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1.  Systemic arteriosclerosis and eating behavior in Japanese type 2 diabetic patients with visceral fat accumulation 
Visceral 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.
The study subjects were 75 Japanese type 2 diabetes mellitus (age: 64.8 ± 11.5 years, mean ± SD). 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 (n = 53) and without (n = 22) 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.
The 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.
Type 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.
PMCID: PMC4301666  PMID: 25592402
Type 2 diabetes; Visceral fat accumulation; Adiponectin; Systemic arteriosclerosis; Vascular ultrasonography; Eating behavior
2.  Combination of Root Surface Modification with BMP-2 and Collagen Hydrogel Scaffold Implantation for Periodontal Healing in Beagle Dogs  
Objective : Biomodification of the root surface plays a major role in periodontal wound healing. Root surface modification with bone morphogenetic protein (BMP) stimulates bone and cementum-like tissue formation; however, severe ankylosis is simultaneously observed. Bio-safe collagen hydrogel scaffolds may therefore be useful for supplying periodontal ligament cells and preventing ankylosis. We examined the effects of BMP modification in conjunction with collagen hydrogel scaffold implantation on periodontal wound healing in dogs. Material and Methods: The collagen hydrogel scaffold was composed of type I collagen sponge and collagen hydrogel. One-wall infrabony defects (5 mm in depth, 3 mm in width) were surgically created in six beagle dogs. In the BMP/Col group, BMP-2 was applied to the root surface (loading dose; 1 µg/µl), and the defects were filled with collagen hydrogel scaffold. In the BMP or Col group, BMP-2 coating or scaffold implantation was performed. Histometric parameters were evaluated at 4 weeks after surgery. Results: Single use of BMP stimulated formation of alveolar bone and ankylosis. In contrast, the BMP/Col group frequently enhanced reconstruction of periodontal attachment including cementum-like tissue, periodontal ligament and alveolar bone. The amount of new periodontal ligament in the BMP/Col group was significantly greater when compared to all other groups. In addition, ankylosis was rarely observed in the BMP/Col group. Conclusion: The combination method using root surface modification with BMP and collagen hydrogel scaffold implantation facilitated the reestablishment of periodontal attachment. BMP-related ankylosis was suppressed by implantation of collagen hydrogel.
PMCID: PMC4319209
Ankylosis; bone morphogenetic protein-2; collagen hydrogel; dog; one-wall infrabony defects; periodontal attachment; periodontal wound healing; regenerative scaffold.
3.  Adipose Hypothermia in Obesity and Its Association with Period Homolog 1, Insulin Sensitivity, and Inflammation in Fat 
PLoS ONE  2014;9(11):e112813.
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.
PMCID: PMC4232416  PMID: 25397888
4.  Long-term impact of liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, on body weight and glycemic control in Japanese type 2 diabetes: an observational study 
Liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, has been shown to possess pleiotropic effects including body weight reduction. However, long-term effect of liraglutide on body weight and glycemic control has not been elucidated in Japanese type 2 diabetes (T2D) subjects. Present study investigates whether liraglutide treatment maintains the body weight-decreasing and glucose-lowering effects for 2 years in Japanese T2D subjects.
The enrolled subjects were 86 T2D patients [age; 59.8 ± 12.8 years, duration of diabetes; 15.8 ± 9.5 years, glycated hemoglobin (HbA1c); 8.5 ± 1.5%, body mass index (BMI); 27.3 ± 5.4 kg/m2 (15.8 - 46.5 kg/m2), mean ± SD]. Among 86 subjects, liraglutide was introduced in 25 inpatients and 61 outpatients, and 46 subjects were followed for 2 years. Clinical parameters were measured at baseline and 3, 6, 9, 12, and 24 months after liraglutide introduction. The increase in liraglutide dosage and the additional usage of glucose-lowering agents depended on each attending physician.
At 1 year after liraglutide introduction, 69 patients (80.2%) decreased body weight and 58 patients (67.4%) improved glycemic control. Body mass index (BMI) was changed 27.3 ± 5.4 kg/m2 to 25.9 ± 4.8 kg/m2 and percent reduction of body weight was significant and maintained over 4% at 2 years after liraglutide introduction. HbA1c was significantly decreased from 8.5 ± 1.5% to 7.7 ± 1.2% for 2 years. Liraglutide treatment tended to ameliorate lipid profile and hepatic enzymes. Stepwise regression analysis demonstrated that baseline BMI and previous insulin dose were positively associated with body weight reduction and baseline HbA1c was positively associated with reduction of HbA1c at 2 years after liraglutide introduction.
Long-term liraglutide treatment effectively maintained the reduction of body weight and the fair glycemic control, and also improved lipid profile and liver enzymes in Japanese T2D subjects.
PMCID: PMC4167135  PMID: 25237400
Liraglutide; Glucagon-like peptide-1; Obesity; Diabetes; Metabolic syndrome; Eating behavior
5.  Comparative study of bioactivity of collagen scaffolds coated with graphene oxide and reduced graphene oxide 
Graphene oxide (GO) is a single layer carbon sheet with a thickness of less than 1 nm. GO has good dispersibility due to surface modifications with numerous functional groups. Reduced graphene oxide (RGO) is produced via the reduction of GO, and has lower dispersibility. We examined the bioactivity of GO and RGO films, and collagen scaffolds coated with GO and RGO.
GO and RGO films were fabricated on a culture dish. Some GO films were chemically reduced using either ascorbic acid or sodium hydrosulfite solution, resulting in preparation of RGO films. The biological properties of each film were evaluated by scanning electron microscopy (SEM), atomic force microscopy, calcium adsorption tests, and MC3T3-E1 cell seeding. Subsequently, GO- and RGO-coated collagen scaffolds were prepared and characterized by SEM and compression tests. Each scaffold was implanted into subcutaneous tissue on the backs of rats. Measurements of DNA content and cell ingrowth areas of implanted scaffolds were performed 10 days post-surgery.
The results show that GO and RGO possess different biological properties. Calcium adsorption and alkaline phosphatase activity were strongly enhanced by RGO, suggesting that RGO is effective for osteogenic differentiation. SEM showed that RGO-modified collagen scaffolds have rough, irregular surfaces. The compressive strengths of GO- and RGO-coated scaffolds were approximately 1.7-fold and 2.7-fold greater, respectively, when compared with the non-coated scaffold. Tissue ingrowth rate was 39% in RGO-coated scaffolds, as compared to 20% in the GO-coated scaffold and 16% in the non-coated scaffold.
In summary, these results suggest that GO and RGO coatings provide different biological properties to collagen scaffolds, and that RGO-coated scaffolds are more bioactive than GO-coated scaffolds.
PMCID: PMC4103921  PMID: 25050063
GO; RGO; tissue engineering; regenerative scaffold; cell ingrowth; biomaterials
6.  Possible Involvement of Opa-Interacting Protein 5 in Adipose Proliferation and Obesity 
PLoS ONE  2014;9(2):e87661.
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.
PMCID: PMC3916335  PMID: 24516558
7.  A Novel Role for Adipose Ephrin-B1 in Inflammatory Response 
PLoS ONE  2013;8(10):e76199.
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.
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.
PMCID: PMC3787942  PMID: 24098442
8.  Low serum eicosapentaenoic acid / arachidonic acid ratio in male subjects with visceral obesity 
Visceral fat accumulation is caused by over-nutrition and physical inactivity. Excess accumulation of visceral fat associates with atherosclerosis. Polyunsaturated fatty acids have an important role in human nutrition, but imbalance of dietary long-chain polyunsaturated fatty acids, especially low eicosapentaenoic acid (EPA) / arachidonic acid (AA) ratio, is associated with increased risk of cardiovascular disease. The present study investigated the correlation between EPA, docosahexaenoic acid (DHA), AA parameters and clinical features in male subjects.
The study subjects were 134 Japanese with diabetes, hypertension and/or dyslipidemia who underwent measurement of visceral fat area (eVFA) by the bioelectrical impedance method and serum levels of EPA, DHA and AA. EPA/AA ratio correlated positively with age, and negatively with waist circumference and eVFA. Stepwise regression analysis demonstrated that age and eVFA correlated significantly and independently with serum EPA/AA ratio. Serum EPA/AA ratio, but not serum DHA/AA and (EPA+DHA)/AA ratios, was significantly lower in subjects with eVFA ≥100 cm2, compared to those with eVFA <100 cm2 (p=0.049). Subjects with eVFA ≥100 cm2 were significantly more likely to have the metabolic syndrome and history of cardiovascular diseases, compared to those with eVFA <100 cm2 (p<0.001, p=0.028, respectively).
Imbalance of dietary long-chain polyunsaturated fatty acids (low serum EPA/AA ratio) correlated with visceral fat accumulation in male subjects.
Clinical trial registration number
PMCID: PMC3606329  PMID: 23497138
Arachidonic acid; Eicosapentaenoic acid; Docosahexaenoic acid; Visceral fat; Metabolic syndrome; Obesity
9.  Mediator Subunits MED1 and MED24 Cooperatively Contribute to Pubertal Mammary Gland Development and Growth of Breast Carcinoma Cells 
Molecular and Cellular Biology  2012;32(8):1483-1495.
The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G1/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.
PMCID: PMC3318591  PMID: 22331469
10.  Efficacy of liraglutide, a glucagon-like peptide-1 (GLP-1) analogue, on body weight, eating behavior, and glycemic control, in Japanese obese type 2 diabetes 
We recently reported that short-term treatment with liraglutide (20.0 ± 6.4 days) reduced body weight and improved some scales of eating behavior in Japanese type 2 diabetes inpatients. However, it remained uncertain whether such liraglutide-induced improvement is maintained after discharge from the hospital. The aim of the present study was to determine the long-term effects of liraglutide on body weight, glycemic control, and eating behavior in Japanese obese type 2 diabetics.
Patients with obesity (body mass index (BMI) >25 kg/m2) and type 2 diabetes were hospitalized at Osaka University Hospital between November 2010 and December 2011. BMI and glycated hemoglobin (HbA1c) were examined on admission, at discharge and at 1, 3, and 6 months after discharge. For the liraglutide group (BMI; 31.3 ± 5.3 kg/m2, n = 29), patients were introduced to liraglutide after correction of hyperglycemic by insulin or oral glucose-lowering drugs and maintained on liraglutide after discharge. Eating behavior was assessed in patients treated with liraglutide using The Guideline For Obesity questionnaire issued by the Japan Society for the Study of Obesity, at admission, discharge, 3 and 6 months after discharge. For the insulin group (BMI; 29.1 ± 3.0 kg/m2, n = 28), each patient was treated with insulin during hospitalization and glycemic control maintained by insulin after discharge.
Liraglutide induced significant and persistent weight loss from admission up to 6 months after discharge, while no change in body weight after discharge was noted in the insulin group. Liraglutide produced significant improvements in all major scores of eating behavior questionnaire items and such effect was maintained at 6 months after discharge. Weight loss correlated significantly with the decrease in scores for recognition of weight and constitution, sense of hunger, and eating style.
Liraglutide produced meaningful long-term weight loss and significantly improved eating behavior in obese Japanese patients with type 2 diabetes.
PMCID: PMC3459720  PMID: 22973968
Liraglutide; Glucagon-like peptide-1 (GLP-1); Obesity; Eating behavior; Diabetes; Incretin
11.  Liraglutide is effective in type 2 diabetic patients with sustained endogenous insulin‐secreting capacity 
Aims/Introduction:  Recently, glucagon‐like peptide‐1 (GLP‐1) receptor agonists of liraglutide have become available in Japan. It has not yet been clarified what clinical parameters could discriminate liraglutide‐effective patients from liraglutide‐ineffective patients.
Materials and Methods:  We reviewed 23 consecutive patients with type 2 diabetes admitted to Osaka University Hospital for glycemic control. All of the patients were treated with diet plus insulin (or plus oral antidiabetic drugs) to improve fasting plasma glucose (FPG) and postprandial glucose below 150 and 200 mg/dL, respectively. After insulin secretion and insulin resistance were evaluated, insulin was replaced by liraglutide. The efficacy of liraglutide was determined according to whether glycemic control was maintained at the target levels.
Results:  Liraglutide was effective in 13 of 23 patients. There were significant differences in the parameters of insulin secretion, including fasting C‐peptide (F‐CPR), C‐peptide index (CPI), insulinogenic index (I.I.) and urine C‐peptide (U‐CPR), between liraglutide‐effective and ‐ineffective patients. The duration of diabetes was significantly shorter in liraglutide‐effective patients than in liraglutide‐ineffective patients. In receiver operating characteristic analyses, the cut‐off value for predicting the efficacy of liraglutide was 0.14 for I.I., 1.1 for CPI, 1.5 ng/mL for F‐CPR, 33.3 μg/day for U‐CPR and 19.5 years for duration of type 2 diabetes.
Conclusions:  Insulin secretion evaluated by F‐CPR, CPI, I.I., U‐CPR and the duration of type 2 diabetes were useful parameters for predicting the efficacy of liraglutide in patients with type 2 diabetes. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2011.00168.x, 2011)
PMCID: PMC4014952  PMID: 24843579
Glucagon‐like peptide‐1; Incretin; Type 2 diabetes
12.  High prevalence of gastroesophageal reflux symptoms in type 2 diabetics with hypoadiponectinemia and metabolic syndrome 
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.
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).
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.
PMCID: PMC3293023  PMID: 22277344
gastroesophageal reflux symptom; metabolic syndrome; visceral fat; adiponectin
13.  Short-term effects of liraglutide on visceral fat adiposity, appetite, and food preference: a pilot study of obese Japanese patients with type 2 diabetes 
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.
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/m2, 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.
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.
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.
PMCID: PMC3260096  PMID: 22132774
liraglutide; glucagon-like peptide-1; obesity; eating behavior
14.  The Transcriptional Mediator Subunit MED1/TRAP220 in Stromal Cells Is Involved in Hematopoietic Stem/Progenitor Cell Support through Osteopontin Expression▿  
Molecular and Cellular Biology  2010;30(20):4818-4827.
MED1/TRAP220, a subunit of the transcriptional Mediator/TRAP complex, is crucial for various biological events through its interaction with distinct activators, such as nuclear receptors and GATA family activators. In hematopoiesis, MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow (BM) cells cocultured with MED1 knockout (Med1−/−) mouse embryonic fibroblasts (MEFs) was significantly suppressed compared to the control. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) was attenuated for BM cells cocultured with Med1−/− MEFs. The vitamin D receptor (VDR)- and Runx2-mediated expression of OPN, as well as Mediator recruitment to the Opn promoter, was specifically attenuated in the Med1−/− MEFs. Addition of OPN to these MEFs restored the growth of cocultured BM cells and the number of LTC-ICs, both of which were attenuated by the addition of the anti-OPN antibody to Med1+/+ MEFs and to BM stromal cells. Consequently, MED1 in niche appears to play an important role in supporting HSPCs by upregulating VDR- and Runx2-mediated transcription on the Opn promoter.
PMCID: PMC2950551  PMID: 20713445

Results 1-14 (14)