Publications by authors named "Keisuke Matsuda"

21 Publications

  • Page 1 of 1

Epithelial folding determines the final shape of beetle horns.

Curr Opin Genet Dev 2021 Apr 10;69:122-128. Epub 2021 Apr 10.

Department of Entomology, Washington State University, Pullman, WA, 99163 USA. Electronic address:

The elaborate ornaments and weapons of sexual selection, such as the vast array of horns observed in scarab beetles, are some of the most striking outcomes of evolution. How these novel traits have arisen, develop, and respond to condition is governed by a complex suite of interactions that require coordination between the environment, whole-animal signals, cell-cell signals, and within-cell signals. Endocrine factors, developmental patterning genes, and sex-specific gene expression have been shown to regulate beetle horn size, shape, and location, yet no overarching mechanism of horn shape has been described. Recent advances in microscopy and computational analyses combined with a functional genetic approach have revealed that patterning genes combined with intricate epithelial folding and movement are responsible for the final shape of a beetle head horn.
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http://dx.doi.org/10.1016/j.gde.2021.03.003DOI Listing
April 2021

Development of a valve type semi-closed extracorporeal circulation system.

J Artif Organs 2021 Feb 3. Epub 2021 Feb 3.

School of Clinical Engineering, Faculty of Health and Medical Care, Saitama Medical University, 1397-1, Yamane, Hidaka, Saitama, 350-1241, Japan.

In Japan, perfusionists who work on other clinical tasks are involved in cardiopulmonary bypass. Moreover, the number of cases they can perform is limited. In view of this situation, valve type semi-closed extracorporeal circulation (VACC) was developed as a system that enables extracorporeal circulation (ECC) regardless of perfusionists' experience. The VACC circuit is based on a conventional open-type ECC circuit. A safety valve is installed at the outlet of the reservoir. It is closed by lowering the reservoir pressure below the venous circuit pressure (Pv), thereby providing a closed-type ECC in which the reservoir is separated from the venous circuit (V-circuit). A closed-type ECC needs means to cope with negative pressure generated in the V-circuit and to remove air mixed in the V-circuit. Water experiments to verify the safety of the VACC were conducted. In experiments simulating low venous return, when the Pv dropped, the safety valve opened so that the V-circuit was connected to the reservoir, and the excessive negative pressure was relieved. In the VACC circuit, a bubble trap is installed in the V-circuit, and the air is degassed to the reservoir by a roller pump (D-pump). A water experiment was conducted to verify the principle of the constant degassing method using the D-pump. It verified that the blood storage volume could be maintained constant even if the D-pump is continuously driven. The VACC system provides handling of air mixed in the V-circuit and safety in the case of low venous return.
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http://dx.doi.org/10.1007/s10047-021-01249-5DOI Listing
February 2021

Computational analyses decipher the primordial folding coding the 3D structure of the beetle horn.

Sci Rep 2021 Jan 13;11(1):1017. Epub 2021 Jan 13.

Pattern Formation Laboratory, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan.

The beetle horn primordium is a complex and compactly folded epithelial sheet located beneath the larval cuticle. Only by unfolding the primordium can the complete 3D shape of the horn appear, suggesting that the morphology of beetle horns is encoded in the primordial folding pattern. To decipher the folding pattern, we developed a method to manipulate the primordial local folding on a computer and clarified the contribution of the folding of each primordium region to transformation. We found that the three major morphological changes (branching of distal tips, proximodistal elongation, and angular change) were caused by the folding of different regions, and that the folding mechanism also differs according to the region. The computational methods we used are applicable to the morphological study of other exoskeletal animals.
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http://dx.doi.org/10.1038/s41598-020-79757-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806817PMC
January 2021

Genetical control of 2D pattern and depth of the primordial furrow that prefigures 3D shape of the rhinoceros beetle horn.

Sci Rep 2020 10 29;10(1):18687. Epub 2020 Oct 29.

Ecological Genetics Laboratory, Department of Genomics and Evolutionary Biology, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.

The head horn of the Asian rhinoceros beetle develops as an extensively folded primordium before unfurling into its final 3D shape at the pupal molt. The information of the final 3D structure of the beetle horn is prefigured in the folding pattern of the developing primordium. However, the developmental mechanism underlying epithelial folding of the primordium is unknown. In this study, we addressed this gap in our understanding of the developmental patterning of the 3D horn shape of beetles by focusing on the formation of furrows at the surface of the primordium that become the bifurcated 3D shape of the horn. By gene knockdown analysis via RNAi, we found that knockdown of the gene Notch disturbed overall horn primordial furrow depth without affecting the 2D furrow pattern. In contrast, knockdown of CyclinE altered 2D horn primordial furrow pattern without affecting furrow depth. Our results show how the depth and 2D pattern of primordial surface furrows are regulated at least partially independently during beetle horn development, and how both can alter the final 3D shape of the horn.
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http://dx.doi.org/10.1038/s41598-020-75709-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596553PMC
October 2020

The GPR40 Full Agonist SCO-267 Improves Liver Parameters in a Mouse Model of Nonalcoholic Fatty Liver Disease without Affecting Glucose or Body Weight.

J Pharmacol Exp Ther 2020 10 27;375(1):21-27. Epub 2020 Jul 27.

SCOHIA PHARMA, Inc., Kanagawa, Japan

Full agonism of G-protein-coupled receptor 40 (GPR40)/free fatty acid 1 receptor improves glycemic control in diabetic rodents. However, the effects of GPR40 full agonism on liver parameters are largely unknown. In the present study, we examined the effects of a GPR40 full agonist, SCO-267, on liver parameters in a nondiabetic mouse model with early-stage nonalcoholic fatty liver disease (NAFLD). SCO-267 was orally administered to mice, which were fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD), a mouse model for NAFLD. An oral dose of SCO-267 increased levels of circulating glucagon and glucagon-like peptide-1 in CDAHFD-fed mice. In a chronic-dose experiment, effects of SCO-267 were compared with those of a dipeptidyl peptidase-4 inhibitor (alogliptin) and a sodium glucose cotransporter 2 inhibitor (dapagliflozin). SCO-267 decreased liver triglyceride content, weight, collagen content, and plasma alanine aminotransferase (ALT) levels without affecting food intake or glucose levels in CDAHFD-fed mice. Furthermore, SCO-267 decreased levels of liver thiobarbituric acid reactive substances (TBARS), markers of oxidative stress. Alogliptin and dapagliflozin had no effect on liver weight or levels of triglyceride, collagen, plasma ALT, and liver TBARS. SCO-267 elevated mRNA levels of molecules with roles in mitochondrial function and -oxidation while inhibiting those with roles in lipogenesis, inflammation, reactive oxygen species generation, and fibrosis in the liver, all of which were less evident with alogliptin and dapagliflozin. This is the first study to show that the GPR40 full agonist SCO-267 improves liver parameters without affecting glucose or body weight in a mouse model of NAFLD. SIGNIFICANCE STATEMENT: Full agonism of GPR40/free fatty acid 1 receptor signaling stimulates islet and gut hormone secretions. The present study is the first to show the treatment effects of GPR40 full agonism on liver parameters in a mouse model for nonalcoholic fatty liver disease.
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http://dx.doi.org/10.1124/jpet.120.000046DOI Listing
October 2020

Structure and development of the complex helmet of treehoppers (Insecta: Hemiptera: Membracidae).

Zoological Lett 2020 24;6. Epub 2020 Feb 24.

4Faculty of Environmental Earth Sciences, Hokkaido University, Sapporo, Hokkaido Japan.

Some insects possess complex three-dimensional (3D) structures that develop under the old cuticle prior to the last imaginal molt. Adult treehoppers (Insecta: Hemiptera: Auchenorrhyncha: Membracidae) have one such complex 3D structure, known as a helmet, on their dorsal side. The adult helmet likely forms inside the nymphal pronotum during the final instar nymphal stage. Previous morphological studies have reported that the adult helmet is a large, bi-layered, plywood-like structure, whereas the nymphal pronotum is a monolayer, sheath-like structure. The adult helmet is much larger than nymphal helmet. Thus, the emergence of the adult helmet involves two structural transitions: a transition from a monolayer, sheath-like pronotum to a bi-layer, plywood-like helmet, and a transition in size from small to large. However, when, how, and in what order these transitions occur within the nymphal cuticle is largely unknown. To determine how adult helmet development occurs under the nymphal cuticle, in the present study we describe the morphology of the final adult helmet and investigate developmental trajectories of the helmet during the final instar nymphal stage. We used micro-CT, scanning electron microscope and paraffin sections for morphological observations, and used as a model species. We found that the structural transition (from monolayer, sheath-like structure to bi-layer, roof-like structure) occurs through the formation of a "miniature" of the adult helmet during the middle stage of development and that subsequently, extensive folding and furrows form, which account for the increase in size. We suggest that the making of a "miniature" is the key developmental step for the formation of various 3D structures of treehopper helmets.
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http://dx.doi.org/10.1186/s40851-020-00155-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041272PMC
February 2020

Healthcare-associated infections in intensive care units in Taiwan, South Korea, and Japan: recent trends based on national surveillance reports.

Antimicrob Resist Infect Control 2018 7;7:129. Epub 2018 Nov 7.

1College of Medicine, National Taiwan University, Taipei, Taiwan.

Background: Sustainable systematic interventions are important for infection prevention and control (IPC). Data from surveillance of healthcare-associated infections (HAI) provides feedback for implementation of IPC programs. To address the paucity of such data in Asia, we searched for national HAI surveillance and IPC programs in this region.

Methods: Data were analysed from open access national surveillance reports of three Asian countries: Taiwan, South Korea and Japan from 2008 to 2015. National IPC programs were identified.

Results: There were differences among the countries in surveillance protocols, hospital coverage rates, and national IPC policies and programs. Nevertheless, there was a 53.0% reduction in overall HAI over the 8-year period. This consisted of a decrease from 9.34 to 5.03 infections per 1000 patient-days in Taiwan, from 7.56 to 2.76 in Korea, and from 4.41 to 2.74 in Japan (Poisson regression, all  < 0.05). Across the three countries, and were the major pathogens for urinary tract infection. and were common bloodstream pathogens. For pneumonia, , , , and were the predominant pathogens, with considerable country differences. There was a 64.6% decrease in the number of isolates of methicillin-resistant , 38.4% decrease in carbapenem-resistant and 49.2% decrease in carbapenem-resistant (CRAB) in Taiwan (all  < 0.05), and similarly in Korea with the exception of CRAB (30.5 and 50.4% reduction, respectively, both  < 0.05.

Conclusion: We found a significant decrease in HAI across the three countries in association with sequential multifaceted interventions such as hand hygiene, care bundles, and antimicrobial stewardships. Further regional collaboration could be forged to develop joint strategies to prevent HAI.
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http://dx.doi.org/10.1186/s13756-018-0422-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223041PMC
September 2019

Anisotropy of cell division and epithelial sheet bending via apical constriction shape the complex folding pattern of beetle horn primordia.

Mech Dev 2018 08 18;152:32-37. Epub 2018 Jun 18.

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan; Faculty of Environmental Earth Sciences, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan. Electronic address:

Insects can dramatically change their outer morphology at molting. To prepare for this drastic transformation, insects generate new external organs as folded primordia under the old cuticle. At molting, these folded primordia are physically extended to form their final outer shape in a very short time. Beetle horns are a typical example. Horn primordia are derived from a flat head epithelial sheet, on which deep furrows are densely added to construct the complex folded structure. Because the 3D structure of the pupa horn is coded in the complex furrow pattern, it is indispensable to know how and where the furrows are set. Here, we studied the mechanism of furrow formation using dachsous (ds) gene knocked down beetles that have shorter and fatter adult horns. The global shape of the beetle horn primordia is mushroom like, with dense local furrows across its surface. Knockdown of ds by RNAi changed the global shape of the primordia, causing the stalk region become apparently thicker. The direction of cell division is biased in wildtype horns to make the stalk shape thin and tall. However, in ds knocked down beetles, it became random, resulting in the short and thick stalk shape. On the other hand, a fine and dense local furrow was not significantly affected by the ds knockdown. In developing wildtype horn primordia, we observed that, before the local furrow is formed, the apical constriction signal emerged at the position of the future furrow, suggesting the pre-pattern for the fine furrow pattern. According to the results, we propose that development of complex horn primordia can be roughly divided to two distinct processes, 1) development of global primordia shape by anisotropic cell division, and 2) local furrow formation via actin-myosin dependent apical constriction of specific cells.
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http://dx.doi.org/10.1016/j.mod.2018.06.003DOI Listing
August 2018

Discovery of benzimidazole derivatives as orally active renin inhibitors: Optimization of 3,5-disubstituted piperidine to improve pharmacokinetic profile.

Bioorg Med Chem 2018 07 27;26(12):3261-3286. Epub 2018 Apr 27.

Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan. Electronic address:

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio.
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http://dx.doi.org/10.1016/j.bmc.2018.04.051DOI Listing
July 2018

Complex furrows in a 2D epithelial sheet code the 3D structure of a beetle horn.

Sci Rep 2017 10 24;7(1):13939. Epub 2017 Oct 24.

Graduate School of Frontier Bioscience, Osaka University, Suita, Osaka, 565-0871, Japan.

The external organs of holometabolous insects are generated through two consecutive processes: the development of imaginal primordia and their subsequent transformation into the adult structures. During the latter process, many different phenomena at the cellular level (e.g. cell shape changes, cell migration, folding and unfolding of epithelial sheets) contribute to the drastic changes observed in size and shape. Because of this complexity, the logic behind the formation of the 3D structure of adult external organs remains largely unknown. In this report, we investigated the metamorphosis of the horn in the Japanese rhinoceros beetle Trypoxylus dichotomus. The horn primordia is essentially a 2D epithelial cell sheet with dense furrows. We experimentally unfolded these furrows using three different methods and found that the furrow pattern solely determines the 3D horn structure, indicating that horn formation in beetles occurs by two distinct processes: formation of the furrows and subsequently unfolding them. We postulate that this developmental simplicity offers an inherent advantage to understanding the principles that guide 3D morphogenesis in insects.
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http://dx.doi.org/10.1038/s41598-017-14170-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655322PMC
October 2017

Adiponectin association with T-cadherin protects against neointima proliferation and atherosclerosis.

FASEB J 2017 04 6;31(4):1571-1583. Epub 2017 Jan 6.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan.

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. , 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 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.
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http://dx.doi.org/10.1096/fj.201601064RDOI Listing
April 2017

Visualized macrophage dynamics and significance of S100A8 in obese fat.

Proc Natl Acad Sci U S A 2015 Apr 6;112(16):E2058-66. Epub 2015 Apr 6.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan;

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.
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http://dx.doi.org/10.1073/pnas.1409480112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413348PMC
April 2015

Positive feedback regulation between adiponectin and T-cadherin impacts adiponectin levels in tissue and plasma of male mice.

Endocrinology 2015 Mar 16;156(3):934-46. Epub 2014 Dec 16.

Departments of Metabolic Medicine (K.M., Y.F., N.M., T.M., A.H., R.S., Y.T., S.M., M.Y., K.I., H.N., S.K., T.F., I.S.) and Metabolism and Atherosclerosis (A.H., S.K., T.F.), Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; and 3Sanford-Burnham Medical Research Institute (B.R.), National Institutes of Health-Designated Cancer Center, Development, Aging, and Regeneration Program, La Jolla, California 92037.

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.
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http://dx.doi.org/10.1210/en.2014-1618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330303PMC
March 2015

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

PLoS One 2014 14;9(11):e112813. Epub 2014 Nov 14.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112813PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4232416PMC
December 2015

Ultrastructural localization of adiponectin protein in vasculature of normal and atherosclerotic mice.

Sci Rep 2014 May 8;4:4895. Epub 2014 May 8.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan 565-0871.

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.
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http://dx.doi.org/10.1038/srep04895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013939PMC
May 2014

Possible involvement of Opa-interacting protein 5 in adipose proliferation and obesity.

PLoS One 2014 6;9(2):e87661. Epub 2014 Feb 6.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087661PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3916335PMC
January 2015

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

Biochem Biophys Res Commun 2014 Feb 22;444(2):224-9. Epub 2014 Jan 22.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, 2-2-B5 Yamada-oka, Suita, Osaka 565-0871, Japan.

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.
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http://dx.doi.org/10.1016/j.bbrc.2014.01.043DOI Listing
February 2014

A novel role for adipose ephrin-B1 in inflammatory response.

PLoS One 2013 1;8(10):e76199. Epub 2013 Oct 1.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076199PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3787942PMC
July 2014

Gene expression levels of S100 protein family in blood cells are associated with insulin resistance and inflammation (Peripheral blood S100 mRNAs and metabolic syndrome).

Biochem Biophys Res Commun 2013 Apr 15;433(4):450-5. Epub 2013 Mar 15.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, 2-2-B5 Yamada-oka, Suita, Osaka 565-0871, Japan.

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 4×44K 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.
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http://dx.doi.org/10.1016/j.bbrc.2013.02.096DOI Listing
April 2013

Monooxygenation by a thermophilic cytochrome P450 via direct electron donation from NADH.

Metallomics 2011 Apr 28;3(4):389-95. Epub 2011 Feb 28.

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.

The catalysis of cytochrome P450s requires two-electron donation for the activation of an oxygen molecule. Here, we report the enzymatic catalysis of cytochrome P450, CYP119A2 (P450st), from a thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7, with NAD(P)H as an electron donor and no redox partners and the crystallographic analysis of P450st at high resolution. P450st can catalyse styrene epoxidation with either NADH or NADPH as an electron donor. The P450st reaction with NADH exhibited a sequential mechanism. X-ray crystallography at a resolution of 1.94 Å revealed a sufficiently large heme pocket for NAD(P)H binding and a novel contiguous channel from the active site to bulk solvent in the distal heme pocket. The narrow channel may transfer protons or water to the heme pocket even when a bulky compound, such as NAD(P)H, binds in the pocket. In addition, the F/G loop region (Leu151-Glu156), located around the substrate channel, was deleted in the mutant and constructed to improve the accessibility of NAD(P)H to the heme pocket. Kinetic properties of the Δ151-156 mutant were compared with those of the wild-type P450st. The K(m) value of the mutant was about 2 times lower than that of the wild-type. The results indicated that NAD(P)H could provide the electrons for P450st within the heme pocket.
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http://dx.doi.org/10.1039/c0mt00079eDOI Listing
April 2011

Aberrant CpG methylation of the imprinting control region KvDMR1 detected in assisted reproductive technology-produced calves and pathogenesis of large offspring syndrome.

Anim Reprod Sci 2010 Dec 7;122(3-4):303-12. Epub 2010 Oct 7.

Ishikawa Prefectural Livestock Research Center, Hodatsushimizu, Ishikawa 929-1325, Japan.

Although somatic cell nuclear transfer (NT) and in vitro fertilization (IVF) have the potential to produce genetically superior livestock, considerable numbers of abnormally large animals, including sheep and cattle affected by "large offspring syndrome" (LOS), have been produced by these assisted reproductive technologies (ART). Interestingly, these phenotypes are reminiscent of Beckwith-Wiedemann syndrome (BWS) in humans, which is an imprinting disorder characterized by pre- and/or postnatal overgrowth. The imprinting control region KvDMR1, which regulates the coordinated expression of growth control genes such as Cdkn1c, is known to be aberrantly hypomethylated in BWS. Therefore, we hypothesized that aberrant imprinting in this region could contribute to LOS. In this study, we analyzed the DNA methylation status of the Kcnq1ot1/Cdkn1c and Igf2/H19 domains on bovine chromosome 29 and examined the coordinated expression of imprinted genes surrounding them in seven calves derived by NT (which showed signs of developmental abnormality), two calves conceived by IVF (both developmentally abnormal), and three conventional calves that died of unrelated causes. Abnormal hypomethylation status at an imprinting control region of Kcnq1ot1/Cdkn1c domain was observed in two of seven NT-derived calves and one of two IVF-derived calves in almost all organs. Moreover, increased expression of Kcnq1ot1 and diminished expression of Cdkn1c were observed by RT-PCR analysis. This study is the first to describe the abnormal hypomethylation of the KvDMR1 domain and subsequent changes in the gene expression of Kcnq1ot1 and Cdkn1c in a subset of calves produced by ART. Our findings provide strong evidence for a role of altered imprinting control in the development of LOS in bovines.
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http://dx.doi.org/10.1016/j.anireprosci.2010.09.008DOI Listing
December 2010