factor accumulation was not related with the increase of CAD risk in subjects with VFA <100 cm(2). CONCLUSIONS: Coexistence of visceral fat and risk factor accumulations is strongly associated with CAD in Japanese.

High serum S100A8/A9 levels and high cardiovascular complication rate in type 2 diabetics with ultrasonographic low carotid plaque density.

AIMS: S100A8/A9 complex is an inflammation-associated biomarker, which binds toll-like receptor 4 and was associated with the receptor for advanced glycation end-products. S100A8 and S100A9 were accumulated in atherosclerotic lesions. High serum levels of S100A8/A9 are associated with acute coronary syndrome and atherosclerosis in type 2 diabetes mellitus (T2DM). However, association between serum S100A8/A9 levels and vulnerable plaque remains unclear. The present study investigated the relation between serum S100A8/A9 levels and relative plaque density (RPD) of the carotid artery determined by ultrasonography in T2DM. METHODS: The study subjects were 72 consecutive T2DM outpatients (males/females=42/30), who underwent the carotid artery ultrasonography. RPD in the carotid artery was calculated by the formula; RPD=[density of the carotid plaque/density of vessel lumen]. Serum levels of adiponectin and S100A8/A9 were measured. RESULTS: The median RPD was 2.1. Patients with low RPD (≤2.1) were significantly more likely to have metabolic syndrome, nephropathy, coronary artery disease, and peripheral artery disease, and higher levels of S100A8/A9, S100A8/A9-to-adiponectin ratio, and uric acid, compared to those with high RPD (>2.1). CONCLUSIONS: T2DM patients with low RPD had higher prevalence of metabolic syndrome, cardiovascular diseases and higher serum S100A8/A9 levels, compared to those with high RPD.

Metabolic syndrome correlates with polyvascular lesions detected by systemic vascular ultrasonography in Japanese people with type 2 diabetes.

Atherosclerosis is a systemic disease of blood vessels. We investigated clinical characteristics of Japanese type 2 diabetic patients with polyvascular lesions detected by systemic vascular ultrasonography. The results showed that the metabolic syndrome correlated with polyvascular lesions detected by systemic vascular ultrasonography in Japanese type 2 diabetics.

Contribution of glucocorticoid-mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction.

AIMS: Mineralocorticoid receptor (MR) blockade ameliorated insulin resistance with improvements in adipocytokine dysregulation, inflammation, and excess of reactive oxygen species (ROS) in obese adipose tissue and adipocytes, but its mechanism has not been clarified. The 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), producing active glucocorticoids, is highly expressed in adipocytes and glucocorticoids bind to MR with higher affinity than to glucocorticoid receptor (GR). We investigated whether glucocorticoids effect on adipocytokines and ROS through MR in adipocytes. In addition, fat distributions of MR and GR were investigated in human subjects. METHODS AND RESULTS: Corticoid receptors and their target genes were examined in adipose tissue of obese db/db mice. 3T3-L1 adipocytes were treated with glucocorticoids, H(2)O(2), MR antagonist eplerenone (EP), GR antagonist RU486 (RU), MR-siRNA, and/or N-acetylcysteine. Human adipose tissues were obtained from seven patients who underwent abdominal surgery. The mRNA levels of MR and its target gene were higher in db/db mice than in control db/m+mice. In 3T3-L1 adipocytes, glucocorticoids, similar to H(2)O(2), caused the dysregulation of mRNA levels of various genes related to adipocytokines and the increase of intracellular ROS. Such changes were rectified by MR blockade, not by GR antagonist. In human fat, MR mRNA level was increased in parallel with the increase of body mass index (BMI) and its increase was more significant in visceral fat, while there were no apparent correlations of GR mRNA level to BMI or fat distribution. CONCLUSION: Glucocorticoid-MR pathway may contribute to the obesity-related adipocytokine dysregulation and adipose ROS.

High prevalence of gastroesophageal reflux symptoms in type 2 diabetics with hypoadiponectinemia and metabolic syndrome.

BACKGROUND: The prevalence of gastroesophageal reflux disease (GERD) has been increasing worldwide. Abdominal obesity or visceral fat accumulation rather than simple obesity is associated with GERD. Previous reports demonstrated the association between GERD and type 2 diabetes mellitus (T2DM). Signification of visceral fat accumulation and adiponectin in T2DM patients with GERD remains unclear. The present study investigated the relationships between GERD symptoms, visceral fat accumulation and adiponectin in subjects with T2DM. FINDINGS: The study (ADMIT study) subjects were 66 Japanese T2DM outpatients, who answered the questionnaire regarding GERD symptoms in Frequency Scale for the Symptoms of GERD (FSSG), and were measured visceral fat area by bioelectrical impedance analysis. Patients with FSSG scores of more than 8 were considered as positive. The prevalence of FSSG score ≥ 8 and average FSSG score in T2DM subjects with the metabolic syndrome (Mets) were significantly higher compared to those without Mets. The prevalence of FSSG score ≥ 8 and average FSSG score in T2DM subjects with low levels of serum adiponectin were significantly higher compared to those with high levels of serum adiponectin. Moreover, the combination of Mets and hypoadiponectinemia had a multiplicative effect on GERD symptom score (p = 0.047). CONCLUSIONS: Our study showed that the coexistence of MetS and low levels of serum adiponectin was associated with the higher prevalence of FSSG score ≥ 8 and the higher scores of GERD symptom in subjects with T2DM. TRIAL REGISTRATION: UMIN 000002271.

Absolute value of visceral fat area measured on computed tomography scans and obesity-related cardiovascular risk factors in large-scale Japanese general population (the VACATION-J study).

BACKGROUND: The management of cardiovascular risk factors is important for prevention of atherosclerotic cardiovascular diseases (ACVD). Visceral fat accumulation plays an important role in the clustering of cardiovascular risk factors, leading to ACVD. The present study investigated the gender- and age-specific relationship between obesity-related cardiovascular risk factor accumulation and computed tomography (CT)-measured fat distribution in a large-scale Japanese general population. METHODS AND RESULTS: Fat distribution was measured on CT scans in 12,443 subjects (males/females = 10,080/2,363), who underwent medical health check-up at 9 centers in Japan. The investigated obesity-related cardiovascular risk factors were hyperglycemia, dyslipidemia, and elevated blood pressure. Visceral fat area (VFA) for all males and old females showed almost symmetric distribution, while that of young females showed skewed distribution with a marked left shift. Only a small proportion of young females had large visceral fat and cardiovascular risk accumulation. The mean number of risk factors exceeded 1.0 at around 100 cm(2) for VFA in all groups, irrespective of gender, age (cut-off age 55), and BMI (cut-off BMI 25 kg/m(2)). CONCLUSIONS: In this large-scale Japan-wide general population study, an absolute VFA value of about 100 cm(2) equated with obesity-related cardiovascular risk factor accumulation, irrespective of gender, age, and BMI.

Short-term effects of liraglutide on visceral fat adiposity, appetite, and food preference: a pilot study of obese Japanese patients with type 2 diabetes.

BACKGROUND: To examine the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, on visceral fat adiposity, appetite, food preference, and biomarkers of cardiovascular system in Japanese patients with type 2 diabetes. METHODS: The study subjects were 20 inpatients with type 2 diabetes treated with liraglutide [age; 61.2 ± 14.0 years, duration of diabetes; 16.9 ± 6.6 years, glycated hemoglobin (HbA1c); 9.1 ± 1.2%, body mass index (BMI); 28.3 ± 5.2 kg/m(2), mean ± SD]. After improvement in glycemic control by insulin or oral glucose-lowering agents, patients were switched to liraglutide. We assessed the estimated visceral fat area (eVFA) by abdominal bioelectrical impedance analysis, glycemic control by the 75-g oral glucose tolerance test (OGTT) and eating behavior by the Japan Society for the Study of Obesity questionnaire. RESULTS: Treatment with liraglutide (dose range: 0.3 to 0.9 mg/day) for 20.0 ± 6.4 days significantly reduced waist circumference, waist/hip ratio, eVFA. It also significantly improved the scores of eating behavior, food preference and the urge for fat intake and tended to reduce scores for sense of hunger. Liraglutide increased serum C-peptide immunoreactivity and disposition index. CONCLUSIONS: Short-term treatment with liraglutide improved visceral fat adiposity, appetite, food preference and the urge for fat intake in obese Japanese patients with type 2 diabetes.

Qualitative score of systemic arteriosclerosis by vascular ultrasonography as a predictor of coronary artery disease in type 2 diabetes.

OBJECTIVE: Patients with type 2 diabetes mellitus (T2DM) are at risk of polyvascular comorbidities and poor prognosis. Non-invasive techniques for early prediction of coronary artery disease (CAD) are desirable to prevent cardiovascular events in these patients. The aim of the present study was to investigate the association between CAD and systemic arteriosclerosis by qualitative vascular ultrasonography. METHODS: The study subjects were 102 consecutive outpatients with T2DM [males/females = 60/42, age: mean ± SD 67 ± 9 (range, 40-85) years] evaluated by vascular ultrasonography for arteriosclerosis in the abdominal aorta, carotid, renal, and common iliac arteries. The total number of detected arteriosclerotic vascular lesions in the four arteries was determined. CAD was diagnosed by two cardiologists using either stress electrocardiography, myocardial scintigraphy, multi-detector row computed tomography or coronary angiography. RESULTS: Multiple arteriosclerotic vascular lesions (>1) were detected in 64 (63%) patients. The total systemic vascular score was significantly higher in patients with CAD than those without (average score 2.7 versus 1.0, p < 0.0001). None of the CAD patients had a total score of 0. Age- and sex-adjusted multiple logistic regression analysis identified total score of ≥ 2 as the only predictor of CAD (p < 0.001). The sensitivity, specificity, positive and negative predictive values for total systemic vascular score in the prediction of CAD were 98%, 77%, 83%, and 97%, respectively, which were better than those for carotid mean and maximum intima-media thickness. CONCLUSION: Non-invasive qualitative evaluation of systemic arteriosclerosis by the total systemic vascular score is potentially useful for the early prediction of CAD in T2DM patients.

Dynamic changes of adiponectin and S100A8 levels by the selective peroxisome proliferator-activated receptor-gamma agonist rivoglitazone.

OBJECTIVE: Accumulating evidence indicates that the regimen to increase adiponectin will provide a novel therapeutic strategy for metabolic syndrome. Here, we tested the effect of a potent and selective peroxisome proliferator-activated receptor-γ agonist, rivoglitazone (Rivo), a newly synthesized thiazolidinedione derivative, on adiponectin, insulin resistance, and atherosclerosis. METHODS AND RESULTS: ob/ob mice, apolipoprotein E knockout (apoE KO) mice, and apoE and adiponectin double knockout mice were administered pioglitazone, Rivo, or no compound. Remarkable elevation of plasma adiponectin was observed, especially in Rivo-treated ob/ob mice. Rivo ameliorated insulin resistance in ob/ob mice and reduced atherosclerotic areas in apoE KO mice compared with the pioglitazone group but failed to decrease atherosclerotic areas in double knockout mice. Among adipose mRNAs, adipose S100A8, which activates Toll-like receptor 4-dependent signal transduction cascades and locates upstream of inflammation, was markedly increased in ob/ob mice, and its increase was completely reversed by Rivo treatment. In RAW264.7 macrophage cells and 3T3-L1 adipocytes, Rivo significantly reduced S100A8 mRNA levels. CONCLUSIONS: The peroxisome proliferator-activated receptor-γ agonist Rivo remarkably enhanced adiponectin in plasma and decreased adipose S100A8 mRNA levels in obese mice. Rivo treatment apparently ameliorated insulin resistance in ob/ob mice and reduced atherosclerosis in apoE KO mice, partly through adiponectin.

Impairment of decidualization in SRC-deficient mice.

Many signaling events induced by ovarian steroid hormones, cytokines, and growth factors are involved in the process of decidualization of human and rodent endometrium. We have reported previously that tyrosine kinase activation of SRC functionally participates in decidualization of human endometrial stromal cells. To address its essential role in decidualization, we examined, using wild-type and Src knockout mice, whether the process of decidualization was impaired in the absence of SRC. Immunohistochemistry using an antibody specific for the active form of SRC revealed that the active SRC was expressed prominently in the decidualizing stromal cells of the pregnant wild-type mouse. Moreover, the active SRC was upregulated in the uterine horn with artificially stimulated decidual reaction. In comparison with wild-type and Src heterozygous mice, the uterus of Src null mice showed no apparent decidual response following artificial stimulation. Ovarian steroid-induced decidualization in vitro, as determined by morphological changes and expression of decidual/trophoblast prolactin-related protein and prostaglandin-endoperoxide synthase 2 (also known as Cox2), both of which are decidualization markers, did not occur in a timely fashion in endometrial stromal cells isolated from the uteri of SRC-deficient mice compared to those from wild-type and Src heterozygous mice. Our results collectively suggest that SRC is an indispensable signaling component for maximal decidualization in mice.

Effectiveness of high trough levels of cyclosporine for 5 months in a case of steroid-dependent nephrotic syndrome with severe steroid toxicity.

Glucocorticoid treatment for steroid-dependent nephrotic syndrome (NS) is associated with severe adverse effects, such as bone fractures and epidural lipomatosis. Furthermore, a high trough level of cyclosporine (CsA) over an extended period of time is known to induce CsA nephropathy. We present a girl with steroid-dependent NS and steroid-induced vertebral compression fractures and epidural lipomatosis who was treated with a high-dose of prednisolone after experiencing several relapses. A high CsA trough level (between 147 and 225 ng/mL) over a period of only 5 months was effective in improving the vertebral compression fractures, alleviating the epidural lipomatosis by enabling the discontinuation of prednisolone treatment. Thus, high trough levels of CsA over a short period of time may enable prednisolone to be discontinued in cases of steroid-dependent NS without causing any clinical, histological, serum and/or urinary CsA-related adverse effects.

Pyrazolo pyrimidine-type inhibitors of SRC family tyrosine kinases promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro.

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.

Involvement of histone acetylation in ovarian steroid-induced decidualization of human endometrial stromal cells.

Histone acetyltransferases and histone deacetylases (HDACs) determine the acetylation status of histones, regulating gene transcription. Decidualization is the progestin-induced differentiation of estrogen-primed endometrial stromal cells (ESCs), which is crucial for implantation and maintenance of pregnancy. We here show that trichostatin A (TSA), a specific HDAC inhibitor, enhances the up-regulation of decidualization markers such as insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin in a dose-dependent manner that is directed by 17beta-estradiol (E(2)) plus progesterone (P(4)) in cultured ESCs, but not glandular cells, both isolated from human endometrium. Morphological changes resembling decidual transformation were also augmented by co-addition of TSA. Acid urea triton gel analysis and immunoblot using acetylated histone type-specific antibodies demonstrated that treatment with E(2) plus P(4) significantly increased the levels of acetylated H3 and H4 whose increment was augmented by co-treatment with TSA. Chromatin immunoprecipitation assay revealed that treatment with E(2) plus P(4) increased the amount of proximal progesterone-responsive region of IGFBP-1 promoter associated with acetylated H4, which was dramatically enhanced by co-addition of TSA. Taken together, our results suggest that histone acetylation is deeply involved in differentiation of human ESCs and that TSA has a potential as an enhancer of decidualization through promotion of progesterone action.

Expression and subcellular distribution of the active form of c-Src tyrosine kinase in differentiating human endometrial stromal cells.

Decidual growth factors and locally produced cytokines are thought to activate specific phosphorylation signalling pathway(s), thereby eliciting a variety of decidual functions. We have previously reported the activation of c-Src tyrosine kinase during ovarian steroid-induced decidualization of cultured human endometrial stromal cells. As chicken c-Src is known to be activated upon dephosphorylation of tyrosine 527 (Y527, corresponding to Y530 in human), we here employed a monoclonal antibody, clone 28, directed against the active form of human c-Src whose Y530 is dephosphorylated, and investigated whether c-Src became dephosphorylated at Y530 and thereby activated during decidualization. We found that the active form of c-Src was up-regulated and demonstrated increased kinase activity during in-vitro decidualization. Immunohistochemistry revealed that decidual cells in early pregnancy decidua were intensely stained with clone 28 when compared with the stromal cells in the non-pregnant endometrium. Moreover, the active form of c-Src translocated from a perinuclear region to the cytoplasm upon decidualization. Thus, the Y530 dephosphorylation, kinase activation, and subcellular translocation of c-Src may be intracellular signalling events associated with decidualization in vivo as well as in vitro.

Protein tyrosine phosphorylation signaling in the differentiation of human endometrial stromal cells.

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is a critical element in signal transduction pathways regulating cell growth, differentiation, apoptosis, and tumorigenesis. The differentiation of human endometrial stromal cells (decidualization) is crucial for successful embryo implantation and maintenance of pregnancy; however, little is known about the molecular events involving tyrosine phosphorylation, PTKs, and PTPs in the process of decidualization. We have previously reported that the tyrosine kinase activity of c-Src belonging to the Src family kinase is increased together with altered tyrosine phosphorylation of several cellular proteins in the in vitro model of decidualization. Focal adhesion kinase (FAK) and paxillin are known to form a complex with c-Src at the focal contacts and to participate in the integrin-mediated signal transduction as c-Src substrates. Those focal adhesion proteins, however, are not hyperphosphorylated on tyrosine during decidualization. Moreover, the loss of focal adhesions and the disorganization of the actin-based cytoskeleton were observed in decidualized stromal cells, suggesting that the escape from regulation by c-Src may be in part due to the decidualization-induced disruption of the interaction between the focal adhesion proteins and c-Src. These findings collectively indicate that decidual c-Src may activate signaling pathway(s) different from the integrin-mediated signaling cascade involving FAK and paxillin. This review summarizes our recent studies on the tyrosine phosphorylation signaling pathway(s) in decidualization.