研究者詳細 - 佐藤　達也

論文 - 佐藤　達也

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Effects of contraction frequency during high-intensity training on fatigue resistance and aerobic adaptations in mouse skeletal muscle.

Azuma Naito, Tatsuya Sato, Iori Kimura, Nao Tokuda, Nao Yamauchi, Hiroyori Fusagawa, Takashi Yamada

Journal of applied physiology (Bethesda, Md. : 1985) 2024年12月 [査読有り] [国際誌]

担当区分：筆頭著者

In high-intensity and sprint interval training, the frequency of contractions is typically higher compared to moderate-intensity continuous training, but it remains unclear whether this contributes to the effective increase in fatigue resistance mechanisms. Here, we investigated the role of contraction frequency in high-intensity training on endurance adaptations of mouse skeletal muscle. Male C57BL/6 mice were divided into groups based on high (0.25 s contraction every 0.5 s) and low (0.25 s contraction every 4.5 s) contraction frequencies, with either 360 contractions per session (Hi360 and Lo360) or 30 contractions per session (Hi30 and Lo30). The plantar flexor muscles were stimulated using in vivo supramaximal electrical stimulation, where all muscle fibers were maximally activated, every other day for 5 weeks. In both the Hi360 and Lo360 groups, where force production declined to less than 40% of the initial value during the training session, muscle endurance, as well as mitochondrial content and respiratory capacity, were increased to a similar extent. In contrast, the rate of torque decline during the training session was more pronounced in the Hi30 group compared to the Lo30 group. In response, the Hi30 group, but not the Lo30 group, exhibited increased fatigue resistance and mitochondrial respiration, which was accompanied by increased PGC-1α expression and an activation of AMPK/Ulk1 pathway. These data suggest that the frequency of contractions is a critical factor in determining the efficient enhancement of mitochondrial respiratory capacity and muscle endurance through high-intensity training, presumably due to promotion of mitochondrial quality control.

A silent interplay between elevated intraocular pressure, glaucoma, and hypertension.

Tatsuya Sato, Araya Umetsu, Marenao Tanaka, Hiroshi Ohguro, Masato Furuhashi

Hypertension research : official journal of the Japanese Society of Hypertension 2024年12月 [査読有り] [国際誌]

担当区分：筆頭著者

High Intraocular Pressure Is Independently Associated With New-Onset Systemic Hypertension Over a 10-Year Period

Araya Umetsu, Marenao Tanaka, Tatsuya Sato, Yukinori Akiyama, Keisuke Endo, Kazuma Mori, Hirofumi Ohnishi, Megumi Watanabe, Hiroshi Ohguro, Nagisa Hanawa, Masato Furuhashi

Circulation Journal ( Japanese Circulation Society ) 2024年07月 [査読有り]

担当区分：筆頭著者

FABP4 Is an Indispensable Factor for Regulating Cellular Metabolic Functions of the Human Retinal Choroid.

Hiroshi Ohguro, Megumi Watanabe, Tatsuya Sato, Nami Nishikiori, Araya Umetsu, Megumi Higashide, Toshifumi Ogawa, Masato Furuhashi

Bioengineering (Basel, Switzerland) 11 ( 6 ) 2024年06月 [査読有り] [国際誌]

担当区分：責任著者

The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells. In HOCF cells, both mitochondrial and glycolytic functions were significantly decreased to trace levels by BMS309403 in a dose-dependent manner. In the RNA sequencing analysis, 67 substantially up-regulated and 94 significantly down-regulated differentially expressed genes (DEGs) were identified in HOCF cells treated with BMS309403 and those not treated with BMS309403. The results of Gene Ontology enrichment analysis and ingenuity pathway analysis (IPA) revealed that the DEGs were most likely involved in G-alpha (i) signaling, cAMP-response element-binding protein (CREB) signaling in neurons, the S100 family signaling pathway, visual phototransduction and adrenergic receptor signaling. Furthermore, upstream analysis using IPA suggested that NKX2-1 (thyroid transcription factor1), HOXA10 (homeobox A10), GATA2 (gata2 protein), and CCAAT enhancer-binding protein A (CEBPA) were upstream regulators and that NKX homeobox-1 (NKX2-1), SFRP1 (Secreted frizzled-related protein 1) and TREM2 (triggering receptor expressed on myeloid cells 2) were causal network master regulators. The findings in this study suggest that intraocularly present FABP4 originates from the ocular choroid and may be a critical regulator for the cellular homeostasis of non-adipocyte HOCF cells.

The Specific ROCK2 Inhibitor KD025 Alleviates Glycolysis through Modulating STAT3-, CSTA- and S1PR3-Linked Signaling in Human Trabecular Meshwork Cells.

Megumi Watanabe, Tatsuya Sato, Araya Umetsu, Toshifumi Ogawa, Nami Nishikiori, Megumi Suzuki, Masato Furuhashi, Hiroshi Ohguro

Biomedicines 12 ( 6 ) 2024年05月 [査読有り] [国際誌]

担当区分：筆頭著者

To investigate the biological significance of Rho-associated coiled-coil-containing protein kinase (ROCK) 2 in the human trabecular meshwork (HTM), changes in both metabolic phenotype and gene expression patterns against a specific ROCK2 inhibitor KD025 were assessed in planar-cultured HTM cells. A seahorse real-time ATP rate assay revealed that administration of KD025 significantly suppressed glycolytic ATP production rate and increased mitochondrial ATP production rate in HTM cells. RNA sequencing analysis revealed that 380 down-regulated and 602 up-regulated differentially expressed genes (DEGs) were identified in HTM cells treated with KD025 compared with those that were untreated. Gene ontology analysis revealed that DEGs were more frequently related to the plasma membrane, extracellular components and integral cellular components among cellular components, and related to signaling receptor binding and activity and protein heterodimerization activity among molecular functions. Ingenuity Pathway Analysis (IPA) revealed that the detected DEGs were associated with basic cellular biological and physiological properties, including cellular movement, development, growth, proliferation, signaling and interaction, all of which are associated with cellular metabolism. Furthermore, the upstream regulator analysis and causal network analysis estimated IL-6, STAT3, CSTA and S1PR3 as possible regulators. Current findings herein indicate that ROCK2 mediates the IL-6/STAT3-, CSTA- and S1PR3-linked signaling related to basic biological activities such as glycolysis in HTM cells.

Tirzepatide ameliorates eating behaviors regardless of prior exposure to glucagon-like peptide receptor agonists in Japanese patients with type 2 diabetes mellitus

Toru Suzuki, Tatsuya Sato, Marenao Tanaka, Keisuke Endo, Kei Nakata, Toshifumi Ogawa, Itaru Hosaka, Yukinori Akiyama, Araya Umetsu, Masato Furuhashi

Journal of Diabetes and its Complications ( Elsevier BV ) 108779 - 108779 2024年05月 [査読有り]

担当区分：筆頭著者

3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions

Keisuke Endo, Tatsuya Sato, Araya Umetsu, Megumi Watanabe, Fumihito Hikage, Yosuke Ida, Hiroshi Ohguro, Masato Furuhashi

Heliyon ( Elsevier BV ) 9 ( 10 ) e20713 - e20713 2023年10月 [査読有り]

担当区分：筆頭著者

Skeletal muscle endurance declines with impaired mitochondrial respiration and inadequate supply of acetyl-CoA during muscle fatigue in 5/6 nephrectomized rats.

Hiroyori Fusagawa, Tatsuya Sato, Takashi Yamada, Yuki Ashida, Iori Kimura, Azuma Naito, Nao Tokuda, Nao Yamauchi, Nobutoshi Ichise, Yoshinori Terashima, Izaya Ogon, Atsushi Teramoto, Toshihiko Yamashita, Noritsugu Tohse

Journal of applied physiology (Bethesda, Md. : 1985) 2023年08月 [査読有り] [国際誌]

担当区分：筆頭著者

Chronic kidney disease (CKD)-related cachexia increases the risks of reduced physical activity and mortality. However, the physiological phenotype of skeletal muscle fatigue and changes in intramuscular metabolites during muscle fatigue in CKD-related cachexia remain unclear. In the present study, we performed detailed muscle physiological evaluation, analysis of mitochondrial function, and comprehensive analysis of metabolic changes before and after muscle fatigue in a 5/6 nephrectomized rat model of CKD. Wistar rats were randomized to a sham-operation (Sham) group that served as a control group or a 5/6 nephrectomy (Nx) group. Eight weeks after the operation, in situ torque and force measurements in plantar flexor muscles in Nx rats using electrical stimulation revealed a significant decrease in muscle endurance during subacute phase related to mitochondrial function. Muscle mass was reduced without changes in the proportions of fiber type-specific myosin heavy chain isoforms in Nx rats. Pyruvate-malate-driven state 3 respiration in isolated mitochondria were impaired in Nx rats. Protein expression levels of mitochondrial respiratory chain complexes III and V were decreased in Nx rats. Metabolome analysis revealed that the increased supply of acetyl CoA in response to fatigue was blunted in Nx rats. These findings suggest that CKD deteriorates skeletal muscle endurance in association with mitochondrial dysfunction and inadequate supply of acetyl-CoA during muscle fatigue.

Prostanoid FP and EP2 Receptor Agonists Induce Epithelial and Subepithelial Fibrogenetic Changes in Human Conjunctival Fibroblasts in Different Manners.

Yuri Tsugeno, Tatsuya Sato, Megumi Watanabe, Masato Furuhashi, Hiroshi Ohguro

Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics 39 ( 6 ) 404 - 414 2023年07月 [査読有り] [国際誌]

担当区分：筆頭著者

Purpose: To examine the effects of prostanoid FP and EP2 receptor agonists, PGF2α and Omidenepag (OMD), respectively, on the transforming growth factor beta (TGF-β2) induced conjunctival fibrogenesis. Methods: Two-dimension (2D) and three-dimension (3D) cultures of these fibroblasts were subjected to following analyses: (1) planar proliferation evaluated by transendothelial electron resistance (TEER) measurements, (2) real-time metabolic analyses, (3) subepithelial proliferation evaluated by 3D spheroid' size and stiffness measurements, and (4) the mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: TGF-β2 induced increase in the planar proliferation was significantly decreased or enhanced by PGF2α or OMD, respectively. The proportion of oxygen consumption required to drive ATP synthesis compared with that driving proton leakage was increased by PGF2α and OMD independently with TGF-β2. In contrast, maximal mitochondrial respiration was decreased by PGF2α and OMD, and the OMD-induced effect was further enhanced by the presence of TGF-β2. In addition, the TGF-β2 dependent increase in the glycolytic capacity was cancelled by PGF2α and/or OMD. Alternatively, subepithelial proliferation, as evidenced by the stiffness of the 3D spheroids, was substantially increased by both PGF2α and OMD, and these were differently modulated by TGF-β2. The expression of several related factors as above fluctuated among the conditions for both 2D and 3D and TGF-β2 untreated or treated cultures. Conclusion: The present findings indicate that the prostanoid FP or the EP2 receptor agonist may solely and differently induce the planar and subepithelial proliferation of HconF cells and these were also modulated by TGF-β2.

Potential favorable action of sodium-glucose cotransporter-2 inhibitors on sudden cardiac death: a brief overview.

Tatsuya Sato, Hidemichi Kouzu, Toshiyuki Yano, Ichiro Sakuma, Masato Furuhashi, Noritsugu Tohse

Frontiers in cardiovascular medicine 10 1159953 - 1159953 2023年05月 [査読有り] [国際誌]

担当区分：筆頭著者 , 責任著者

The primary pharmacological action of sodium-glucose co-transporter 2 (SGLT2) inhibitors is to inhibit the reabsorption of glucose and sodium ions from the proximal tubules of the kidney and to promote urinary glucose excretion. Notably, several clinical trials have recently demonstrated potent protective effects of SGLT2 inhibitors in patients with heart failure (HF) or chronic kidney disease (CKD), regardless of the presence or absence of diabetes. However, the impact of SGLT2 inhibitors on sudden cardiac death (SCD) or fatal ventricular arrhythmias (VAs), the pathophysiology of which is partly similar to that of HF and CKD, remains undetermined. The cardiorenal protective effects of SGLT2 inhibitors have been reported to include hemodynamic improvement, reverse remodeling of the failing heart, amelioration of sympathetic hyperactivity, correction of anemia and impaired iron metabolism, antioxidative effects, correction of serum electrolyte abnormalities, and antifibrotic effects, which may lead to prevent SCD and/or VAs. Recently, as possible direct cardiac effects of SGLT2 inhibitors, not only inhibition of Na+/H+ exchanger (NHE) activity, but also suppression of late Na+ current have been focused on. In addition to the indirect cardioprotective mechanisms of SGLT2 inhibitors, suppression of aberrantly increased late Na+ current may contribute to preventing SCD and/or VAs via restoration of the prolonged repolarization phase in the failing heart. This review summarizes the results of previous clinical trials of SGLT2 inhibitors for prevention of SCD, their impact on the indices of electrocardiogram, and the possible molecular mechanisms of their anti-arrhythmic effects.

Optimized protocol for quantification of mitochondrial non-heme and heme iron content in mouse tissues and cultured cells

Tatsuya Sato, Hsiang-Chun Chang, Konrad T. Sawicki, Hossein Ardehali

STAR Protocols 2023年03月 [査読有り]

担当区分：筆頭著者

Case report: Transient lactate elevation by intravenous insulin infusion therapy for diabetic ketoacidosis in a patient with mitochondrial DNA 3243 A > G mutation: A glycolysis rebooting syndrome?

Wataru Ohwada, Hidemichi Kouzu, Tatsuya Sato, Kahomi Sazawa, Azumi Matsui, Nobutaka Nagano, Masayuki Koyama, Noriko Ogasawara, Akifumi Takada, Toshiyuki Yano, Masato Furuhashi

Frontiers in cardiovascular medicine 10 1144925 - 1144925 2023年 [査読有り] [国際誌]

担当区分：責任著者

Mitochondrial disease, most cases of which are caused by mitochondrial DNA (mtDNA) mutation, is present with multiple phenotypes including diabetes mellitus, sensorineural hearing loss, cardiomyopathy, muscle weakness, renal dysfunction, and encephalopathy, depending on the degree of heteroplasmy. While mitochondria play an important role in intracellular glucose and lactate metabolism in insulin-sensitive tissues such as muscles, appropriate strategies for glycemic control have not yet been established in a patient with mitochondrial disease, which is often complicated by myopathy. Here, we describe the history of a 40-year-old man with mtDNA 3243A > G who had sensorineural hearing loss, cardiomyopathy, muscle wasting, and diabetes mellitus with stage 3 chronic kidney disease. He developed mild diabetic ketoacidosis (DKA) in the process of treatment for poor glycemic control with severe latent hypoglycemia. According to the standard therapy for DKA, he was treated with continuous intravenous insulin infusion therapy, which unexpectedly resulted in an abrupt and transient elevation in blood lactate levels without exacerbation of heart failure and kidney function. Since blood lactate levels are determined by the balance between lactate production and consumption, an abrupt and transient lactate elevation following intravenous insulin injection therapy may reflect not only enhanced glycolysis in insulin-sensitive tissues with mitochondrial dysfunction but also decreased lactate consumption in the sarcopenic skeletal muscle and failing heart. Intravenous insulin infusion therapy in patients with mitochondrial disease may unmask derangements of intracellular glucose metabolism in response to insulin signaling.

Addition of ROCK Inhibitors Alleviates Prostaglandin-Induced Inhibition of Adipogenesis in 3T3L-1 Spheroids.

Yosuke Ida, Tatsuya Sato, Araya Umetsu, Megumi Watanabe, Masato Furuhashi, Fumihito Hikage, Hiroshi Ohguro

Bioengineering (Basel, Switzerland) 9 ( 11 ) 2022年11月 [査読有り] [国際誌]

担当区分：筆頭著者

To elucidate the additive effects of the ROCK inhibitors (ROCK-i), ripasudil (Rip) and Y27632 on bimatoprost acid (BIM-A), a prostaglandin analog (PG), on adipose tissue, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells, the most well characterized cells in the field of lipid research, were used. The cells were subjected to a variety of analyses including lipid staining, real-time cellular metabolic analysis, the mRNA expressions of genes related to adipogenesis and extracellular matrices (ECMs) as well as the sizes and physical properties of the 3D spheroids by a micro-squeezer. BIM-A induced strong inhibitory effects on most of the adipogenesis-related changes in the 2D and 3D cultured 3T3-L1 cells, including (1) the enlargement and softening of the 3D spheroids, (2) a dramatic enhancement in lipid staining and the expression of adipogenesis-related genes, and (3) a decrease in mitochondrial and glycolytic metabolic function. By adding ROCK-i to the BIM-A, most of these BIM-A-induced effects were cancelled. The collective findings reported herein suggest that ROCK-i eliminated the PG-induced suppression of adipogenesis in the 3T3-L1 cells, accompanied by the formation of enlarged 3D spheroids. Such effects of adding ROCK-i to a PG in preadipocytes on cellular properties appear to be associated with the suppression of PG-induced adverse effects, and provide additional insight into our understanding of lipid-related research.

FGF-2 enhances fibrogenetic changes in TGF-β2 treated human conjunctival fibroblasts.

Yuri Tsugeno, Masato Furuhashi, Tatsuya Sato, Megumi Watanabe, Araya Umetsu, Soma Suzuki, Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro

Scientific reports 12 ( 1 ) 16006 - 16006 2022年09月 [査読有り] [国際誌]

担当区分：筆頭著者

The objective of the current study was to examine the effects of fibroblast growth factor-2 (FGF-2) on conjunctival fibrogenesis that was induced by the presence of transforming growth factor-β2 (TGF-β2). Two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) were used for this purpose. The 2D and 3D cultured HconF were characterized by transendothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D), real-time metabolic analyses (2D), size and stiffness measurements (3D), and the mRNA expression of extracellular matrix molecules, their modulators, Tissue inhibitor of metalloproteinases and matrix metalloproteinases and ER-stress related genes (2D and 3D). FGF-2 significantly increased planar proliferation, as evidenced by TEER values and FITC dextran permeability, and shifted glucose metabolism to the energetic phenotype of 2D HconF cells, and the stiffness of the 3D spheroids, and these effects were further enhanced in the presence of TGF-β2. Analyses of the expression of possible candidate molecules involved in cell architecture and stress indicated that some additive effects caused by both factors were also recognized in some of these molecules. The findings reported herein indicate that the FGF-2, either along or additively with TGF- β2 increased the fibrogenetic changes on the plane as well as in the spatial space of HconF cells.

Case report: Successful combination therapy with double-filtration plasmapheresis and rituximab under the condition of the use of a sensor-augmented pump for type B insulin resistance syndrome.

Arata Osanami, Masatoshi Kanda, Tatsuya Sato, Chikako Akazawa, Shuhei Baba, Hiroaki Komatsu, Kazuyuki Murase, Tomohisa Yamashita, Toshiyuki Yano

Frontiers in endocrinology 13 997296 - 997296 2022年09月 [査読有り] [国際誌]

担当区分：責任著者

Type B insulin resistance syndrome (TBIR) is a rare disease characterized by refractory diabetes due to severe insulin resistance caused by anti-insulin receptor autoantibodies, and a standard treatment regimen for TBIR has not been established, leading to therapeutic difficulties and high mortality. Since TBIR is known to be associated with autoimmune diseases such as systemic lupus erythematosus (SLE), glucocorticoids are often used as key immunosuppressive agents. However, glucocorticoids have the potential to exacerbate the pathophysiology of TBIR by worsening insulin sensitivity, which leads to hyperglycemia and muscle wasting. Here, we report a case history of a 66-year-old man who was diagnosed as having TBIR in combination with SLE and Sjögren's syndrome with marked hyperglycemia, ketosis, and muscle wasting. He was successfully treated with combination therapy of double-filtration plasmapheresis (DFPP) and administration of the anti-CD20 monoclonal antibody rituximab without induction of glucocorticoid therapy while using a sensor-augmented insulin pump (SAP) to prevent hypoglycemia. Remission of diabetes was achieved without severe hypoglycemic events and his circulating insulin receptor antibodies became negative after seven months of initiation of these treatments. Based on the successful clinical courses of this case, our report suggests the possibility of an effective therapeutic regimen with DFPP and rituximab under the condition of the use of an SAP for a patient with TBIR without induction of glucocorticoids.

Hypoxia Differently Affects TGF-β2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells.

Soma Suzuki, Tatsuya Sato, Megumi Watanabe, Megumi Higashide, Yuri Tsugeno, Araya Umetsu, Masato Furuhashi, Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro

International journal of molecular sciences 23 ( 10 ) 2022年05月 [査読有り] [国際誌]

担当区分：筆頭著者

The hypoxia associated with the transforming growth factor-β2 (TGF-β2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O2 gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-β2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-β2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-β2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-β2 increased proton leakage. The findings reported herein indicate that the TGF-β2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.

Aging is associated with increased brain iron through cortex-derived hepcidin expression.

Tatsuya Sato, Jason Solomon Shapiro, Hsiang-Chun Chang, Richard A Miller, Hossein Ardehali

eLife 11 2022年01月 [査読有り] [国際誌]

担当区分：筆頭著者

Iron is an essential molecule for biological processes, but its accumulation can lead to oxidative stress and cellular death. Due to its oxidative effects, iron accumulation is implicated in the process of aging and neurodegenerative diseases. However, the mechanism for this increase in iron with aging, and whether this increase is localized to specific cellular compartment(s), are not known. Here, we measured the levels of iron in different tissues of aged mice, and demonstrated that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain cortex exhibits an increase in both the cytosolic and mitochondrial non-heme iron. This increase in brain iron is associated with elevated levels of local hepcidin mRNA and protein in the brain. We also demonstrate that the increase in hepcidin is associated with increased ubiquitination and reduced levels of the only iron exporter, ferroportin-1 (FPN1). Overall, our studies provide a potential mechanism for iron accumulation in the brain through increased local expression of hepcidin, and subsequent iron accumulation due to decreased iron export. Additionally, our data support that aging is associated with mitochondrial and cytosolic iron accumulation only in the brain and not in other tissues.

Enhanced glucose metabolism through activation of HIF-1α covers the energy demand in a rat embryonic heart primordium after heartbeat initiation.

Tatsuya Sato, Nobutoshi Ichise, Takeshi Kobayashi, Hiroyori Fusagawa, Hiroya Yamazaki, Taiki Kudo, Noritsugu Tohse

Scientific reports 12 ( 1 ) 74 - 74 2022年01月 [国際誌]

担当区分：筆頭著者 , 責任著者

The initiation of heartbeat is an essential step in cardiogenesis in the heart primordium, but it remains unclear how intracellular metabolism responds to increased energy demands after heartbeat initiation. In this study, embryos in Wistar rats at embryonic day 10, at which heartbeat begins in rats, were divided into two groups by the heart primordium before and after heartbeat initiation and their metabolic characteristics were assessed. Metabolome analysis revealed that increased levels of ATP, a main product of glucose catabolism, and reduced glutathione, a by-product of the pentose phosphate pathway, were the major determinants in the heart primordium after heartbeat initiation. Glycolytic capacity and ATP synthesis-linked mitochondrial respiration were significantly increased, but subunits in complexes of mitochondrial oxidative phosphorylation were not upregulated in the heart primordium after heartbeat initiation. Hypoxia-inducible factor (HIF)-1α was activated and a glucose transporter and rate-limiting enzymes of the glycolytic and pentose phosphate pathways, which are HIF-1α-downstream targets, were upregulated in the heart primordium after heartbeat initiation. These results suggest that the HIF-1α-mediated enhancement of glycolysis with activation of the pentose phosphate pathway, potentially leading to antioxidant defense and nucleotide biosynthesis, covers the increased energy demand in the beating and developing heart primordium.

Ultrastructural Assessment and Proteomic Analysis in Myofibrillogenesis in the Heart Primordium After Heartbeat Initiation in Rats.

Nobutoshi Ichise, Tatsuya Sato, Hiroyori Fusagawa, Hiroya Yamazaki, Taiki Kudo, Izaya Ogon, Noritsugu Tohse

Frontiers in physiology 13 907924 - 907924 2022年 [査読有り] [国際誌]

担当区分：筆頭著者 , 責任著者

Myofibrillogenesis is an essential process for cardiogenesis and is closely related to excitation-contraction coupling and the maintenance of heartbeat. It remains unclear whether the formation of myofibrils and sarcomeres is associated with heartbeat initiation in the early embryonic heart development. Here, we investigated the association between the ultrastructure of myofibrils assessed by transmission electron microscopy and their proteomic profiling assessed by data-independent acquisition mass spectrometry (DIA-MS) in the rat heart primordia before and after heartbeat initiation at embryonic day 10.0, when heartbeat begins in rats, and in the primitive heart tube at embryonic day 11.0. Bundles of myofilaments were scattered in a few cells of the heart primordium after heartbeat initiation, whereas there were no typical sarcomeres in the heart primordia both before and after heartbeat initiation. Sarcomeres with Z-lines were identified in cells of the primitive heart tube, though myofilaments were not aligned. DIA-MS proteome analysis revealed that only 43 proteins were significantly upregulated by more than 2.0 fold among a total of 7,762 detected proteins in the heart primordium after heartbeat initiation compared with that before heartbeat initiation. Indeed, of those upregulated proteins, 12 (27.9%) were constituent proteins of myofibrils and 10 (23.3%) were proteins that were accessories and regulators for myofibrillogenesis, suggesting that upregulated proteins that are associated with heartbeat initiation were enriched in myofibrillogenesis. Collectively, our results suggest that the establishment of heartbeat is induced by development of bundles of myofilaments with upregulated proteins associated with myofibrillogensis, whereas sarcomeres are not required for the initial heartbeat.

Commentary: Assessment of Hypertension Using Clinical Electrocardiogram Features: A First-Ever Review.

Tatsuya Sato

Frontiers in medicine 8 691330 - 691330 2021年 [査読有り] [国際誌]

担当区分：筆頭著者 , 最終著者 , 責任著者

Response to exercise-induced blood pressure elevation is blunted in wrist-cuff automated oscillometric measurement in healthy young college students.

Tatsuya Sato, Nobutoshi Ichise, Yoshinori Terashima, Aoi Kato, Hiroya Yamazaki, Shunsuke Jimbo, Noritsugu Tohse

Physiological reports 8 ( 17 ) e14570 2020年09月 [査読有り] [国際誌]

担当区分：筆頭著者 , 責任著者

BACKGROUND: A wrist-cuff automated oscillometric device is portable and useful for self-monitoring of blood pressure (BP) at home and outdoors when an upper arm device is not available. Although the height of the forearm in wrist BP measurement is acknowledged to be the major cause of measurement error, it remains unclear whether exercise affects subsequent wrist BP measurement. METHODS AND RESULTS: Ninety-seven healthy college students (median age of 20 years with an age range of 19 to 36 years, 70.1% males) participated in this study. Care was taken to keep the position of the wrist at a level near the upper arm level in BP measurement. At rest, BP measured by a wrist-cuff oscillometric device (Omron HEM-6183) was generally acceptable when it was compared with BP measured by an upper arm oscillometric device (Omron HEM-7130-HP) and with BP measured by the auscultatory method using a mercury sphygmomanometer. However, the ratio of systolic BP measured by oscillometric devices just after a two-step exercise test to that before exercise on the wrist (1.22 ± 0.14) was significantly lower than the ratio on the upper arm (1.27 ± 0.14), and the difference was significantly correlated with exercise-induced increase in pulse rate (Spearman's ρ = 0.23), suggesting a possible role of autonomic nerve activity in the blunted response to exercise-induced BP elevation in wrist BP measurement. CONCLUSIONS: The results indicate that the blunted response to exercise-induced BP elevation should be considered in wrist BP measurement when using a wrist-cuff oscillometric device.

Reply to the comment of Hirota et al. on "Accuracy of flash glucose monitoring in insulin-treated patients with type 2 diabetes".

Tatsuya Sato, Hiroto Oshima, Kei Nakata, Yukishige Kimura, Toshiyuki Yano, Masato Furuhashi, Masaya Tannno, Takayuki Miki, Tetsuji Miura

Journal of diabetes investigation 11 ( 1 ) 256 - 256 2020年01月 [査読有り] [国内誌]

担当区分：筆頭著者

Longitudinal impact of dapagliflozin treatment on ventricular repolarization heterogeneity in patients with type 2 diabetes.

Tatsuya Sato, Takayuki Miki, Shinya Furukawa, Bunzo Matsuura, Yoichi Hiasa, Hirofumi Ohnishi, Masaya Tanno, Tetsuji Miura

Journal of diabetes investigation 10 ( 6 ) 1593 - 1594 2019年11月 [査読有り] [国内誌]

担当区分：筆頭著者

Accuracy of flash glucose monitoring in insulin-treated patients with type 2 diabetes.

Tatsuya Sato, Hiroto Oshima, Kei Nakata, Yukishige Kimura, Toshiyuki Yano, Masato Furuhashi, Masaya Tanno, Takayuki Miki, Tetsuji Miura

Journal of diabetes investigation 10 ( 3 ) 846 - 850 2019年05月 [査読有り] [国内誌]

担当区分：筆頭著者

The present study evaluated the accuracy of interstitial glucose measurements by flash glucose monitoring (FGM) and continuous glucose monitoring (CGM). Five diabetes patients simultaneously underwent FGM (FreeStyle Libre Pro) and CGM (iPro™2), and their glucose levels were compared with venous blood and capillary blood glucose levels. The range of daily venous blood glucose levels (30 measurements) was 70-245 mg/dL, with a median of 138 mg/dL. There were good correlations of glucose levels measured by FGM (r2 = 0.90, mean absolute relative difference 8.2 ± 5.6%), CGM (r2 = 0.86, mean absolute relative difference 9.2 ± 9.1%) and capillary blood (r2 = 0.87, mean absolute relative difference 7.2 ± 7.2%) with venous blood glucose levels. The accuracy of FGM measurements was also shown against CGM, with 99.9% of the FGM values (1,279 measurements) being within the Parkes error grid zones A and B. The results suggest that the accuracy of FGM is similar to that of CGM, and that FGM is a useful tool for determining daily glucose profile.

mRNA-binding protein tristetraprolin is essential for cardiac response to iron deficiency by regulating mitochondrial function.

Tatsuya Sato, Hsiang-Chun Chang, Marina Bayeva, Jason S Shapiro, Lucia Ramos-Alonso, Hidemichi Kouzu, Xinghang Jiang, Ting Liu, Sumeyye Yar, Konrad T Sawicki, Chunlei Chen, María Teresa Martínez-Pastor, Deborah J Stumpo, Paul T Schumacker, Perry J Blackshear, Issam Ben-Sahra, Sergi Puig, Hossein Ardehali

Proceedings of the National Academy of Sciences of the United States of America 115 ( 27 ) E6291-E6300 2018年07月 [査読有り] [国際誌]

担当区分：筆頭著者

Cells respond to iron deficiency by activating iron-regulatory proteins to increase cellular iron uptake and availability. However, it is not clear how cells adapt to conditions when cellular iron uptake does not fully match iron demand. Here, we show that the mRNA-binding protein tristetraprolin (TTP) is induced by iron deficiency and degrades mRNAs of mitochondrial Fe/S-cluster-containing proteins, specifically Ndufs1 in complex I and Uqcrfs1 in complex III, to match the decrease in Fe/S-cluster availability. In the absence of TTP, Uqcrfs1 levels are not decreased in iron deficiency, resulting in nonfunctional complex III, electron leakage, and oxidative damage. Mice with deletion of Ttp display cardiac dysfunction with iron deficiency, demonstrating that TTP is necessary for maintaining cardiac function in the setting of low cellular iron. Altogether, our results describe a pathway that is activated in iron deficiency to regulate mitochondrial function to match the availability of Fe/S clusters.

Effect of sodium-glucose co-transporter-2 inhibitors on impaired ventricular repolarization in people with Type 2 diabetes.

Tatsuya Sato, Takayuki Miki, Hirofumi Ohnishi, Tomohisa Yamashita, Akifumi Takada, Toshiyuki Yano, Masaya Tanno, Akihito Tsuchida, Tetsuji Miura

Diabetic medicine : a journal of the British Diabetic Association 34 ( 10 ) 1367 - 1371 2017年10月 [査読有り] [国際誌]

担当区分：筆頭著者

AIMS: To test the hypothesis that treatment with a sodium-glucose co-transporter-2 inhibitor would reverse ventricular repolarization heterogeneity, a predictor of cardiovascular mortality, in people with Type 2 diabetes. METHODS: We retrospectively analysed changes in indices of ventricular repolarization before and after treatment with a sodium-glucose co-transporter-2 inhibitor in 46 people with Type 2 diabetes. RESULTS: Sodium-glucose co-transporter-2 inhibitor treatment reduced HbA1c concentration [62±13 mmol/mol (7.7±1.2%) vs 59±16 mmol/mol (7.5±1.4%)], body weight (77.8±13.9 vs 74.7±12.5 kg) and systolic blood pressure (133±18 vs 126±12 mmHg) in the study participants. Heart rate and QTc interval were not changed by sodium-glucose co-transporter-2 inhibitor treatment, but QTc dispersion was significantly reduced (median, 48.8 vs 44.2 ms). Sodium-glucose co-transporter-2 inhibitor treatment reversed QTc dispersion more in participants who had larger QTc dispersion before the treatment. Changes in systolic blood pressure (Spearman's ρ= 0.319; P=0.031), but not in HbA1c concentration, were correlated with changes in QTc dispersion after sodium-glucose co-transporter-2 inhibitor treatment. CONCLUSIONS: The findings suggest that sodium-glucose co-transporter-2 inhibitor treatment reverses ventricular repolarization heterogeneity in people with Type 2 diabetes, independently of its effect on glycaemic control. The favourable effect on ventricular repolarization heterogeneity could be the mechanism by which empaglifozin reduced cardiovascular events in a recent study.

Type 2 diabetes induces subendocardium-predominant reduction in transient outward K+ current with downregulation of Kv4.2 and KChIP2.

Tatsuya Sato, Takeshi Kobayashi, Atsushi Kuno, Takayuki Miki, Masaya Tanno, Hidemichi Kouzu, Takahito Itoh, Satoko Ishikawa, Takashi Kojima, Tetsuji Miura, Noritsugu Tohse

American journal of physiology. Heart and circulatory physiology 306 ( 7 ) H1054-65 2014年04月 [査読有り] [国際誌]

担当区分：筆頭著者 , 責任著者

In the present study, we examined if and how cardiac ion channels are modified by type 2 diabetes mellitus (T2DM). Subendocardial (Endo) myocytes and subepicardial (Epi) myocytes were isolated from left ventricles of Otsuka-Long-Evans-Tokushima Fatty rats (OLETF) rats, a rat model of T2DM, and Otsuka-Long-Evans-Tokushima (LETO) rats (nondiabetic control rats). Endo and Epi myocytes were used for whole cell patch-clamp recordings and for protein and mRNA analyses. Action potential durations in Endo and Epi myocytes were longer in OLETF rats than in LETO rats, and the difference was larger in Endo myocytes. Steady-state transient outward K+ current (Ito) density was reduced in Endo but not Epi myocytes of OLETF rats compared with LETO rats, although the contribution of the fast component of Ito recovery from inactivation was smaller in both Endo and Epi myocytes of OLETF rats than in LETO rats. Kv4.2 protein was reduced only in Endo myocytes in OLETF rats, although voltage-gated K+ channel-interacting protein 2 (KChIP2) protein levels in both Endo and Epi myocytes were lower in OLETF rats than in LETO rats. Corresponding regional differences in mRNA levels of KChIP2 and Kv4.2 were observed between OLETF and LETO rats. mRNA levels of Iroquois homeobox 5 in Endo myocytes were 53% higher in OLETF rats than in LETO rats. Densities of inward rectifier K+ current and L-type Ca2+ current and mRNA levels of Kv4.3 and Kv1.4 were similar in OLETF and LETO rats. In conclusion, T2DM induces Endo-predominant prolongation of the action potential duration via a reduction of the fast component of Ito recovery from inactivation and reduced steady-state Ito, in which downregulation of Kv4.2 and KChIP2 may be involved. Increased Iroquois homeobox 5 expression may underlie Kv4.2 downregulation in T2DM.

Histopathology of the pancreas in fulminant type 1 diabetes after 23-year follow-up: a case report.

Tatsuya Sato, Takayuki Miki, Naoto Murakami, Yoshihiko Hirohashi, Hidemichi Kouzu, Masato Furuhashi, Masaya Tanno, Satoshi Yuda, Shigeyuki Saitoh, Tadashi Hasegawa, Tetsuji Miura

Pathology international 62 ( 12 ) 827 - 9 2012年12月 [査読有り] [国際誌]

担当区分：筆頭著者

Roles of phospho-GSK-3β in myocardial protection afforded by activation of the mitochondrial K ATP channel.

Yoshiaki Terashima, Tatsuya Sato, Toshiyuki Yano, Ole Maas, Takahito Itoh, Takayuki Miki, Masaya Tanno, Atsushi Kuno, Kazuaki Shimamoto, Tetsuji Miura

Journal of molecular and cellular cardiology 49 ( 5 ) 762 - 70 2010年11月 [査読有り] [国際誌]

担当区分：筆頭著者

The aim of this study was to determine the roles of glycogen synthase kinase-3β (GSK-3β) in cardioprotection by activation of the mitochondrial ATP-sensitive K(+) channel (mK(ATP) channel). In isolated rat hearts, an mK(ATP) activator, diazoxide, and a GSK-3β inhibitor, SB216763, similarly limited infarct size and the combination of these agents did not afford further protection. The protection by pre-ischemic treatment with diazoxide was abolished by inhibition of protein kinase C-ε (PKC-ε) or phosphatidylinositol-3-kinase (PI3K) upon reperfusion. Infusion of a GSK-3β inhibitor (LiCl), but not diazoxide, during reperfusion limited infarct size. Inhibition of PKC-ε or PI3K did not affect the protection by LiCl. Diazoxide infusion alone did not induce GSK-3β phosphorylation. However, diazoxide infusion before ischemia increased mitochondrial phospho-GSK-3β level and reduced cyclophilin-D (CypD) binding to adenine nucleotide translocase (ANT) at 10 min after reperfusion. This diazoxide-induced GSK-3β phosphorylation was inhibited by blockade of the mK(ATP) channel before ischemia and by blockade of PKC-ε, PI3K or the adenosine A2b receptor at the time of reperfusion. Inhibition of GSK-3β by LiCl during reperfusion increased phospho-GSK-3β but had no significant effect on CypD-ANT binding. These results suggest that GSK-3β phosphorylation at the time of reperfusion by a PKC-ε, PI3K- and A2b receptor-dependent mechanism contributes to prevention of myocardial necrosis by pre-ischemic activation of the mK(ATP) channel. Inhibition of CypD-ANT interaction may contribute to mK(ATP)-induced myocardial protection, though it is not the sole mechanism of phospho-GSK-3β-mediated cytoprotection.

Incorporation of Metabolic Dysfunction-Associated Steatotic Liver Disease in the Health Stage of Cardiovascular-Kidney-Metabolic Syndrome Improves Predictive Ability for Coronary Artery Disease in a Japanese General Population.

Wataru Kawaharata, Marenao Tanaka, Tatsuya Sato, Yukinori Akiyama, Itaru Hosaka, Hiroki Aida, Rie Matsumori, Kei Nakata, Keitaro Nishizawa, Toru Suzuki, Hidemichi Kouzu, Nagisa Hanawa, Masato Furuhashi

Journal of the American Heart Association e043173 2025年08月 [査読有り] [国際誌]

BACKGROUND: Cardiovascular-kidney-metabolic (CKM) syndrome is a recently proposed condition encompassing metabolic dysfunction, chronic kidney disease, and cardiovascular diseases including coronary artery disease (CAD). Although concomitant metabolic dysfunction-associated steatotic liver disease (MASLD) exacerbates CKM syndrome, MASLD is not included in the original stratification (stages 0-3) for predicting cardiovascular disease (stage 4). METHODS: We investigated whether the addition of MASLD in the original health stage of CKM syndrome improves the risk stratification of CAD in Japanese participants who underwent annual health checkups. A total of 18 358 participants were categorized by the original health stages (stages 0-3) or modified health stages incorporating MASLD without chronic kidney disease in stage 2 and MASLD with chronic kidney disease in stage 3 to analyze the predictive ability for CAD during a 10-year period. RESULTS: Kaplan-Meier survival curve analysis showed that the modified classification more effectively stratified the risk of CAD than did the original CKM health stage. In Cox hazard proportional analyses after adjustment of confounders, hazard ratios in the original and modified stages 2/3 were 1.63 (95% CI, 1.27-2.11; P<0.001), 2.41 (95% CI, 0.34-17.4; P=0.381), 1.58 (95% CI, 1.23-2.05; P<0.001), and 2.56 (95% CI, 1.66-3.95; P<0.001), respectively. Risk discrimination significantly improved in the modified classification evaluated by integrated discrimination improvement. Furthermore, machine learning-mediated feature importance analyses using random forest and extreme gradient boosting identified MASLD as a key predictor of CAD with a high value of the Shapley Additive Explanations, indicating strong contribution in models. CONCLUSIONS: The modified health stage classification of CKM syndrome incorporating MASLD improves the accuracy of predicting new onset of CAD in a Japanese general population.

Association of Difference Between eGFR From Cystatin C and Creatinine and Serum GDF-15 With Adverse Outcomes in Diabetes Mellitus.

Tomohito Gohda, Nozomu Kamei, Marenao Tanaka, Masato Furuhashi, Tatsuya Sato, Mitsunobu Kubota, Michiyoshi Sanuki, Takeo Koshida, Shinji Hagiwara, Yusuke Suzuki, Maki Murakoshi

Journal of cachexia, sarcopenia and muscle 16 ( 4 ) e70011 2025年08月 [査読有り] [国際誌]

BACKGROUND: Protein catabolism and chronic inflammation drive sarcopenia and frailty in individuals with diabetes mellitus and chronic kidney disease (CKD). The difference between estimated glomerular filtration rates derived from cystatin C and creatinine (eGFRcys and eGFRcr, respectively), termed eGFRdiff, along with growth differentiation factor-15 (GDF-15) levels, have emerged as markers of metabolic and inflammatory dysregulation. Lower eGFRdiff and elevated GDF-15 levels are associated with sarcopenia, frailty, CKD progression and mortality. However, their interplay and respective impacts on CKD progression and mortality remain unclear. METHODS: A total of 638 Japanese individuals with diabetes mellitus were stratified into tertiles based on eGFRdiff. Serum GDF-15 levels were measured using enzyme-linked immunosorbent assays. The relationships between eGFRdiff and GDF-15 were assessed using Spearman's correlation coefficients. Multivariate ordered logistic regression was used to evaluate the association between eGFRdiff and GDF-15 tertiles, with GDF-15 as the dependent variable and eGFRdiff as the independent variable, adjusting for covariates including age, sex, urinary albumin-to-creatinine ratio (UACR) and eGFRcr or eGFRcys. Cox proportional hazards models with restricted cubic splines were used to examine associations between eGFRdiff and GDF-15 (independent variables) with CKD progression (≥ 30% decline in eGFRcr from baseline) and mortality (dependent variables). These models were adjusted for age, sex, glycated haemoglobin, UACR and eGFRcr. RESULTS: The median age was 65 years (interquartile range: 58-73), and 53.9% of participants were male. Over median follow-up periods of 5.3 years for CKD progression and 5.4 years for mortality, 75 participants (11.8%) experienced CKD progression and 44 (6.9%) died. GDF-15 levels inversely correlated with eGFRdiff (r = -0.35, p < 0.001). Higher eGFRdiff values were associated with lower odds of being in a higher GDF-15 tertile (odds ratio 0.86; 95% confidence interval [CI]: 0.76-0.97; p = 0.01). Both lower eGFRdiff and higher GDF-15 levels were independently associated with adverse outcomes: CKD progression (GDF-15, hazard ratio [HR] 1.36, 95% CI: 1.02-1.81, p < 0.05; eGFRdiff, HR 0.67, 95% CI: 0.58-0.78, p < 0.0001) and mortality (GDF-15, HR 2.35, 95% CI: 1.63-3.41, p < 0.0001; eGFRdiff: 0.80, 95% CI: 0.65-0.99, p < 0.05). CONCLUSIONS: Both eGFRdiff and GDF-15 were independently associated with adverse outcomes in individuals with diabetes mellitus. GDF-15 showed a stronger association with mortality, whereas eGFRdiff was more strongly linked to CKD progression. These findings underscore the potential utility of these markers in risk stratification for diabetes-related complications and may guide individualized interventions in clinical practice.

Plasma hepcidin level is elevated by water immersion‐induced central fatigue via hepatic inflammatory response in male and female rats

Takuro Karaushi, Toshifumi Ogawa, Hiroyori Fusagawa, Taiki Kudo, Yuito Inoue, Takashi Yamada, Nobutoshi Ichise, Tatsuya Sato, Noritsugu Tohse

Physiological Reports 13 ( e70468 ) 2025年08月 [査読有り]

担当区分：責任著者

The health stage of cardiovascular-kidney-metabolic (CKM) syndrome is useful for predicting all-cause mortality in patients with type 2 diabetes: a cohort study in a period prior to the standard use of recent pharmacotherapy.

Keitaro Nishizawa, Marenao Tanaka, Tatsuya Sato, Tomohito Gohda, Nozomu Kamei, Maki Murakoshi, Yukinori Akiyama, Wataru Kawaharata, Hiroki Aida, Hidemichi Kouzu, Naoya Yama, Mitsunobu Kubota, Michiyoshi Sanuki, Yusuke Suzuki, Masato Furuhashi

Journal of diabetes and its complications 39 ( 10 ) 109146 - 109146 2025年07月 [査読有り] [国際誌]

AIM: Cardiovascular-kidney-metabolic (CKM) syndrome is a recently defined systemic condition linking cardiovascular disease, chronic kidney disease and metabolic disorders including type 2 diabetes (T2D). Although the CKM staging has been proposed for integrated risk assessment, its association with all-cause mortality in patients with T2D remains unclear. We investigated the prognosis in patients with T2D assigned by the CKM health stage. METHODS: A total of 632 Japanese patients with T2D were enrolled. The primary endpoint was all-cause death. RESULTS: The numbers of the recruited patients with stages 2, 3 and 4 were 353 (55.9 %), 116 (18.3 %) and 163 (25.8 %), respectively. During a median follow-up of 64 months (35,327 person-months), 62 patients (9.8 %) died. Kaplan-Meier survival curves analysis showed significant differences in cumulative mortality among CKM health stages (log-rank test: P < 0.001) with higher cumulative mortality in stages 3 and 4 than in stage 2. Multivariable Cox proportional hazard models after adjustment of age, sex, body mass index, current smoking habit, cancer, relevant medications and hemoglobin A1c showed that adjusted hazard ratios (HRs) [95 % confidence intervals] for all-cause death were significantly higher in patients with stages 3 (2.25[1.08-4.69]) and those with stage 4 (2.87[1.41-5.84]) than in those with stage 2 as the reference. After additional adjustment of N-terminal pro-brain natriuretic peptide and estimated glomerular filtration rate among definition criteria for staging, the association of stages with all-cause death remained statistically significant in only stage 4 (2.16[1.02-4.56]). CONCLUSION: The CKM health staging is useful for predicting all-cause mortality in Japanese patients with T2D.

Association among circulating erythropoietin-producing hepatoma A2, progranulin, and kidney function in individuals with diabetes.

Maki Murakoshi, Nozomu Kamei, Kenichiro Abe, Takumi Iwasawa, Kazunori Kato, Marenao Tanaka, Tatsuya Sato, Masato Furuhashi, Mitsunobu Kubota, Michiyoshi Sanuki, Yusuke Suzuki, Tomohito Gohda

Journal of diabetes investigation 2025年07月 [査読有り] [国内誌]

BACKGROUND: Erythropoietin-producing hepatoma A2 (EphA2), a receptor for progranulin (PGRN), which is a growth factor associated with metabolic disorders, is implicated in inflammation and atherosclerotic diseases. Since both EphA2 and PGRN play roles in diabetic kidney disease (DKD) and cardiovascular disease (CVD), this study examined their association with renal function and CVD markers in individuals with diabetes. In addition, the diagnostic value of EphA2 and PGRN in predicting renal impairment was evaluated. METHODS: Circulating EphA2 and PGRN levels were measured using enzyme-linked immunosorbent assay in 735 participants with diabetes. Clinical data, including biometric parameters, physiological measurements, and comorbidities, were collected for analysis. RESULTS: Both EphA2 and PGRN levels positively correlated with older age, elevated blood pressure, higher urinary albumin-to-creatinine ratio (UACR), and brain natriuretic peptide (BNP) levels but negatively correlated with estimated glomerular filtration rate (eGFR). Multivariate logistic regression analysis revealed that EphA2 was an independent predictor of eGFR <60 mL/min/1.73 m2, even after adjusting for UACR and CVD risk factors and glycated hemoglobin. Although PGRN was also independently associated with eGFR <60 mL/min/1.73 m2, its association was weaker than that of EphA2. Conversely, when UACR ≥30 mg/g was used as the dependent variable, PGRN emerged as a stronger independent determinant than EphA2, even after adjusting for eGFR and CVD risk factors. CONCLUSIONS: EphA2 and PGRN levels are significantly associated with renal function in individuals with diabetes. These findings suggest that EphA2 and PGRN could serve as novel biomarkers for kidney impairment, independent of established CVD markers in this population.

Treatment with pemafibrate ameliorates fatty liver index and atherogenic lipid profiles in Japanese patients with type 2 diabetes mellitus.

Toru Suzuki, Tatsuya Sato, Marenao Tanaka, Kei Nakata, Keisuke Endo, Hiroki Aida, Wataru Kawaharata, Itaru Hosaka, Araya Umetsu, Toshifumi Ogawa, Yukinori Akiyama, Masato Furuhashi

Frontiers in endocrinology 16 1496671 - 1496671 2025年07月 [査読有り] [国際誌]

担当区分：筆頭著者

BACKGROUND: Pemafibrate, a selective peroxisome proliferator-activated receptor α modulator, ameliorates hypertriglyceridemia. We investigated the effects of pemafibrate on steatotic liver disease (SLD) in relation to various atherogenic lipid profiles. METHODS: Thirty-nine Japanese patients with both type 2 diabetes mellitus (T2DM) and hypertriglyceridemia (men/women: 24/15, mean age: 58.2 years, median duration of diabetes: 5.0 years) were treated with 0.2 mg/day of pemafibrate for 12 months (M). SLD was estimated by fatty liver index (FLI), which is calculated by using waist circumference, body mass index and levels of triglycerides and γ-glutamyl transpeptidase. RESULTS: Treatment with pemafibrate significantly increased mean levels of high-density lipoprotein cholesterol (HDL-C) (baseline/3M/6M/12M: 46/55/55/54 mg/dL) and decreased median levels of triglycerides (baseline/3M/6M/12M: 211/112/99/98 mg/dL), non-HDL-C (146/128/125/121 mg/dL), small dense low-density lipoprotein cholesterol (45/33/30/30 mg/dL) and remnant-like particle cholesterol (8.1/2.6/2.3/2.4 mg/dL). There was no significant change in hemoglobin A1c level over time. FLI (mean ± standard deviation: 68.1 ± 21.9 vs. 39.6 ± 25.0, P < 0.001), but not FIB-4 index as a marker of hepatic fibrosis (median [interquartile range]: 1.04 [0.78-1.39] vs. 1.01 [0.68-1.36], P = 0.909), was significantly decreased by treatment with pemafibrate for 12M, and the proportion of patients with metabolic dysfunction-associated SLD (MASLD) was significantly decreased from 92.3% (baseline) to 61.5% (12M). CONCLUSIONS: Pemafibrate ameliorates MASLD estimated by FLI in addition to various atherogenic lipid profiles in Japanese hypertriglyceridemia patients with T2DM in the past mean 5 years. An early intervention with pemafibrate might contribute to prevention of the development of MASLD and atherosclerotic cardiovascular disease.

Association of the Glycated Albumin-to-Glycated Haemoglobin Ratio With Mortality in Type 2 Diabetes: A Retrospective Cohort Analysis.

Tomohito Gohda, Nozomu Kamei, Marenao Tanaka, Masato Furuhashi, Tatsuya Sato, Mitsunobu Kubota, Michiyoshi Sanuki, Risako Mikami, Koji Mizutani, Yusuke Suzuki, Maki Murakoshi

Endocrinology, diabetes & metabolism 8 ( 4 ) e70072 2025年07月 [査読有り] [国際誌]

INTRODUCTION: The glycated albumin-to-glycated haemoglobin (GA/HbA1c) ratio is a potential marker of glycaemic variability; however, its association with adverse clinical outcomes in type 2 diabetes remains unclear. We aimed to determine whether the GA/HbA1c ratio is a better predictor of mortality and chronic kidney disease (CKD) progression than GA alone in type 2 diabetes. METHODS: This retrospective cohort analysis included 571 Japanese participants with type 2 diabetes who were stratified into tertiles based on their GA/HbA1c ratio. Cox proportional hazards models assessed associations between the GA/HbA1c ratio and mortality or CKD progression (≥ 30% decline in the estimated glomerular filtration rate [eGFR]), adjusting for age, sex, urinary albumin-to-creatinine ratio, eGFR, body mass index, haemoglobin and serum albumin. RESULTS: In this cohort, the median age was 67 years, and 53.9% were male. During the median follow-up of 5.4 and 5.3 years for mortality and CKD progression, respectively, 40 (7.0%) participants died and 70 (12.3%) experienced CKD progression. For mortality, the GA/HbA1c ratio demonstrated a U-shaped association: although both the lowest (T1) and highest (T3) tertiles showed higher mortality risks than the middle tertile (T2), this association was significant for only T3 (hazard ratio, 1.46; 95% CI, 1.05-2.04). Neither GA nor HbA1c alone was significantly associated with mortality. For CKD progression, GA alone showed a U-shaped association, with both T1 and T3 exhibiting non-significantly higher risks than T2. Neither the GA/HbA1c ratio nor HbA1c alone was associated with CKD progression. CONCLUSIONS: In individuals with type 2 diabetes, a higher GA/HbA1c ratio was associated with an increased risk of mortality but not with CKD progression. However, given the retrospective design and limited sample size, these findings should be interpreted with caution and confirmed in larger, prospective studies.

Mapping research trends in obsessive-compulsive disorder before and after the COVID-19 pandemic: a bibliometric analysis focusing on its molecular mechanisms.

Yuito Inoue, Nobutoshi Ichise, Wataru Ukai, Jun Shinozaki, Toshifumi Ogawa, Takuro Karaushi, Marenao Tanaka, Yukinori Akiyama, Masato Furuhashi, Atsushi Kuno, Tatsuya Sato

Frontiers in psychiatry 16 1615497 - 1615497 2025年07月 [査読有り] [国際誌]

担当区分：責任著者

Obsessive-compulsive disorder (OCD) is a psychiatric disorder that primarily develops during adolescence, and is characterized by obsessive thoughts and compulsive behaviors. Although multiple factors including heredity, environment, and abnormalities in neural networks and synapses are involved in the onset and exacerbation of OCD, their underlying molecular mechanisms have not been fully elucidated. In addition, recent studies have demonstrated that the novel coronavirus disease (COVID-19) pandemic worsened OCD phenotypes. Hence, this global crisis may have changed the field of molecular-focused OCD research. We conducted a brief bibliometric analysis to investigate changes in prevalent topics in molecular-focused OCD research before (2015-2019) and after (2020-2025) the COVID-19 pandemic using Web of Science and VOSviewer. "Schizophrenia" and "metaanalysis" remained highly ranked terms in molecular-focused OCD research. In terms of neurotransmitters, the term "serotonin" became more prevalent than "dopamine" after the COVID-19 pandemic. In addition, research interest shifted toward younger populations, and there was a noticeable increase in terms related to neural networks such as "connectivity". However, only a few specific molecular mechanisms or cellular physiological pathways by which COVID-19 exacerbates OCD have been identified. To address this gap, an additional post hoc analysis focusing on inflammation-related terms was conducted, revealing the emergence of "oxidative stress" and "c-reactive protein" in studies published after the COVID-19 pandemic. The findings of this study highlight several potential clues for elucidating the pathophysiology of OCD and identifying aggravating factors such as COVID-19, while also emphasizing the importance of continued molecular-focused research to establish novel therapeutic targets.

Small Dense Low-Density Lipoprotein Cholesterol and Renal Function: A Missing Link in Cardiovascular Risk Assessment for Chronic Kidney Disease Patients - Reply.

Marenao Tanaka, Tatsuya Sato, Wataru Kawaharata, Hiroki Aida, Masato Furuhashi

Circulation journal : official journal of the Japanese Circulation Society 89 ( 6 ) 848 - 849 2025年05月 [査読有り] [国内誌]

Fatty Acid Metabolism Regulators Have Pivotal Roles in the Pathogenesis of Ovarian Carcinoma

Megumi Watanabe, Motoki Matsuura, Tatsuya Sato, Makoto Usami, Tsuyoshi Saito, Masato Furuhashi, Kohichi Takada, Hiroshi Ohguro

International Journal of Molecular Sciences 2025年05月 [査読有り]

The serum γ-Glutamyltransferase Level is Associated with the Development of Hypertension in Alcohol Infrequent Drinkers but not in Frequent Drinkers.

Kazuma Mori, Marenao Tanaka, Tatsuya Sato, Yukinori Akiyama, Hirofumi Ohnishi, Nagisa Hanawa, Masato Furuhashi

Internal medicine (Tokyo, Japan) ( Japanese Society of Internal Medicine ) 2025年04月 [査読有り] [国内誌]

Background The controversy persists regarding whether the serum level of γ-glutamyltransferase (GGT), a marker of liver damage, is associated with hypertension irrespective of alcohol intake. Methods We investigated the relationship between the GGT level and new-onset hypertension during a 10-year follow-up period in Japanese individuals who underwent annual health examinations (n=28,990). After excluding subjects without systolic blood pressure and GGT data and those with hypertension at baseline, a total of 18,618 subjects (men/women: 11,262/7,356, mean age: 44 years) were enrolled. Results During the follow-up period, 2,753 men (24.4%) and 837 women (11.4%) developed hypertension. When the subjects were divided by quartiles of GGT at baseline (Q1-Q4), multivariable Cox proportional hazard model analyses after adjustment for age, sex, systolic blood pressure, body mass index, levels of uric acid, estimated glomerular filtration rate, family history of hypertension, habits of current smoking and alcohol drinking, and diagnosis of diabetes mellitus and dyslipidemia showed that hazard risks (HRs) for the development of hypertension were significantly higher in the Q2, Q3, and Q4 groups than in the Q1 group. A significant interaction was observed between alcohol drinking habits and the GGT level at baseline for the development of hypertension (p=0.022), and adjusted HRs were similarly significant in alcohol infrequent drinkers (≤5 days/week). However, the GGT level was not significantly associated with the development of hypertension in frequent alcohol drinkers (≥6 days/week). Conclusions A high GGT level is an independent predictor of new-onset hypertension in infrequent alcohol drinkers but not in frequent drinkers.

Can small dense LDL cholesterol be estimated from the lipid profile?

Tatsuya Sato, Marenao Tanaka, Masato Furuhashi

Current opinion in lipidology 2025年04月 [査読有り] [国際誌]

担当区分：筆頭著者

PURPOSE OF REVIEW: Small dense low-density lipoprotein cholesterol (sdLDL-C) is recognized for its strong atherosclerogenic potential. However, its direct measurement remains impractical in clinical settings due to its high cost, time constraints, and labor-intensive nature. This review discusses the benefits and limitations of estimating sdLDL-C using conventional lipid fractions, highlighting recent advancements in estimation methods. RECENT FINDINGS: Sampson et al. proposed a novel equation for estimating sdLDL-C based on conventional lipid parameters, offering a more accessible alternative to direct measurement. Recent studies, including ours, demonstrated that this estimation method achieves sufficiently high accuracy for overall application. However, its accuracy can be improved by incorporating machine learning. Furthermore, sdLDL-C estimated by Sampson's equation has been shown to be a superior risk marker for hypertension, an intermediate phenotype of atherosclerosis, and ischemic heart disease, a major cardiovascular event, compared to conventional lipid profiles alone, although further research is needed to determine whether estimated sdLDL-C is equivalent to directly measured sdLDL-C in risk assessment. SUMMARY: Estimated sdLDL-C presents a promising alternative to direct measurement. While estimated sdLDL-C levels can serve a risk marker for cardiovascular diseases, further research is needed to refine estimation models and explore their integration into clinical practice.

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) Is an Independent Risk Factor for the Development of Ischemic Heart Disease ― A 10-Year Cohort Study ―

Toshifumi Ogawa, Tatsuya Sato, Marenao Tanaka, Yukinori Akiyama, Kei Nakata, Hidemichi Kouzu, Kazuma Mori, Hiroki Aida, Wataru Kawaharata, Itaru Hosaka, Toru Suzuki, Nagisa Hanawa, Masato Furuhashi

Circulation Reports ( Japanese Circulation Society ) 2025年04月 [査読有り]

担当区分：筆頭著者

High Level of Estimated Small Dense Low-Density Lipoprotein Cholesterol as an Independent Risk Factor for the Development of Ischemic Heart Disease Regardless of Low-Density Lipoprotein Cholesterol Level - A 10-Year Cohort Study.

Keisuke Endo, Marenao Tanaka, Tatsuya Sato, Masafumi Inyaku, Kei Nakata, Wataru Kawaharata, Hiroki Aida, Itaru Hosaka, Yukinori Akiyama, Nagisa Hanawa, Masato Furuhashi

Circulation journal : official journal of the Japanese Circulation Society 2025年03月 [査読有り] [国内誌]

BACKGROUND: We previously reported that a high level of small dense low-density lipoprotein cholesterol (sdLDL-C) calculated by the Sampson equation was independently associated with the development of ischemic heart disease (IHD), but it remains unclear whether the effect depends on the level of low-density lipoprotein cholesterol (LDL-C). METHODS AND RESULTS: We investigated the associations of new onset of IHD with categorized groups of high (H-) and low (L-) levels of estimated sdLDL-C and LDL-C using 25thpercentile levels of sdLDL-C level (25.2 mg/dL) and LDL-C (100 mg/dL) as cutoff values in 17,963 Japanese individuals (men/women: 11,508/6,455, mean age: 48 years) who underwent annual health checkups. During a 10-year follow-up period, 570 subjects (men/women: 449/121) had new development of IHD. Multivariable Cox proportional hazard analyses after adjustment of age, sex, smoking habit, hypertension and diabetes mellitus at baseline showed that the hazard ratio (HR) [95% confidence interval (CI)] for new onset of IHD was significantly higher in subjects with H-sdLDL-C/H-LDL-C (1.49 [1.06-2.08]) and subjects with H-sdLDL-C/L-LDL-C (1.49 [1.00-2.22]) than in subjects with L-sdLDL-C/L-LDL-C as the reference. CONCLUSIONS: A high level of sdLDL-C estimated by the Sampson equation was a predominant predictor for the development of IHD, regardless of the level of LDL-C, in a general Japanese population.

Elevated urinary fatty acid-binding protein 4 level predicts future renal dysfunction and poor prognosis in Japanese patients with diabetes: a longitudinal cohort study

Marenao Tanaka, Tatsuya Sato, Tomohito Gohda, Nozomu Kamei, Maki Murakoshi, Erika Ishiwata, Kei Nakata, Yukinori Akiyama, Keisuke Endo, Wataru Kawaharata, Hiroki Aida, Toru Suzuki, Mitsunobu Kubota, Michiyoshi Sanuki, Yusuke Suzuki, Masato Furuhashi

Clinical Kidney Journal ( Oxford University Press (OUP) ) 2025年03月 [査読有り]

担当区分：筆頭著者

Abstract Background Fatty acid-binding protein 4 (FABP4) is an adipokine secreted from adipocytes and macrophages and is also expressed in injured, but not normal, glomerular endothelial cells. Elevated levels of urinary FABP4 (U-FABP4) have been reported to be associated with glomerular damage and increased proteinuria. Methods The associations of levels of U-FABP4 at baseline with future events including renal dysfunction defined by a 30% decline in estimated glomerular filtration rate (eGFR) and all-cause death were investigated in 660 patients with diabetes (type 1/2: 57/603). Results During a follow-up period (median: 62 months), 90 patients (13.6%) developed renal dysfunction, and 66 patients (10.0%) died (median follow-up period: 65 months). Kaplan-Meier survival curves showed that there were significant differences in cumulative incidences for a 30% decline in eGFR and all-cause death in patients divided by the tertiles of U-FABP4 level. Furthermore, multivariable Cox proportional hazard models with a restricted cubic spline showed that hazard ratios for a 30% decline in eGFR and all-cause death increased with a higher level of logarithmically transformed (Log) U-FABP4 after adjustment for age, sex, type of diabetes, body mass index, current smoking habit, duration of diabetes, comorbidities of hypertension and dyslipidemia, eGFR and the categorical classification of urinary albumin-creatinine ratio. The addition of Log U-FABP4 to traditional risk factors significantly increased the discriminatory capacities for renal dysfunction in net reclassification improvement (NRI) and integrated discrimination improvement and for all-cause death in NRI. Conclusion U-FABP4 is a predictive biomarker for future renal dysfunction and poor prognosis in patients with diabetes.

The Combination of PPARα Agonist GW7647 and Imeglimin Has Potent Effects on High-Glucose-Induced Cellular Biological Responses in Human Retinal Pigment Epithelium Cells.

Nami Nishikiori, Megumi Watanabe, Megumi Higashide, Araya Umetsu, Toshifumi Ogawa, Masato Furuhashi, Hiroshi Ohguro, Tatsuya Sato

Bioengineering (Basel, Switzerland) 12 ( 3 ) 2025年03月 [査読有り] [国際誌]

担当区分：責任著者

BACKGROUND: Hyperglycemic changes in the cellular biological properties of retinal pigment epithelium cells are involved in the pathophysiology of diabetic retinopathy (DR). To assess the effects of the new anti-diabetic agent imeglimin (Ime) on DR, the pharmacological effects of Ime and those of metformin (Met) in combination with the PPARα agonist GW7646 (GW) on adult retinal pigment epithelium (ARPE19) cells cultured in high-glucose conditions were compared. METHODS: Cell viability, levels of reactive oxygen species (ROS), monolayer barrier function measured by transepit very much helial electrical resistance (TEER), and metabolic functions determined by an extracellular flux analyzer were evaluated. RESULTS: While glucose concentrations did not alter cell viability regardless of the presence of Met or Ime, levels of ROS were significantly increased by the high-glucose conditions, and increased levels of ROS were significantly alleviated by the combination of Ime and GW but not by Met alone. Similarly, TEER values were increased by high-glucose conditions, but the effects of high-glucose conditions were dramatically enhanced by the combination of Ime and GW. Furthermore, a metabolic assay showed that an energetic shift was induced by the combination of Ime and GW, whereas energy status became quiescent with Met or Ime alone. CONCLUSIONS: The collective results suggest that Ime in combination with GW has synergetic effects on high-glucose-induced cellular biological changes in ARPE19 cells.

Construction of Predictive Models for Cardiovascular Mortality by Machine Learning Approaches in Patients Who Underwent Transcatheter Aortic Valve Implantation

Shunsaku Otomo, Itaru Hosaka, Marenao Tanaka, Naoto Murakami, Nobuaki Kokubu, Atsuko Muranaka, Ryo Nishikawa, Naoki Hachiro, Ryota Kawamura, Jun Nakata, Nobutaka Nagano, Yukinori Akiyama, Tatsuya Sato, Yutaka Iba, Toshiyuki Yano, Nobuyoshi Kawaharada, Masato Furuhashi

Circulation Reports ( Japanese Circulation Society ) 2025年03月 [査読有り]

Expression and Secretion of Intraocular Fatty Acid-Binding Protein 4 (ioFABP4) and 5 (ioFABP5) Are Regulated by Glucose Levels and Fatty Acids

Hiroshi Ohguro, Megumi Higashide, Erika Ishiwata, Fumihito Hikage, Megumi Watanabe, Nami Nishikiori, Tatsuya Sato, Masato Furuhashi

International Journal of Molecular Sciences 2025年02月 [査読有り]

Unraveling Novel Subsets of Lymphocytes Involved in Sac Expansion in the Tertiary Lymphoid Structure Within an Abdominal Aortic Aneurysm.

Itaru Hosaka, Ippei Ikegami, Takuma Mikami, Tatsuya Sato, Toshifumi Ogawa, Kei Mukawa, Marenao Tanaka, Keisuke Endo, Yukinori Akiyama, Akihito Ohkawa, Junji Nakazawa, Tsuyoshi Shibata, Tomohiro Nakajima, Yutaka Iba, Chikara Shiiku, Satoshi Sumino, Ryuji Koshima, Kenichi Takano, Shingo Ichimiya, Nobuyoshi Kawaharada, Masato Furuhashi

Journal of the American Heart Association e040279 2025年02月 [査読有り] [国際誌]

BACKGROUND: Chronic inflammation is involved in the development of abdominal aortic aneurysm (AAA). A tertiary lymphoid structure (TLS) within vascular lesions has recently been focused on for its role in modulation of inflammation in local tissues. We aimed to elucidate the relationships between TLS and pathophysiology of AAA. METHODS: Abdominal aortic samples obtained from 37 patients with AAA (men/women: 34/3, age: 72.8±9.9 years) and 15 autopsied patients who died from non-aortic events (men/women: 11/4, age: 65.5±9.8 years) were investigated. RESULTS: TLSs in AAA lesions were confirmed by focal infiltration of CD3-positive cells surrounding germinal center-like structures containing CD20-positive cells between the tunica adventitia and tunica media layers. The formation of a TLS was significantly more prevalent in AAA patients than in autopsied patients. The number of TLSs in AAA lesions was positively correlated with sac diameter (r=0.357, P=0.035) and the amount of intraluminal thrombosis (r=0.466, P=0.005). T cells and B cells were predominant cellular populations among CD45+ cells in AAA lesions. There was a significantly positive correlation between the proportions of interfollicular T follicular helper (CD3+CD4+CD45RA-CXCR5+PD-1+) cells and double negative B (CD3-CD19+IgD-CD27-) cells, and they were positively correlated with sac diameter, intraluminal thrombosis, and serum lipids. Deposited single-cell RNA-sequencing data for AAA showed that T follicular helper cells and double negative B cells were associated with lipid metabolism, T cell activation/proliferation and inflammation. CONCLUSIONS: The formation of a TLS in AAA lesions is associated with sac diameter and intraluminal thrombosis in connection with interfollicular T follicular helper cells and double negative B cells, which may contribute to the pathophysiology of AAA and might be novel therapeutic targets for the development of AAA.

High-Glucose-Induced Metabolic and Redox Alterations Are Distinctly Modulated by Various Antidiabetic Agents and Interventions Against FABP5/7, MITF and ANGPTL4 in Melanoma A375 Cells

Nami Nishikiori, Hiroshi Ohguro, Megumi Watanabe, Megumi Higashide, Toshifumi Ogawa, Masato Furuhashi, Tatsuya Sato

International Journal of Molecular Sciences ( MDPI AG ) 26 ( 3 ) 1014 - 1014 2025年01月 [査読有り]

担当区分：責任著者

Hyperglycemia-induced effects on cellular metabolic properties and reactive oxygen species (ROS) generation play pivotal roles in the pathogenesis of malignant melanoma (MM). This study assessed how metabolic states, ROS production, and related gene expression are modulated by antidiabetic agents. The anti-diabetic agents metformin (Met) and imeglimin (Ime), inhibitors of fatty acid-binding proteins 5/7 (MF6) and microphthalmia-associated transcription factor (MITF) (ML329), and siRNA-mediated knockdown of angiopoietin-like protein 4 (ANGPTL4), which affect mitochondrial respiration, ROS production, and related gene expression, were tested in A375 (MM cell line) cells cultured in low (5.5 mM) and high glucose (50 mM) conditions. Cellular metabolic functions were significantly and differently modulated by Met, Ime, MF6, or ML329 and knockdown of ANGPTL4. High glucose significantly enhanced ROS production, which was alleviated by Ime but not by Met. Both MF6 and ML329 reduced ROS levels under both low and high glucose conditions. Knockdown of ANGPTL4 enhanced the change in glucose-dependent ROS production. Gene expression related to mitochondrial respiration and the pathogenesis of MM was significantly modulated by different glucose conditions, antidiabetic agents, MF6, and ML329. These findings suggest that glucose-dependent changes in cellular metabolism and redox status are differently modulated by antidiabetic agents, inhibition of fatty acid-binding proteins or MITF, and ANGPTL4 knockdown in A375 cells.

Effects of linsitinib on M22 and IGF:1-treated 3D spheroids of human orbital fibroblasts.

Fumihito Hikage, Megumi Suzuki, Tatsuya Sato, Araya Umetsu, Toshifumi Ogawa, Nami Nishikiori, Masato Furuhashi, Hiroshi Ohguro, Megumi Watanabe

Scientific reports 15 ( 1 ) 384 - 384 2025年01月 [査読有り] [国際誌]

To elucidate the role of IGF1R inhibition in the pathogenesis of Graves' orbitopathy (GO), the effects of linsitinib (Lins) on a recombinant human TSHR antibody (M22) and IGF1 to activate TSHR and IGF1R of human orbital fibroblasts (HOFs) obtained from patients without GO (HOFs) and patients with GO (GHOFs) were studied using in vitro three-dimensional (3D) spheroid models in addition to their 2D planar cell culture. For this purpose, we evaluated 1) cellular metabolic functions by using a seahorse bioanalyzer (2D), 2) physical properties including size and stiffness of 3D spheroids, and mRNA expression of several extracellular matrix (ECM) proteins, their modulators (CCL2 LOX, CTGF, MMPs), ACTA2 and inflammatory cytokines (IL1β, IL6). Administration of IGF1 and M22 induced increases of cellular metabolic functions with the effect on HOFs being much more potent than the effect on GHOFs, suggesting that IGF1R and TSHR of GHOFs may already be stimulated. Lins had effects similar to those of IGF1/M22 on cellular biological functions of HOFs but not on those of GHOFs. As for physical properties of 3D GHOFs spheroids, stiffness but not size was significantly increased by IGF1 and/or M22. In contrast, Lins significantly inhibited the M22-induced increase in stiffness despite the fact that Lins alone had no effect. The mRNA expression levels of several genes of ECM proteins and most of the other genes also fluctuated similarly to the changes in stiffness of 3D spheroids despite the fact that Lins induced up-regulation of inflammatory cytokines and MMP3. The findings presented herein indicate that IGF1R inhibition by Lins may beneficially affect GO-related fibrogenesis.

Machine learning-based analyses of contributing factors for the development of hypertension: a comparative study.

Marenao Tanaka, Yukinori Akiyama, Kazuma Mori, Itaru Hosaka, Keisuke Endo, Toshifumi Ogawa, Tatsuya Sato, Toru Suzuki, Toshiyuki Yano, Hirofumi Ohnishi, Nagisa Hanawa, Masato Furuhashi

Clinical and experimental hypertension (New York, N.Y. : 1993) 47 ( 1 ) 2449613 - 2449613 2025年01月 [査読有り] [国際誌]

OBJECTIVES: Sufficient attention has not been given to machine learning (ML) models using longitudinal data for investigating important predictors of new onset of hypertension. We investigated the predictive ability of several ML models for the development of hypertension. METHODS: A total of 15 965 Japanese participants (men/women: 9,466/6,499, mean age: 45 years) who received annual health examinations were randomly divided into a training group (70%, n = 11,175) and a test group (30%, n = 4,790). The predictive abilities of 58 candidates including fatty liver index (FLI), which is calculated by using body mass index, waist circumference and levels of γ-glutamyl transferase and triglycerides, were investigated by statistics analogous to the area under the curve (AUC) in receiver operating characteristic curve analyses using ML models including logistic regression, random forest, naïve Bayes, extreme gradient boosting and artificial neural network. RESULTS: During a 10-year period (mean period: 6.1 years), 2,132 subjects (19.1%) in the training group and 917 subjects (19.1%) in the test group had new onset of hypertension. Among the 58 parameters, systolic blood pressure, age and FLI were identified as important candidates by random forest feature selection with 10-fold cross-validation. The AUCs of ML models were 0.765-0.825, and discriminatory capacity was significantly improved in the artificial neural network model compared to that in the logistic regression model. CONCLUSIONS: The development of hypertension can be simply and accurately predicted by each ML model using systolic blood pressure, age and FLI as selected features. By building multiple ML models, more practical prediction might be possible.

Urinary fatty acid-binding protein 4 is a promising biomarker for glomerular damage in patients with diabetes mellitus.

Marenao Tanaka, Tatsuya Sato, Tomohito Gohda, Nozomu Kamei, Maki Murakoshi, Erika Ishiwata, Keisuke Endo, Wataru Kawaharata, Hiroki Aida, Kei Nakata, Yukinori Akiyama, Mitsunobu Kubota, Michiyoshi Sanuki, Toru Suzuki, Yusuke Suzuki, Masato Furuhashi

Journal of diabetes investigation 2024年12月 [査読有り] [国内誌]

AIMS/INTRODUCTION: Fatty acid-binding protein (FABP) 4, which acts as an adipokine secreted by adipocytes, macrophages, and capillary endothelial cells, is expressed in injured glomerular cells. It has been reported that urinary (U-) FABP4 is associated with renal dysfunction and proteinuria in several glomerular kidney diseases. However, the clinical significance of U-FABP4 in diabetic kidney disease (DKD) remains undetermined. MATERIALS AND METHODS: Immunohistological analyses of FABP4 and FABP1 (liver-type FABP), an established biomarker for impaired proximal tubules, were performed in the kidneys of patients with DKD and nonobese diabetic mice (KK-Ta/Akita mice). The associations between U-FABP4 and U-FABP1 with kidney function and metabolic indices were also investigated in patients with type 1 diabetes (n = 57, mean age: 61 years) and patients with type 2 diabetes (n = 608, mean age: 65 years). RESULTS: In both patients with diabetes and diabetic mice, FABP4 was expressed in injured glomeruli with increased markers of endoplasmic reticulum stress in addition to peritubular capillaries, whereas FABP1 was mainly expressed in proximal tubules. Levels of U-FABP4 and U-FABP1 were independently associated with each other, and both levels were independently associated with estimated glomerular filtration rate (eGFR) and urinary albumin-to-creatinine ratio (UACR) after adjustment of age, sex, type of diabetes, duration of diabetes, and systolic blood pressure in patients with diabetes. CONCLUSIONS: Urinary level of FABP4 derived from injured glomeruli with increased endoplasmic reticulum stress is independently associated with eGFR and UACR, suggesting a promising biomarker for glomerular damage in patients with diabetes.

Pemafibrate Induces a Low Level of PPARα Agonist-Stimulated mRNA Expression of ANGPTL4 in ARPE19 Cell

Hiroshi Ohguro, Nami Nishikiori, Tatsuya Sato, Megumi Watanabe, Megumi Higashide, Masato Furuhashi

Bioengineering (Basel, Switzerland) 11 ( 12 ) 2024年12月 [査読有り]

RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial–Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts

Araya Umetsu, Yosuke Ida, Tatsuya Sato, Megumi Higashide, Nami Nishikiori, Masato Furuhashi, Hiroshi Ohguro, Megumi Watanabe

Biomedicines 12 ( 12 ) 2024年12月 [査読有り]

LINC02154 promotes cell cycle and mitochondrial function in oral squamous cell carcinoma.

Takeshi Niinuma, Hiroshi Kitajima, Tatsuya Sato, Toshifumi Ogawa, Kazuya Ishiguro, Masahiro Kai, Eiichiro Yamamoto, Yui Hatanaka, Iyori Nojima, Mutsumi Toyota, Akira Yorozu, Shohei Sekiguchi, Noritsugu Tohse, Masato Furuhashi, Hiroshi Ohguro, Akihiro Miyazaki, Hiromu Suzuki

Cancer science 2024年11月 [査読有り] [国際誌]

Long noncoding RNAs (lncRNAs) play pivotal roles in the development of human malignancies, though their involvement in oral squamous cell carcinoma (OSCC) remains incompletely understood. Using The Cancer Genome Atlas (TCGA) dataset, we analyzed expression of 7840 lncRNAs in primary head and neck squamous cell carcinoma (HNSCC) and found that upregulation of LINC02154 is associated with a poorer prognosis. LINC02154 knockdown in OSCC cell lines induced cell cycle arrest and apoptosis, and significantly attenuated tumor growth in vitro and in vivo. Notably, depletion of LINC02154 downregulated FOXM1, a master regulator of cell cycle-related genes. RNA pulldown and mass spectrometry analyses identified a series of proteins that could potentially interact with LINC02154, including HNRNPK and LRPPRC. HNRNPK stabilizes FOXM1 expression by interacting with the 3'-UTR of FOXM1 mRNA, which suggests LINC02154 and HNRNPK promote cell cycling by regulating FOXM1 expression. Additionally, LINC02154 positively regulates HNRNPK expression by inhibiting microRNAs targeting HNRPNK. Moreover, LINC02154 affects mitochondrial function by interacting with LRPPRC. Depletion of LINC02154 suppressed expression of mitochondrial genes, including MTCO1 and MTCO2, and inhibited mitochondrial respiratory function in OSCC cells. These results suggest that LINC02154 exerts its oncogenic effects by modulating the cell cycle and oxidative phosphorylation in OSCC, highlighting LINC02154 as a potential therapeutic target.

mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners.

Megumi Watanabe, Tatsuya Sato, Toshiyuki Yano, Megumi Higashide, Toshifumi Ogawa, Nami Nishikiori, Masato Furuhashi, Hiroshi Ohguro

Biomedicines 12 ( 11 ) 2024年11月 [査読有り] [国際誌]

Background/Objectives: Mammalian target of rapamycin (mTOR) inhibition may have been suggested to have a beneficial effect on the glaucomatous human trabecular meshwork (HTM). To study the effects of the mTOR inhibitors rapamycin (Rapa) and Torin1 on the glaucomatous HTM, transforming growth factor-β2 (TGF-β2)-treated two-dimensionally (2D) and three-dimensionally (3D) cultured HTM cells were used. Methods: We evaluated (1) the levels of autophagy via Western blot analysis using a specific antibody against microtubule-associated protein 1 light chain 3 (LC3), (2) barrier capacity based on transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) permeability (2D), (3) cellular metabolic functions (2D), (4) the size and stiffness of spheroids, and (5) the mRNA expression of ECM proteins. Results: TGF-β2-induced inhibition of autophagy was significantly inhibited by Rapa and Torin1. Rapa and Torin1 substantially decreased barrier capacity in both TGF-β2-untreated and TGF-β2-treated HTM cells. Cellular metabolic analysis indicated that Rapa, but not Torin1, substantially enhanced both mitochondrial and glycolytic functions of TGF-β2-untreated HTM cells. In the physical properties of spheroids, TGF-β2 resulted in the formation of down-sized and stiffened spheroids. mTOR inhibitors decreased the size but not the stiffness of TGF-β2-untreated spheroids and significantly reduced the TGF-β2-related increase in the stiffness but not the size of spheroids. The diverse effects of mTOR inhibitors on TGF-β2-untreated and TGF-β2-treated spheroids were also observed in the mRNA expression of extracellular matrix proteins. Conclusions: The results taken together suggest that mTOR inhibitors significantly influence the biological aspects of both a single layer and multiple layers of the TGF-β2-treated HTM and untreated HTM.

TGF-β effects on adipogenesis of 3T3-L1 cells differ in 2D and 3D cell culture conditions.

Araya Umetsu, Megumi Watanabe, Tatsuya Sato, Megumi Higashide, Nami Nishikiori, Masato Furuhashi, Hiroshi Ohguro

FEBS open bio 2024年10月 [査読有り] [国際誌]

The TGF-β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF-β, TGF-β-1~3. To further elucidate their role, two-dimensionally (2D) and three-dimensionally (3D) cultured 3T3-L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis-related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3-L1 spheroids. In the 2D cultured 3T3-L1 cells, mRNA expression levels of adipogenesis-related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF-β isoforms, with TGF-β2 having the greater effects. Consistent with these results, treatment with TGF-β2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3-L1 cells. However, the inhibitory effect of TGF-β on adipogenesis decreased under 3D spheroid culture conditions and TGF-β isoforms did not affect adipogenesis-induced (a) enlargement and downsizing of 3T3-L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up-regulation of the mRNA expression of adipogenesis-related genes. The findings presented herein suggest that the three TGF-β isoforms have different suppressive effects on adipogenesis-related cellular properties of 2D cultured 3T3-L1 cells and that their effects decrease under 3D spheroid culture conditions.

Unexpected and Synergistical Effects of All-Trans Retinoic Acid and TGF-β2 on Biological Aspects of 2D and 3D Cultured ARPE19 Cells.

Megumi Higashide, Megumi Watanabe, Tatsuya Sato, Toshifumi Ogawa, Araya Umetsu, Soma Suzuki, Masato Furuhashi, Hiroshi Ohguro, Nami Nishikiori

Biomedicines 12 ( 10 ) 2024年09月 [査読有り] [国際誌]

Objectives: To study the effects of all-trans retinoic acid (ATRA) on TGF-β2-induced effects of human retinal pigment epithelium cells under normoxia and hypoxia conditions. Methods: Two-dimensionally (2D) and three-dimensionally (3D) cultured ARPE19 cells were subjected to cellular functional analyses by transepithelial electrical resistance (TEER) and an extracellular flux assay (2D), measurement of levels of reactive oxygen species (ROS), gene expression analyses of COL1, αSMA, Zo-1, HIF1α, and PGC1α (2D), and physical property analyses (3D). Results: Under a normoxia condition, treatment with 100 nM ATRA substantially decreased barrier function regardless of the presence of 5 ng/mL TGF-β2 in 2D ARPE19 monolayer cells. Under a hypoxia condition, treatment with ATRA conversely increased barrier function, but the effect was masked by a marked increase in effects induced by TGF-β2. Although ATRA alone did not affect cellular metabolism and ROS levels in 2D ARPE cells, treatment with ATRA under a hypoxia condition did not affect ROS levels but shifted cellular metabolism from mitochondrial respiration to glycolysis. The changes of cellular metabolism and ROS levels were more pronounced with treatment of both ATRA and TGF-β2 independently of oxygen conditions. Changes in mRNA expressions of some of the above genes suggested the involvement of synergistical regulation of cellular functions by TGF-β2 and hypoxia. In 3D ARPE spheroids, the size was decreased and the stiffness was increased by either treatment with TGF-β2 or ATRA, but these changes were unexpectedly modulated by both ATRA and TGF-β2 treatment regardless of oxygen conditions. Conclusions: The findings reported herein indicate that TGF-β2 and hypoxia synergistically and differentially induce effects in 2D and 3D cultured ARPE19 cells and that their cellular properties are significantly altered by the presence of ATRA.

Application of Single Cell Type-Derived Spheroids Generated by Using a Hanging Drop Culture Technique in Various In Vitro Disease Models: A Narrow Review.

Hiroshi Ohguro, Megumi Watanabe, Tatsuya Sato, Nami Nishikiori, Araya Umetsu, Megumi Higashide, Toshiyuki Yano, Hiromu Suzuki, Akihiro Miyazaki, Kohichi Takada, Hisashi Uhara, Masato Furuhashi, Fumihito Hikage

Cells 13 ( 18 ) 2024年09月 [査読有り] [国際誌]

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases.

Contribution of MLKL to the development of doxorubicin-induced cardiomyopathy and its amelioration by rapamycin.

Masaki Shimizu, Wataru Ohwada, Toshiyuki Yano, Hidemichi Kouzu, Tatsuya Sato, Toshifumi Ogawa, Arata Osanami, Yuki Toda, Hiroshi Nagahama, Masaya Tanno, Tetsuji Miura, Atsushi Kuno, Masato Furuhashi

Journal of pharmacological sciences 156 ( 1 ) 9 - 18 2024年09月 [査読有り] [国内誌]

Necroptosis, necrosis characterized by RIPK3-MLKL activation, has been proposed as a mechanism of doxorubicin (DOX)-induced cardiomyopathy. We showed that rapamycin, an mTORC1 inhibitor, attenuates cardiomyocyte necroptosis. Here we examined role of MLKL in DOX-induced myocardial damage and protective effects of rapamycin. Cardiomyopathy was induced in mice by intraperitoneal injections of DOX (10 mg/kg, every other day) and followed for 7 days. DOX-treated mice showed a significant decline in LVEF assessed by cardiac MRI (45.5 ± 5.1% vs. 65.4 ± 4.2%), reduction in overall survival rates, and increases in myocardial RIPK3 and MLKL expression compared with those in vehicle-treated mice, and those changes were prevented by administration of rapamycin (0.25 mg/kg) before DOX injection. In immunohistochemical analyses, p-MLKL signals were detected in the cardiomyocytes of DOX-treated mice, and the signals were reduced by rapamycin. Mlkl+/- and Mlkl-/- mice were similarly resistant to DOX-induced cardiac dysfunction, indicating that a modest reduction in MLKL level is sufficient to prevent the development of DOX-induced cardiomyopathy. However, evidence of cardiomyocyte necrosis assessed by C9 immunostaining, presence of replacement fibrosis, and electron microscopic analyses was negligible in the myocardium of DOX-treated mice. Thus, MLKL-mediated signaling contributes to DOX-induced cardiac dysfunction primarily by a necrosis-independent mechanism, which is inhibitable by rapamycin.

FABP5 Is a Possible Factor for the Maintenance of Functions of Human Non-Pigmented Ciliary Epithelium Cells.

Megumi Higashide, Megumi Watanabe, Tatsuya Sato, Araya Umetsu, Nami Nishikiori, Toshifumi Ogawa, Masato Furuhashi, Hiroshi Ohguro

International journal of molecular sciences 25 ( 17 ) 2024年08月 [査読有り] [国際誌]

To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6. IPA analysis of these DEGs suggested that FABP5 may be involved in essential roles required for cell development, cell survival and cell homeostasis. In support of this possibility, both mitochondrial and glycolytic functions of HNPCE cells, in which mRNA expression of FABP5, but not that of FABP7, was detected, were shown by using a Seahorse XFe96 Bioanalyzer to be dramatically suppressed by FABP ligand 6-induced inhibition of the activity of FABP5. Furthermore, in IPA upstream analysis, various unfolded protein response (UPR)-related factors were identified as upstream and causal network master regulators. Analysis by qPCR analysis showed significant upregulation of the mRNA expression of most of UPR-related factors and aquaporin1 (AQP1). The findings in this study suggest that HNPCE is one of intraocular cells producing FABP5 and may be involved in the maintenance of UPR and AQP1-related functions of HNPCE.

Straightforward and immediate ultrasound-guided kidney biopsy using a guide needle technique to get adequate tissue with reduced procedural time.

Hiroaki Komatsu, Tomohisa Yamashita, Arata Osanami, Chikako Akazawa, Kota Endo, Shun Tsugawa, Ayumu Kimura, Daisuke Miyamori, Koki Abe, Satoko Takahashi, Yufu Gocho, Masayuki Koyama, Tatsuya Sato, Marenao Tanaka, Norihito Moniwa, Masato Furuhashi

Clinical and experimental nephrology 2024年08月 [査読有り] [国内誌]

BACKGROUND: A streamlined and effective renal biopsy technique is essential for all nephrologists, particularly those who are less experienced, such as residents. Herein, we report the efficacy of a Straightforward and Immediate ultrasound-guided kidney biopsy using a Guide Needle (SIGN) technique, which allows operators to insert a biopsy gun through a guide needle placed into the fascia of the posterior abdominal wall. METHODS: A retrospective cross-sectional study was conducted at a nephrology training institution to compare the time spent on the procedure and the number of glomeruli obtained between a group using the SIGN (n = 81) and a group using the conventional ultrasound-guided kidney biopsy technique with a needle guide device (n = 143). RESULTS: The median procedure time in the SIGN group (2 min, interquartile range [IQR]: 1-3 min) was significantly shorter than that in the conventional group (3 min, IQR: 2-4 min) (P < 0.001). Multivariable linear regression and logistic regression analyses adjusted for covariates, including operators (board-certificated nephrologists or nephrology residents), showed that the use of the SIGN technique was independently associated with a high number of glomeruli obtained and a procedure time above 2 min as the median value (odds ratio: 0.17, 95% confidence interval CI 0.09-0.34). The prevalence of complications was comparable between the two groups (P = 0.681). CONCLUSION: The SIGN technique reduces the procedure time and obtains adequate biopsy tissue regardless of the operator's experience. SIGN can be applied in nephrology training programs and used as a standard biopsy technique.

Intraocular fatty acids induce reinforcement of barrier functions on the outer blood-retinal barrier.

Nami Nishikiori, Megumi Watanabe, Tatsuya Sato, Araya Umetsu, Megumi Higashide, Masato Furuhashi, Hiroshi Ohguro

Prostaglandins, leukotrienes, and essential fatty acids 202 102637 - 102637 2024年08月 [査読有り] [国際誌]

The aim of the present study was to elucidate unknown effects of intraocular fatty acids (ioFAs) including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), arachidonic acid (C20:4), eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6) on the outer blood-retinal barrier (oBRB). For this purpose, human retinal pigment epithelium cell line ARPE19 was subjected to analyses for evaluating the following biological phenotypes: (1) cell viability, (2) cellular metabolic functions, (3) barrier functions by trans-epithelial electrical resistance (TEER), and (4) expression of tight junction (TJ) molecules. In the presence of 100 nM ioFAs, no significant effects on cell viability of ARPE19 cells was observed. While treatment with EPA or DHA tended to reduce non-mitochondrial oxygen consumption, most indices in mitochondrial functions were not markedly affected by treatment with ioFAs in ARPE19 cells. On the other hand, ioFAs except for palmitic acid and stearic acid significantly increased basal extracellular acidification rates, suggesting activated glycolysis or increased lactate production. Interestingly, TEER values of planar ARPE19 monolayer were significantly increased by treatment any ioFAs. Consistently, gene expression levels of TJ proteins were increased by treatment with ioFAs. Collectively, the findings presented herein suggest that ioFAs may contribute to reinforcement of barrier functions of the oBRB albeit there are some differences in biological effects depending on the type of ioFAs.

Metabolic dysfunction-associated steatotic liver disease (SLD) and alcohol-associated liver disease, but not SLD without metabolic dysfunction, are independently associated with new onset of chronic kidney disease during a 10-year follow-up period.

Kazuma Mori, Marenao Tanaka, Tatsuya Sato, Yukinori Akiyama, Keisuke Endo, Toshifumi Ogawa, Toru Suzuki, Hiroki Aida, Wataru Kawaharata, Kei Nakata, Itaru Hosaka, Araya Umetsu, Nagisa Hanawa, Masato Furuhashi

Hepatology research : the official journal of the Japan Society of Hepatology 2024年08月 [査読有り] [国際誌]

AIMS: The new nomenclature of steatotic liver disease (SLD) including metabolic dysfunction-associated SLD (MASLD), MASLD and increased alcohol intake (MetALD), and alcohol-associated liver disease (ALD) has recently been proposed. We aimed to elucidate the relationship between each category of SLD and chronic kidney disease (CKD). METHODS: We investigated the effects of various SLDs on the development of CKD, defined as estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 or positive for urinary protein, during a 10-year period in 12 138 Japanese subjects (men / women, 7984/4154; mean age, 48 years) who received annual health examinations including abdominal ultrasonography. RESULTS: The prevalences of SLD without metabolic dysfunction (SLD-MD[-]), MASLD, MetALD, and ALD were 1.7%, 26.3%, 4.9%, and 1.9%, respectively. During the follow-up period, 1963 subjects (16.2%) (men / women, 1374 [17.2%]/589 [14.2%]) had new onset of CKD. Multivariable Cox proportional hazard model analyses after adjustment of age, sex, eGFR, current smoking habit, diabetes mellitus, hypertension, and dyslipidemia showed that the hazard ratios (HR [95% confidence interval]) for the development of CKD in subjects with MASLD (1.20 [1.08-1.33], p = 0.001) and those with ALD (1.41 [1.05-1.88], p = 0.022), but not those with MetALD (1.11 [0.90-1.36], p = 0.332), were significantly higher than the HR in subjects with non-SLD. Interestingly, subjects with SLD-MD[-] had a significantly lower HR (0.61 [0.39-0.96], p = 0.034) than that in subjects with non-SLD. The addition of the novel classification of SLDs into traditional risk factors for the development of CKD significantly improved the discriminatory capacity. CONCLUSIONS: MASLD and ALD, but not SLD-MD[-], are independently associated with the development of CKD.

Associations between circulating levels of FABP4 and TNF receptors are more evident in patients with type 2 diabetes mellitus than in patients with type 1 diabetes mellitus.

Marenao Tanaka, Tomohito Gohda, Nozomu Kamei, Maki Murakoshi, Tatsuya Sato, Mitsunobu Kubota, Michiyoshi Sanuki, Erika Ishiwata, Keisuke Endo, Yusuke Suzuki, Masato Furuhashi

Endocrine connections 2024年08月 [査読有り] [国際誌]

BACKGROUND: Fatty acid-binding protein 4 (FABP4) is an adipokine that plays significant roles in the development of insulin resistance and atherosclerosis. High levels of soluble tumor necrosis factor receptors (TNFRs) including TNFR1 and TNFR2 are associated with renal dysfunction and increased mortality in patients with diabetes mellitus (DM). However, the association between circulating levels of FABP4 and TNFRs remains unclear. METHODS: We investigated the associations of FABP4 with TNFRs and metabolic markers in Japanese patients with type 1 DM (T1DM, n=76, men/women: 31/45) and type 2 DM (T2DM, n=575, men/women: 312/263). RESULTS: FABP4 concentration was positively correlated with levels of TNFR1 and TNFR2 in both patients with T1DM and those with T2DM. Multivariable regression analyses showed that there were independent associations of FABP4 concentration with body mass index (BMI) and estimated glomerular filtration rate (eGFR) after adjustment of age and sex in both patients with T1DM and those with T2DM. FABP4 concentration was independently associated with circulating levels of TNFR1 and TNFR2 after adjustment of the confounders in patients with T2DM but not in those with T1DM. Similarly, levels of TNFR1 and TNFR2 were independently associated with FABP4 concentration after adjustment of age, sex, systolic blood pressure, duration of DM and levels of eGFR, high-density lipoprotein cholesterol and C-reactive protein in patients with T2DM but not in those with T1DM. CONCLUSION: FABP4 concentration is independently associated with levels of TNFRs in patients with DM, but the association is more evident in patients with T2DM than in those with T1DM.

Fatty Acid-Binding Protein 4-Mediated Regulation Is Pivotally Involved in Retinal Pathophysiology: A Review.

Hiroshi Ohguro, Megumi Watanabe, Fumihito Hikage, Tatsuya Sato, Nami Nishikiori, Araya Umetsu, Megumi Higashide, Toshifumi Ogawa, Masato Furuhashi

International journal of molecular sciences 25 ( 14 ) 2024年07月 [査読有り] [国際誌]

Fatty acid-binding proteins (FABPs), a family of lipid chaperone molecules that are involved in intracellular lipid transportation to specific cellular compartments, stimulate lipid-associated responses such as biological signaling, membrane synthesis, transcriptional regulation, and lipid synthesis. Previous studies have shown that FABP4, a member of this family of proteins that are expressed in adipocytes and macrophages, plays pivotal roles in the pathogenesis of various cardiovascular and metabolic diseases, including diabetes mellitus (DM) and hypertension (HT). Since significant increases in the serum levels of FABP4 were detected in those patients, FABP4 has been identified as a crucial biomarker for these systemic diseases. In addition, in the field of ophthalmology, our group found that intraocular levels of FABP4 (ioFABP4) and free fatty acids (ioFFA) were substantially elevated in patients with retinal vascular diseases (RVDs) including proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO), for which DM and HT are also recognized as significant risk factors. Recent studies have also revealed that ioFABP4 plays important roles in both retinal physiology and pathogenesis, and the results of these studies have suggested potential molecular targets for retinal diseases that might lead to future new therapeutic strategies.

Influence of the COVID-19 pandemic on the achievement of guideline targets for HbA1c, blood pressure, and LDL cholesterol in people with diabetes in Japan

Shingo Kuwajima, Takahito Itoh, Tatsuya Sato, Shoya Ino, Satoru Shibata, Kouhei Ohno, Hiroyuki Hotta, Tomoaki Matsumoto, Hitoshi Ooiwa, Hirofumi Kubo, Takayuki Miki

Diabetology International 15 ( 3 ) 507 - 517 2024年07月 [査読有り] [国内誌]

Objective: In this study, we investigated whether the COVID-19 pandemic affected achievement of guideline targets for HbA1c, blood pressure (BP), and low-density lipoprotein (LDL) cholesterol in people with diabetes mellitus (DM). Materials and methods: Data for 556 people with DM who were treated regularly for 4 years before and during the COVID-19 pandemic in Japan were analyzed in this retrospective study. Achieved targets were defined as HbA1c < 7.0%, BP < 130/80 mmHg, and LDL cholesterol < 100 or < 120 mg/dL depending on the presence or absence of coronary artery disease. Results: In 2019, before the start of the COVID-19 pandemic, achievement rates of guideline targets for HbA1c, BP and LDL cholesterol were 53.4%, 45.9% and 75.7%, respectively. In 2020, the achievement rates for HbA1c and BP targets were significantly decreased to 40.8% and 31.3%, respectively. The achievement rates for the HbA1c target gradually recovered to 49.3% in 2021 and to 51.1% in 2022. However, recovery in achieving the BP target was slow, remaining at 40.5% even in 2022. On the other hand, the achievement rate for the LDL cholesterol target was not affected and remained relatively high during the COVID-19 pandemic. Conclusions: The rates of achieving therapeutic targets for HbA1c and BP have not been high enough in people with DM, and the rates were further reduced by lifestyle changes due to the COVID-19 pandemic. Although there has been a trend toward improvement with the lifting of behavioral restrictions, more intensified treatment is necessary to achieve good control.

Lysophosphatidic Acid Modulates TGF-β2-Induced Biological Phenotype in Human Conjunctival Fibroblasts.

Megumi Watanabe, Yuri Tsugeno, Tatsuya Sato, Megumi Higashide, Nami Nishikiori, Araya Umetsu, Toshifumi Ogawa, Masato Furuhashi, Hiroshi Ohguro

Life (Basel, Switzerland) 14 ( 6 ) 2024年06月 [査読有り] [国際誌]

BACKGROUND: Although lysophosphatidic acid (LPA) is known to have multiple pathophysiological roles, its contributions to ocular tissues, especially conjunctival fibrogenesis, remain to be elucidated. METHODS: To study this issue, the effects of LPA on transforming growth factor-β2 (TGF-β2)-induced fibrogenesis of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblasts (HconF) were examined by the following analyses: (1) planar proliferation determined by transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran permeability measurements, (2) real-time metabolic analyses, (3) measurements of the size and stiffness of 3D spheroids, and (4) mRNA expression of extracellular matrix (ECM) molecules and their modulators. RESULTS: LPA had no effect on TGF-β2-induced increase in the planar proliferation of HconF cells. LPA induced a more quiescent metabolic state in 2D HconF cells, but this metabolic suppression by LPA was partially blunted in the presence of TGF-β2. In contrast, LPA caused a substantial decrease in the hardness of 3D HconF spheroids independently of TGF-β2. In agreement with these different LPA-induced effects between 2D and 3D cultured HconF cells, mRNA expressions of ECM and their modulators were differently modulated. CONCLUSION: The findings that LPA induced the inhibition of both TGF-β2-related and -unrelated subepithelial proliferation of HconF cells may be clinically applicable.

TGF-β Isoforms and Local Environments Greatly Modulate Biological Nature of Human Retinal Pigment Epithelium Cells.

Nami Nishikiori, Tatsuya Sato, Toshifumi Ogawa, Megumi Higashide, Araya Umetsu, Soma Suzuki, Masato Furuhashi, Hiroshi Ohguro, Megumi Watanabe

Bioengineering (Basel, Switzerland) 11 ( 6 ) 2024年06月 [査読有り] [国際誌]

To characterize transforming growth factor-β (TGF-β) isoform (TGF-β1~3)-b's biological effects on the human retinal pigment epithelium (RPE) under normoxia and hypoxia conditions, ARPE19 cells cultured by 2D (two-dimensional) and 3D (three-dimensional) conditions were subjected to various analyses, including (1) an analysis of barrier function by trans-epithelial electrical resistance (TEER) measurements; (2) qPCR analysis of major ECM molecules including collagen 1 (COL1), COL4, and COL6; α-smooth muscle actin (αSMA); hypoxia-inducible factor 1α (HIF1α); and peroxisome proliferator-activated receptor-gamma coactivator (PGC1α), a master regulator for mitochondrial respiration;, tight junction-related molecules, Zonula occludens-1 (ZO1) and E-cadherin; and vascular endothelial growth factor (VEGF); (3) physical property measurements of 3D spheroids; and (4) cellular metabolic analysis. Diverse effects among TGF-β isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-β1 and TGF-β3 caused a marked increase in TEER values, and TGF-β2 caused a substantial increase in TEER values under normoxia conditions and hypoxia conditions, respectively; (2) the results of qPCR analysis supported data obtained by TEER; (3) 3D spheroid sizes were decreased by TGF-β isoforms, among which TGF-β1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-β2 and TGF-β3 or by TGF-β1 and TGF-β3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-β isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-β-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-β isoforms and oxygen levels, suggesting that pathological conditions including epithelial-mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.

The Correlation between the Severity of Obstructive Sleep Apnea and Insulin Resistance in a Japanese Population.

Yukako Tomo, Ryo Naito, Yasuhiro Tomita, Satoshi Kasagi, Tatsuya Sato, Takatoshi Kasai

Journal of clinical medicine 13 ( 11 ) 2024年05月 [査読有り] [国際誌]

Background: Repetitive episodes of apnea and hypopnea during sleep in patients with obstructive sleep apnea (OSA) are known to increase the risk of atherosclerosis. Underlying obesity and related disorders, such as insulin resistance, are indirectly related to the development of atherosclerosis. In addition, OSA is independently associated with insulin resistance; however, data regarding this relationship are scarce in Japanese populations. Methods: This study aimed to examine the relationship between the severity of OSA and insulin resistance in a Japanese population. We analyzed the data of consecutive patients who were referred for polysomnography under clinical suspicion of developing OSA and who did not have diabetes mellitus or any cardiovascular disease. Multiple regression analyses were performed to determine the relationship between the severity of OSA and insulin resistance. Results: The data from a total of 483 consecutive patients were analyzed. The median apnea-hypopnea index (AHI) was 40.9/h (interquartile range: 26.5, 59.1) and the median homeostasis model assessment for insulin resistance (HOMA-IR) was 2.00 (interquartile range: 1.25, 3.50). Multiple regression analyses revealed that the AHI, the lowest oxyhemoglobin saturation (SO2), and the percentage of time spent on SO2 < 90% were independently correlated with HOMA-IR (an adjusted R-squared value of 0.01278821, p = 0.014; an adjusted R-squared value of -0.01481952, p = 0.009; and an adjusted R-squared value of 0.018456581, p = 0.003, respectively). Conclusions: The severity of OSA is associated with insulin resistance assessed by HOMA-IR in a Japanese population.

Modulation of Epithelial-Mesenchymal Transition Is a Possible Underlying Mechanism for Inducing Chemoresistance in MIA PaCa-2 Cells against Gemcitabine and Paclitaxel.

Hajime Nakamura, Megumi Watanabe, Kohichi Takada, Tatsuya Sato, Fumihito Hikage, Araya Umetsu, Joji Muramatsu, Masato Furuhashi, Hiroshi Ohguro

Biomedicines 12 ( 5 ) 2024年05月 [査読有り] [国際誌]

To elucidate the currently unknown molecular mechanisms responsible for the similarity and difference during the acquirement of resistance against gemcitabine (GEM) and paclitaxel (PTX) in patients with pancreatic carcinoma, we examined two-dimensional (2D) and three-dimensional (3D) cultures of parent MIA PaCa-2 cells (MIA PaCa-2-PA) and their GEM resistance cell line (MIA PaCa-2-GR) and PTX resistance (MIA PaCa-2-PR). Using these cells, we examined 3D spheroid configurations and cellular metabolism, including mitochondrial and glycolytic functions, with a Seahorse bio-analyzer and RNA sequencing analysis. Compared to the MIA PaCa-2-PA, (1) the formation of the 3D spheroids of MIA PaCa-2-GR or -PR was much slower, and (2) their mitochondrial and glycolytic functions were greatly modulated in MIA PaCa-2-GR or -PR, and such metabolic changes were also different between their 2D and 3D culture conditions. RNA sequencing and bioinformatic analyses of the differentially expressed genes (DEGs) using an ingenuity pathway analysis (IPA) suggested that various modulatory factors related to epithelial -mesenchymal transition (EMT) including STAT3, GLI1, ZNF367, NKX3-2, ZIC2, IFIT2, HEY1 and FBLX, may be the possible upstream regulators and/or causal network master regulators responsible for the acquirement of drug resistance in MIA PaCa-2-GR and -PR. In addition, among the prominently altered DEGs (Log2 fold changes more than 6 or less than -6), FABP5, IQSEC3, and GASK1B were identified as unique genes associated with their antisense RNA or pseudogenes, and among these, FABP5 and GASK1B are known to function as modulators of cancerous EMT. Therefore, the observations reported herein suggest that modulations of cancerous EMT may be key molecular mechanisms that are responsible for inducing chemoresistance against GEM or PTX in MIA PaCa-2 cells.

Deciphering metabolic dysfunction-associated steatotic liver disease: insights from predictive modeling and clustering analysis.

Kazuma Mori, Yukinori Akiyama, Marenao Tanaka, Tatsuya Sato, Keisuke Endo, Itaru Hosaka, Nagisa Hanawa, Naoya Sakamoto, Masato Furuhashi

Journal of gastroenterology and hepatology 2024年04月 [査読有り] [国際誌]

BACKGROUND AND AIM: New nomenclature of steatotic liver disease (SLD) including metabolic dysfunction-associated SLD (MASLD), MASLD and increased alcohol intake (MetALD), and alcohol-associated liver disease (ALD) has recently been proposed. We investigated clustering analyses to decipher the complex landscape of SLD pathologies including the former nomenclature of nonalcoholic fatty liver disease (NAFLD) and metabolic dysfunction-associated fatty liver disease (MAFLD). METHODS: Japanese individuals who received annual health checkups including abdominal ultrasonography (n = 15 788, men/women: 10 250/5538, mean age: 49 years) were recruited. RESULTS: The numbers of individuals with SLD, MASLD, MetALD, ALD, NAFLD, and MAFLD were 5603 (35.5%), 4227 (26.8%), 795 (5.0%), 324 (2.1%), 3982 (25.8%), and 4946 (31.3%), respectively. Clustering analyses using t-distributed stochastic neighbor embedding and K-means to visually represent interconnections in SLDs uncovered five cluster formations. MASLD and NAFLD mainly shared three clusters including (i) low alcohol intake with relatively low-grade obesity; (ii) obesity with dyslipidemia; and (iii) dysfunction of glucose metabolism. Both MetALD and ALD displayed one distinct cluster intertwined with alcohol consumption. MAFLD widely shared all of the five clusters. In machine learning-based analyses using algorithms of random forest and extreme gradient boosting and receiver operating characteristic curve analyses, fatty liver index (FLI), calculated by body mass index, waist circumference, and levels of γ-glutamyl transferase and triglycerides, was selected as a useful feature for SLDs. CONCLUSIONS: The new nomenclature of SLDs is useful for obtaining a better understanding of liver pathologies and for providing valuable insights into predictive factors and the dynamic interplay of diseases. FLI may be a noninvasive predictive marker for detection of SLDs.

Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-β2 or Dexamethasone.

Megumi Watanabe, Tatsuya Sato, Araya Umetsu, Nami Nishikiori, Megumi Higashide, Masato Furuhashi, Hiroshi Ohguro

Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics 2024年03月 [査読有り] [国際誌]

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-β2 or 250 nM dexamethasone (DEX). Methods: Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

Downregulation of Mitochondrial Fusion Protein Expression Affords Protection from Canonical Necroptosis in H9c2 Cardiomyoblasts

Yuki Toda, SANG-BING ONG, Toshiyuki Yano, Atsushi Kuno, Hidemichi Kouzu, Tatsuya Sato, Wataru Ohwada, Yuki Tatekoshi, Toshifumi Ogawa, Masaki Shimizu, Masaya Tanno, Masato Furuhashi

International Journal of Molecular Sciences 2024年03月 [査読有り]

Bob1 maintains T follicular helper cells for long-term humoral immunity

Masahiro Yanagi, Ippei Ikegami, Ryuta Kamekura, Tatsuya Sato, Taiki Sato, Shiori Kamiya, Kosuke Murayama, Sumito Jitsukawa, Fumie Ito, Akira Yorozu, Miho Kihara, Takaya Abe, Hiromi Takaki, Koji Kawata, Katsunori Shigehara, Satsuki Miyajima, Hirotaka Nishikiori, Akinori Sato, Noritsugu Tohse, Ken-ichi Takano, Hirofumi Chiba, Shingo Ichimiya

Communications Biology 7 ( 185 ) 2024年02月 [査読有り]

Platelet-rich plasma does not accelerate the healing of damaged muscle following muscle strain.

Hiroyori Fusagawa, Takashi Yamada, Tatsuya Sato, Yuki Ashida, Atsushi Teramoto, Hiroyuki Takashima, Azuma Naito, Nao Tokuda, Nao Yamauchi, Nobutoshi Ichise, Izaya Ogon, Toshihiko Yamashita, Noritsugu Tohse

Journal of orthopaedic research 2024年01月 [査読有り] [国際誌]

Although platelet-rich plasma (PRP) has been widely used regardless of the severity of muscle strain, there have been very few basic studies in which its effects on muscle injury were examined by using models that accurately mimic the clinical muscle strain injury process. Therefore, the aim of this study was to confirm by physiological and structural analyses whether PRP purified by a general preparation method has a muscle healing effect on muscle damage caused by eccentric contraction (ECC). Male Wistar rats were subjected to muscle injury induced by ECC in bilateral plantar flexor muscles using electrical stimulation and an automatically dorsiflexing footplate. The rats were randomly assigned to three groups by type of injection: phosphate-buffered saline (PBS), leukocyte-poor PRP (LP-PRP), or leukocyte-rich PRP (LR-PRP) injection into gastrocnemius muscles three times at weekly intervals. The platelet concentrations of the LP-PRP and LR-PRP were three to five times higher than that of whole blood. The recovery process of torque strength in the plantar flexor muscle, signal changes in MRI images, and histological evaluation 3 weeks after injury showed no obvious differences among the three groups, and every muscle recovered well from the injury without marked fibrosis. The results that neither LP-PRP nor LR-PRP was found to accelerate healing of muscle injuries suggested that conventional preparation and use of PRP for simple muscle injuries caused by muscle strain should be carefully considered, and further basic research using models that accurately mimic clinical practice should be carried out to determine the optimal use of PRP.

Significant and Various Effects of ML329-Induced MITF Suppression in the Melanoma Cell Line.

Nami Nishikiori, Megumi Watanabe, Tatsuya Sato, Masato Furuhashi, Masae Okura, Tokimasa Hida, Hisashi Uhara, Hiroshi Ohguro

Cancers 16 ( 2 ) 2024年01月 [査読有り] [国際誌]

To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 μM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, including STAT3 as a possible regulator of 3D spheroid formation, KRAS and SOX2 as oncogenic-signaling-related factors, PCG1a as the main regulator of mitochondrial biogenesis, and HIF1a as a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM.

High-intensity interval training using electrical stimulation ameliorates muscle fatigue in chronic kidney disease-related cachexia by restoring mitochondrial respiratory dysfunction.

Hiroyori Fusagawa, Tatsuya Sato, Takashi Yamada, Azuma Naito, Nao Tokuda, Nao Yamauchi, Nobutoshi Ichise, Toshifumi Ogawa, Takuro Karaushi, Atsushi Teramoto, Noritsugu Tohse

Frontiers in physiology 15 1423504 - 1423504 2024年 [査読有り] [国際誌]

BACKGROUND: Exercise, especially high-intensity interval training (HIIT), can increase mitochondrial respiratory capacity and enhance muscular endurance, but its systemic burden makes it difficult to safely and continuously prescribe for patients with chronic kidney disease (CKD)-related cachexia who are in poor general condition. In this study, we examined whether HIIT using electrical stimulation (ES), which does not require whole-body exercise, improves muscle endurance in the skeletal muscle of 5/6 nephrectomized rats, a widely used animal model for CKD-related cachexia. METHODS: Male Wistar rats (10 weeks old) were randomly assigned to a group of sham-operated (Sham) rats and a group of 5/6 nephrectomy (Nx) rats. HIIT was performed on plantar flexor muscles in vivo with supramaximal ES every other day for 4 weeks to assess muscle endurance, myosin heavy-chain isoforms, and mitochondrial respiratory function in Nx rats. A single session was also performed to identify upstream signaling pathways altered by HIIT using ES. RESULTS: In the non-trained plantar flexor muscles from Nx rats, the muscle endurance was significantly lower than that in plantar flexor muscles from Sham rats. The proportion of myosin heavy chain IIa/x, mitochondrial content, mitochondrial respiratory capacity, and formation of mitochondrial respiratory supercomplexes in the plantaris muscle were also significantly decreased in the non-trained plantar flexor muscles from Nx rats than compared to those in plantar flexor muscles from Sham rats. Treatment with HIIT using ES for Nx rats significantly improved these molecular and functional changes to the same degrees as those in Sham rats. Furthermore, a single session of HIIT with ES significantly increased the phosphorylation levels of AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK), pathways that are essential for mitochondrial activation signaling by exercise, in the plantar muscles of both Nx and Sham rats. CONCLUSION: The findings suggest that HIIT using ES ameliorates muscle fatigue in Nx rats via restoration of mitochondrial respiratory dysfunction with activation of AMPK and p38 MAPK signaling. Our ES-based HIIT protocol can be performed without placing a burden on the whole body and be a promising intervention that is implemented even in conditions of reduced general performance status such as CKD-related cachexia.

Lipid Metabolism Regulators Are the Possible Determinant for Characteristics of Myopic Human Scleral Stroma Fibroblasts (HSSFs).

Hiroshi Ohguro, Araya Umetsu, Tatsuya Sato, Masato Furuhashi, Megumi Watanabe

International journal of molecular sciences 25 ( 1 ) 2023年12月 [査読有り] [国際誌]

The purpose of the current investigation was to elucidate what kinds of responsible mechanisms induce elongation of the sclera in myopic eyes. To do this, two-dimensional (2D) cultures of human scleral stromal fibroblasts (HSSFs) obtained from eyes with two different axial length (AL) groups, <26 mm (low AL group, n = 2) and >27 mm (high AL group, n = 3), were subjected to (1) measurements of Seahorse mitochondrial and glycolytic indices to evaluate biological aspects and (2) analysis by RNA sequencing. Extracellular flux analysis revealed that metabolic indices related to mitochondrial and glycolytic functions were higher in the low AL group than in the high AL group, suggesting that metabolic activities of HSSF cells are different depending the degree of AL. Based upon RNA sequencing of these low and high AL groups, the bioinformatic analyses using gene ontology (GO) enrichment analysis and ingenuity pathway analysis (IPA) of differentially expressed genes (DEGs) identified that sterol regulatory element-binding transcription factor 2 (SREBF2) is both a possible upstream regulator and a causal network regulator. Furthermore, SREBF1, insulin-induced gene 1 (INSIG1), and insulin-like growth factor 1 (IGF1) were detected as upstream regulators, and protein tyrosine phosphatase receptor type O (PTPRO) was detected as a causal network regulator. Since those possible regulators were all pivotally involved in lipid metabolisms including fatty acid (FA), triglyceride (TG) and cholesterol (Chol) biosynthesis, the findings reported here indicate that FA, TG and Chol biosynthesis regulation may be responsible mechanisms inducing AL elongation via HSSF.

Validation of Estimated Small Dense Low-Density Lipoprotein Cholesterol Concentration in a Japanese General Population.

Keisuke Endo, Ryo Kobayashi, Makito Tanaka, Marenao Tanaka, Yukinori Akiyama, Tatsuya Sato, Itaru Hosaka, Kei Nakata, Masayuki Koyama, Hirofumi Ohnishi, Satoshi Takahashi, Masato Furuhashi

Journal of atherosclerosis and thrombosis 2023年12月 [査読有り] [国内誌]

AIM: A high level of directly measured small dense low-density lipoprotein cholesterol (sdLDL-C) is a strong risk factor for atherosclerotic cardiovascular disease. A method for estimating sdLDL-C by using Sampson's equation that includes levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), non-HDL-C and triglycerides (TG) has recently been proposed. We investigated the validation and exploration of estimated sdLDL-C level. METHODS: The associations between measured and estimated sdLDL-C levels were investigated in 605 Japanese subjects (men/women: 280/325; mean age: 65±15 years) who received annual health check-ups in the Tanno Sobetsu Study, a population-based cohort. RESULTS: Estimated sdLDL-C level was highly correlated with measured sdLDL-C level in all subjects (R2 =0.701), nondiabetic subjects without any medication (n=254, R 2=0.686) and subjects with diabetes mellitus (n=128, R2=0.721). Multivariable regression analysis showed that levels of non-HDL-C, TG and γ-glutamyl transpeptidase (γGTP) were independent predictors of measured sdLDL-C level. In a stratification of the LDL window, all of the subjects with a combination of high non-HDL-C (≥ 170 mg/dL) and high TG (≥ 150 mg/dL) had high levels of measured and estimated sdLDL-C (≥ 35 mg/dL). Furthermore, machine learning-based estimation of sdLDL-C level by artificial intelligence software, Prediction One, was substantially improved by using components of Sampson's equation (R2=0.803) and by using those components with the addition of γGTP and deletion of TC (R2=0.929). CONCLUSIONS: sdLDL-C level estimated by Sampson's equation can be used instead of measured sdLDL-C level in general practice. By building multiple machine learning models of artificial intelligence, a more accurate and practical estimation of sdLDL-C level might be possible.

Benzalkonium chloride greatly deteriorates the biological activities of human corneal stroma fibroblasts in a concentration-dependent manner.

Araya Umetsu, Yosuke Ida, Tatsuya Sato, Masato Furuhashi, Hiroshi Ohguro, Megumi Watanabe

Graefe's archive for clinical and experimental ophthalmology 2023年12月 [査読有り] [国際誌]

BACKGROUND: Corneal tissues indirectly obtain nutritional needs and oxygen to maintain their homeostasis, and therefore, benzalkonium chloride (BAC) containing ocular instillations for medical therapy may, in turn, induce toxic effects more than expected in corneal tissues, especially the inside stroma layer. METHODS: To evaluate the effects of very low concentrations (10-8%, 10-6%, or 10-4%) of BAC on human corneal stroma, we used two-dimensional (2D) cultures of human corneal stromal fibroblast (HCSF) cells and carried out the following analyses: (1) cell viability measurements, (2) Seahorse cellular bio-metabolism analysis, and (3) the expression of ECM molecules and endoplasmic reticulum (ER) stress-related molecules. RESULTS: In the absence and presence of 10-8%, 10-6%, or 10-4% concentrations of BAC, cell viability deteriorated and this deterioration was dose-dependent. The results showed that maximal mitochondrial respiration was decreased, the mRNA expression of most of ECM proteins was decreased, and ER stress-related molecules were substantially and dose-dependently down-regulated in HCSFs by the BAC treatment. CONCLUSIONS: The findings reported herein indicate that the presence of BAC, even at such low concentrations, is capable of causing the deterioration of cellular metabolic functions and negatively affecting the response to ER stress in HCSF cells resulting in a substantially decreased cellular viability.

Systemic sarcoidosis presenting as a rare combination of interstitial nephritis with necrotizing vasculitis and urinary retention due to prostate involvement: a case report.

Arata Osanami, Tomohisa Yamashita, Shintaro Sakurada, Tatsuya Sato, Yuki Kyoda, Tetsuya Shindo, Hiromi Fujita, Yayoi Ogawa, Masato Furuhashi

BMC nephrology 24 ( 1 ) 370 - 370 2023年12月 [査読有り] [国際誌]

BACKGROUND: Sarcoidosis affects multiple organs and exhibits diverse clinical manifestations. Although tubulointerstitial nephritis is a known feature of renal involvement, necrotizing vasculitis is rare. Furthermore, prostate involvement with urinary retention is unusual in patients with sarcoidosis. Here, we report a case of systemic sarcoidosis with a rare combination of manifestations and different acute kidney injuries. CASE PRESENTATION: A 66-year-old man developed sudden urinary retention and fever. He was diagnosed with prostatitis and admitted to our hospital. An indwelling urethral catheter was inserted, and antimicrobial therapy was initiated; however, the prostatitis was refractory. Computed tomography revealed enlarged mediastinal lymph nodes. Analysis of transbronchoscopic lymph node and prostate biopsies showed epithelioid cell granulomas, suggesting systemic sarcoidosis. During the clinical course, the serum creatinine level rapidly increased to 2.36 mg/dL without oliguria. A kidney biopsy revealed tubulointerstitial injury with moderate lymphohistiocytic infiltration and small-vessel vasculitis in the interstitium. Following oral administration of 60 mg/day prednisolone, the patient's renal function immediately improved, and urinary retention did not recur. CONCLUSIONS: To the best of our knowledge, this is the first reported case of sarcoidosis with two unusual complications. Given its clinical course and pathology, this case is clinically valuable.

Relationship between serum iron level and physical function in heart failure patients is lost by presence of diabetes.

Katsuhiko Ohori, Toshiyuki Yano, Satoshi Katano, Ryohei Nagaoka, Ryo Numazawa, Kotaro Yamano, Yusuke Fujisawa, Hidemichi Kouzu, Nobutaka Nagano, Takefumi Fujito, Ryo Nishikawa, Wataru Ohwada, Tatsuya Sato, Masato Furuhashi

ESC heart failure 2023年12月 [査読有り] [国際誌]

AIMS: Iron deficiency (ID) is common in patients with heart failure (HF) and is reportedly associated with exercise intolerance and impaired quality of life. Iron supplementation therapy in HF patients with ID improves exercise capacity. Conversely, protective roles of iron depletion in the development of diabetes mellitus (DM) and its complications have been proposed. This study aimed to determine the impact of ID on physical function in HF patients with and without DM. METHODS AND RESULTS: We enrolled consecutive patients who were admitted to our institute for HF diagnosis and management. The short physical performance battery (SPPB) was used to evaluate physical function, and low physical function was defined as an SPPB score of <10 points as individuals with SPPB scores of <10 points are most likely to be classified as frail and are at high risk for disability and future adverse events, including death. ID was defined as serum ferritin < 100 or 100-299 ng/mL when transferrin saturation (TSAT) was <20% according to the HF guidelines. Among the 562 HF patients (72 ± 14 years old; 56% male), 329 patients (58%) and 191 patients (34%) had ID and low physical function, respectively. Multivariate logistic regression analysis showed that TSAT as a continuous variable, but not ID, was a predictor of low physical function (odds ratio: 0.980, P = 0.024). Subgroup analysis showed that a significant association between low TSAT and low physical function was lost in HF patients with DM (P for interaction < 0.001). A spline dose-response curve for the relationship between TSAT and risk of low physical function with adjustments for covariates associated with low physical function in non-DM patients was almost linear with an increase in the risk of low physical function as the TSAT increased, but such a relationship was not found in the analyses of DM patients. A lack of close TSAT-SPPB relationship in HF patients with DM was confirmed also in a propensity-score-matched cohort. CONCLUSIONS: TSAT as a continuous variable, but not ID, was independently associated with physical function in HF patients, and a significant association was lost in patients with HF and DM, suggesting a limited impact of iron supplementation therapy in HF patients with DM.

Predictive modeling for the development of diabetes mellitus using key factors in various machine learning approaches

Marenao Tanaka, Yukinori Akiyama, Kazuma Mori, Itaru Hosaka, Kenichi Kato, Keisuke Endo, Toshifumi Ogawa, Tatsuya Sato, Toru Suzuki, Toshiyuki Yano, Hirofumi Ohnishi, Nagisa Hanawa, Masato Furuhashi

Diabetes Epidemiology and Management ( Elsevier BV ) 100191 - 100191 2023年12月 [査読有り]

A high level of thyroid-stimulating hormone is a risk factor for the development of chronic kidney disease in men: a 10-year cohort study.

Keisuke Endo, Marenao Tanaka, Tatsuya Sato, Kazuma Mori, Itaru Hosaka, Takuma Mikami, Araya Umetsu, Yukinori Akiyama, Hirofumi Ohnishi, Nagisa Hanawa, Masato Furuhashi

Hypertension research : official journal of the Japanese Society of Hypertension 2023年10月 [査読有り] [国際誌]

Hypothyroidism has been reported to be associated with chronic kidney disease (CKD). However, the impact of thyroid-stimulating hormone (TSH) on new onset of CKD and its gender dependence remain undetermined. We investigated the association of serum TSH level and the development of CKD defined by estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2 or positive for urine protein in 28,990 Japanese subjects who received annual health examinations. After excluding subjects with no data for serum TSH, urinalysis and eGFR and those with CKD at baseline, a total of 10,392 subjects (men/women: 6802/3590, mean age: 48 years) were recruited. During a 10-year follow-up, 1185 men (6.7%) and 578 women (2.9%) newly developed CKD. Multivariable Cox proportional hazard models after adjustment of age, body mass index, smoking habit, hypertension, diabetes mellitus, dyslipidemia, ischemic heart disease and eGFR (≥ 90 mL/min/1.73 m2) showed that the hazard ratio (HR) for the development of CKD in the high TSH (> 4.2 mU/L) group was significantly higher than that in the low TSH (≤ 4.2 mU/L) group in men (HR [95% confidence interval]: 1.41 [1.09-1.83]) but not in women (1.08 [0.77-1.51]). There was a significant interaction between sex and the category of TSH level for the development of CKD (p = 0.02). The adjusted HR with a restricted cubic spline increased with a higher level of TSH in men but not in women. In conclusion, a high level of TSH is associated with an increased risk for the development of CKD in men but not in women.

Iron drives anabolic metabolism through active histone demethylation and mTORC1.

Jason S Shapiro, Hsiang-Chun Chang, Yuki Tatekoshi, Zibo Zhao, Zohra Sattar Waxali, Bong Jin Hong, Haimei Chen, Justin A Geier, Elizabeth T Bartom, Adam De Jesus, Farnaz K Nejad, Amir Mahmoodzadeh, Tatsuya Sato, Lucia Ramos-Alonso, Antonia Maria Romero, Maria Teresa Martinez-Pastor, Shang-Chuan Jiang, Shiv K Sah-Teli, Liming Li, David Bentrem, Gary Lopaschuk, Issam Ben-Sahra, Thomas V O'Halloran, Ali Shilatifard, Sergi Puig, Joy Bergelson, Peppi Koivunen, Hossein Ardehali

Nature cell biology 25 ( 10 ) 1478 - 1494 2023年09月 [査読有り] [国際誌]

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

SIRT2 inhibition protects against cardiac hypertrophy and ischemic injury.

Xiaoyan Yang, Hsiang-Chun Chang, Yuki Tatekoshi, Amir Mahmoodzadeh, Maryam Balibegloo, Zeinab Najafi, Rongxue Wu, Chunlei Chen, Tatsuya Sato, Jason Solomon Shapiro, Hossein Ardehali

eLife 12 2023年09月 [査読有り] [国際誌]

Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of ischemic injury and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion of Sirt2 (Sirt2-/-) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO), suggesting that SIRT2 exerts maladaptive effects in the heart in response to stress. Similar results were obtained in mice with cardiomyocyte-specific Sirt2 deletion. Mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which results in reduced expression of antioxidant proteins. Deletion of Nrf2 in the hearts of Sirt2-/- mice reversed protection after PO. Finally, treatment of mouse hearts with a specific SIRT2 inhibitor reduced cardiac size and attenuates cardiac hypertrophy in response to PO. These data indicate that SIRT2 has detrimental effects in the heart and plays a role in cardiac response to injury and the progression of cardiac hypertrophy, which makes this protein a unique member of the SIRT family. Additionally, our studies provide a novel approach for treatment of cardiac hypertrophy and injury by targeting SIRT2 pharmacologically, providing a novel avenue for the treatment of these disorders.

TGF-β2 Induces Epithelial-Mesenchymal Transitions in 2D Planer and 3D Spheroids of the Human Corneal Stroma Fibroblasts in Different Manners.

Araya Umetsu, Yosuke Ida, Tatsuya Sato, Masato Furuhashi, Hiroshi Ohguro, Megumi Watanabe

Biomedicines 11 ( 9 ) 2023年09月 [査読有り] [国際誌]

To examine the epithelial-mesenchymal transition (EMT) that is induced on the human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs) were used. In this study, HCSF 2D monolayers and 3D spheroids were characterized by (1) scanning electron microscopy (SEM), (2) trans-endothelial electrical resistance (TEER) measurements and fluorescein isothiocyanate (FITC)-dextran permeability, (3) cellular metabolic measurements, (4) the physical properties of 3D HCSF spheroids, and (5) the extracellular matrix (ECM) molecule gene expressions, including collagen (COL) 1, 4 and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 3, 9 and 14, and several endoplasmic reticulum (ER) stress-related factors. In the 2D HCSFs, TGF-β2 concentration-dependently generated (1) a considerable increase in ECM deposits revealed by SEM, (2) an increase in TEER values and a decrease in FITC-dextran permeability, (3) increases in both mitochondrial and glycolytic functions, and a substantial upregulation of COL1, COL4, FN, αSMA, TIMP1, TIMP, and most ER stress-related genes and the downregulation of COL6 and MMP3. In the case of 3D spheroids, TGF-β2 induced the downsizing and stiffening of 3D spheroids and the upregulation of COL6, MMP14, and most ER stress-related genes. These findings suggest that TGF-β2 significantly induced a number of EMT-associated biological events including planar proliferation, cellular metabolic functions, and the production of ECM molecules in the 2D cultured HCSF cells, but these effects were significantly less pronounced in the case of 3D HCSF spheroids.

Circulating level of β-aminoisobutyric acid (BAIBA), a novel myokine-like molecule, is inversely associated with fat mass in patients with heart failure

Satoshi Katano, Toshiyuki Yano, Hidemichi Kouzu, Ryohei Nagaoka, Ryo Numazawa, Kotaro Yamano, Yusuke Fujisawa, Katsuhiko Ohori, Nobutaka Nagano, Takefumi Fujito, Ryo Nishikawa, Wataru Ohwada, Masaki Katayose, Tatsuya Sato, Atsushi Kuno, Masato Furuhashi

Heart and Vessels ( Springer Science and Business Media LLC ) 2023年09月 [査読有り]

Calculated Small Dense Low-Density Lipoprotein Cholesterol Level is A Predominant Predictor for New Onset of Ischemic Heart Disease.

Masafumi Inyaku, Marenao Tanaka, Tatsuya Sato, Keisuke Endo, Kazuma Mori, Itaru Hosaka, Takuma Mikami, Araya Umetsu, Hirofumi Ohnishi, Yukinori Akiyama, Nagisa Hanawa, Masato Furuhashi

Journal of atherosclerosis and thrombosis 2023年08月 [査読有り] [国内誌]

AIM: A high level of directly measured small dense low-density lipoprotein cholesterol (sdLDL-C) is a strong risk factor for ischemic heart disease (IHD). However, it remains unclear whether estimated sdLDL-C level is a predictor for IHD. We investigated the associations of new onset of IHD with levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), non-HDL-C, triglycerides (TG), LDL-C and calculated sdLDL-C by Sampson's equation. METHODS: After exclusion of subjects with IHD or those with TG ≥ 800 mg/dL, a total of 18,176 subjects (men/women: 11,712/6,464, mean age: 46 years) were recruited among 28,990 Japanese individuals who received annual health checkups. RESULTS: During the 10-year follow-up period, 456 men (3.9%) and 121 women (1.9%) newly developed IHD. Multivariable Cox proportional hazard analyses after adjustment of age, sex, obesity, smoking habit, family history of IHD, estimated glomerular filtration rate, hypertension and diabetes mellitus at baseline showed that the hazard ratio (HR) (1.38 [95% confidence interval: 1.03-1.85]) for new onset of IHD in subjects with the 4th quartile (Q4) of sdLDL-C (≥ 42 mg/dL) was significantly higher than that in subjects with the 1st quartile (Q1) (≤ 24 mg/dL) as the reference, though the adjusted HRs in subjects with Q2-Q4 of TC, HDL-C, non-HDL-C, LDL-C and TG were comparable with those in subjects with Q1 of the respective lipid fractions. The adjusted HR with a restricted cubic spline increased with a higher level of calculated sdLDL-C as a continuous value at baseline. CONCLUSIONS: sdLDL-C level calculated by Sampson's equation is a predominant predictor for the development of IHD in a general Japanese population.

Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts.

Nami Nishikiori, Kohichi Takada, Tatsuya Sato, Sho Miyamoto, Megumi Watanabe, Yui Hirakawa, Shohei Sekiguchi, Masato Furuhashi, Akira Yorozu, Kenichi Takano, Akihiro Miyazaki, Hiromu Suzuki, Hiroshi Ohguro

Cells 12 ( 17 ) 2023年08月 [査読有り] [国際誌]

The current study's objective was to elucidate some currently unknown biological indicators to evaluate the biological nature of cancer-associated fibroblasts (CAFs). For this purpose, four different CAFs, CAFS1, CAFS2, SCC17F and MO-1000, were established using surgical specimens from oral squamous cell carcinomas (OSCC) with different clinical malignant stages (CAFS1 and CAFS2, T2N0M0, stage II; SCC17F and MO-1000, T4aN2bM0, stage IVA). Fibroblasts unrelated to cancer (non-CAFs) were also prepared and used as controls. Initially, confirmation that these four fibroblasts were indeed CAFs was obtained by their mRNA expression using positive and negative markers for the CAF or fibroblasts. To elucidate possible unknown biological indicators, these fibroblasts were subjected to a cellular metabolic analysis by a Seahorse bioanalyzer, in conjugation with 3D spheroid cultures of the cells and co-cultures with a pancreas ductal carcinoma cell line, MIA PaCa-2. The mitochondrial and glycolytic functions of human orbital fibroblasts (HOF) were nearly identical to those of Graves'-disease-related HOF (GOF). In contrast, the characteristics of the metabolic functions of these four CAFs were different from those of human conjunctival fibroblasts (HconF), a representative non-CAF. It is particularly noteworthy that CAFS1 and CAFS2 showed markedly reduced ratios for the rate of oxygen consumption to the extracellular acidification rate, suggesting that glycolysis was enhanced compared to mitochondrial respiration. Similarly, the physical aspects, their appearance and stiffness, of their 3D spheroids and fibroblasts that were induced effects based on the cellular metabolic functions of MIA PaCa-2 were also different between CAFs and non-CAFs, and their levels for CAFS1 or SCC17F were similar to those for CAFS2 or MO-1000 cells, respectively. The findings reported herein indicate that cellular metabolic functions and the physical characteristics of these types of 3D spheroids may be valuable and useful indicators for estimating potential biological diversity among various CAFs.

An increase in calculated small dense low-density lipoprotein cholesterol predicts new onset of hypertension in a Japanese cohort.

Marenao Tanaka, Tatsuya Sato, Keisuke Endo, Masafumi Inyaku, Kazuma Mori, Itaru Hosaka, Takuma Mikami, Yukinori Akiyama, Hirofumi Ohnishi, Nagisa Hanawa, Masato Furuhashi

Hypertension research :official journal of the Japanese Society of Hypertension 2023年08月 [査読有り] [国際誌]

A disorder of lipid metabolism is involved in cardiovascular diseases including hypertension. A high level of small dense low-density lipoprotein cholesterol (sdLDL-C) is a strong risk factor for atherosclerotic cardiovascular disease. However, the association between sdLDL-C and hypertension has not been fully investigated. We investigated the associations between the development of hypertension during a 10-year period and levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), non-HDL-C, triglycerides (TG), and LDL-C and sdLDL-C calculated by using the Sampson equations in 28,990 Japanese subjects who received annual health examinations. After exclusion of subjects with missing data, those with hypertension, and those with TG ≥ 800 mg/dL at baseline, a total of 15,177 subjects (men/women: 9374/5803, mean age: 46 years) were recruited. During the 10-year period, 2379 men (25.4%) and 724 women (12.5%) had new onset of hypertension. Multivariable Cox proportional hazard model analyses showed that levels of HDL-C, non-HDL-C, TG and sdLDL-C, but not levels of TC and LDL-C, were independent risk factors for the development of hypertension after adjustment of age, sex, family history of hypertension, systolic blood pressure, obesity, current smoking habit, alcohol drinking habit, estimated glomerular filtration rate, diagnosis of diabetes mellitus and use of lipid-lowering drugs and that the adjusted risk of sdLDL-C (per 1-standard deviation) was highest (hazard ratio [95% confidence interval: 1.09 [1.05-1.13]). The addition of sdLDL-C to traditional risk factors for hypertension significantly improved the discriminatory capability, which was better than that of other lipid fractions. In conclusion, a high level of calculated sdLDL-C predicts the development of hypertension.

mTOR Inhibitors Modulate the Physical Properties of 3D Spheroids Derived from H9c2 Cells

Megumi Watanabe, Toshiyuki Yano, Tatsuya Sato, Araya Umetsu, Megumi Higashide, Masato Furuhashi, Hiroshi Ohguro

International Journal of Molecular Sciences 2023年07月 [査読有り]

Coexistence of Metabolic Dysfunction-Associated Fatty Liver Disease and Chronic Kidney Disease Is a More Potent Risk Factor for Ischemic Heart Disease.

Daisuke Miyamori, Marenao Tanaka, Tatsuya Sato, Keisuke Endo, Kazuma Mori, Takuma Mikami, Itaru Hosaka, Nagisa Hanawa, Hirofumi Ohnishi, Masato Furuhashi

Journal of the American Heart Association 12 ( 14 ) e030269 2023年07月 [査読有り] [国際誌]

Background Metabolic dysfunction-associated fatty liver disease (MAFLD), defined as fatty liver with overweight/obesity, type 2 diabetes, or metabolic abnormalities, is a newly proposed disease. However, it remains unclear whether the coexistence of MAFLD and chronic kidney disease (CKD) is a more potent risk factor for ischemic heart disease (IHD). Methods and Results We investigated the risk of the combination of MAFLD and CKD for development of IHD during a 10-year follow-up period in 28 990 Japanese subjects who received annual health examinations. After exclusion of subjects without data for abdominal ultrasonography or with the presence of IHD at baseline, a total of 14 141 subjects (men/women: 9195/4946; mean age, 48 years) were recruited. During the 10-year period (mean, 6.9 years), 479 subjects (men/women, 397/82) had new onset of IHD. Kaplan-Meier survival curves showed significant differences in rates of the cumulative incidence of IHD in subjects with and those without MAFLD (n=4581) and CKD (n=990; stages 1/2/3/4-5, 198/398/375/19). Multivariable Cox proportional hazard model analyses showed that coexistence of MAFLD and CKD, but not MAFLD or CKD alone, was an independent predictor for development of IHD after adjustment for age, sex, current smoking habit, family history of IHD, overweight/obesity, diabetes, hypertension, and dyslipidemia (hazard ratio, 1.51 [95% CI, 1.02-2.22]). The addition of the combination of MAFLD and CKD to traditional risk factors for IHD significantly improved the discriminatory capability. Conclusions The coexistence of MAFLD and CKD predicts new onset of IHD better than does MAFLD or CKD alone.

TGF-β Isoforms Affect the Planar and Subepithelial Fibrogenesis of Human Conjunctival Fibroblasts in Different Manners

Megumi Watanabe, Yuri Tsugeno, Tatsuya Sato, Araya Umetsu, Nami Nishikiori, Masato Furuhashi, Hiroshi Ohguro

Biomedicines 2023年07月 [査読有り]

Simultaneous Effects of a Selective EP2 Agonist, Omidenepag, and a Rho-Associated Coiled-Coil Containing Protein Kinase Inhibitor, Ripasudil, on Human Orbital Fibroblasts.

Fumihito Hikage, Megumi Watanabe, Tatsuya Sato, Araya Umetsu, Yuri Tsugeno, Masato Furuhashi, Hiroshi Ohguro

Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics 2023年06月 [査読有り] [国際誌]

Purpose: To assess the combined effects of omidenepag (OMD), a selective EP2 agonist, and ripasudil (Rip), an inhibitor of rho-associated coiled-coil containing protein kinases, on the human orbital adipose tissue, two-dimensional (2D) or three-dimensional (3D) cultures of human orbital fibroblasts (HOFs) were employed. Methods: Cellular metabolic functions (2D), physical (3D), lipid staining (3D), and quantitative polymerase chain reaction for adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) molecules, including collagen (COL)1, 4, and 6, and fibronectin (FN) (3D) were evaluated in the presence of OMD (100 nM) and/or Rip (10 μM). Results: Real-time metabolic analyses revealed that the adipogenic differentiation (DIF+) with OMD significantly shifted an energetic state toward energetic, whereas DIF+ with Rip significantly shifted that toward quiescent. In the case of both drugs upon DIF+, the metabolic effect of OMD was predominant. DIF+ induced enlargement and stiffed 3D spheroid with increased lipid staining and mRNA expression of adipogenesis-related genes, COL4 and COL6, and decreased the expression of COL1. In the presence of OMD and/or Rip to DIF+, (1) the sizes were further increased by Rip and the stiffness was significantly decreased by OMD or Rip and (2) COL4 or AP2 expression was substantially increased by OMD or Rip, respectively. Conclusion: The results presented herein indicate that the metabolic effects of OMD and Rip exerted opposing effects and the effects of OMD toward Ap2 and ECM expressions were distinct from those of Rip, but the effects of OMD toward the physical aspects and adipogenesis of the 3D cultured HOFs were similar to the effects of Rip.

Three-Dimensional Spheroid Configurations and Cellular Metabolic Properties of Oral Squamous Carcinomas Are Possible Pharmacological and Pathological Indicators

Sho Miyamoto, Nami Nishikiori, Tatsuya Sato, Megumi Watanabe, Araya Umetsu, Yuri Tsugeno, Fumihito Hikage, Takashi Sasaya, Hirotaka Kato, Kazuhiro Ogi, Masato Furuhashi, Hiroshi Ohguro, Akihiro Miyazaki

Cancers 15 ( 10 ) 2023年05月 [査読有り] [国際誌]

The objective of the current study was to elucidate the clinicopathological significance and appearance of in vitro three-dimension (3D) spheroid models of oral malignant tumors that were prepared from four pathologically different squamous cell carcinoma (OSCC; low-grade; SSYP and MO-1000, intermediate-grade; LEM2) and oral adenosquamous carcinoma (OASC; high-grade; Mesimo) obtained from patients with different malignant stages. To characterize the biological significance of these cell lines themselves, two-dimensional (2D) cultured cells were subjected to cellular metabolic analysis by a Seahorse bioanalyzer alongside the measurement of the cytotoxicity of cisplatin (CDDP). The appearance of their 3D spheroids was then observed by phase contrast microscopy, and both 2D and 3D cultured cells were subject to trypsin digestion and qPCR analysis of factors related to oncogenic signaling and other related analyses. ATP-linked respiration and proton leaking were significantly different among the four cell lines, and the malignant stages of these cultures were significantly associated with increased ATP-linked respiration and decreased proton leakage. Alternatively, the appearances of these 3D spheroids were also significantly diverse among them, and their differences increased in the order of LEM2, MO-1000, SSYP, and Mesimo. Interestingly, these orders were exactly the same in that the efficacies of CDDP-induced cytotoxicity increased in the same order. qPCR analysis indicated that the levels of expression of oncogenic signaling-related factors varied among these four cell lines, and the values for fibronectin and a key regulator of mitochondrial biogenesis, PGC-1α, were prominently elevated in cultures of the worst malignant Mesimo cells. In addition, although 0.25% trypsin-induced destruction was comparable among all four 2D cultured cells, the values for the 3D spheroids were also substantially varied among these cultures. The findings reported herein indicate that cellular metabolic functions and 3D spheroid architectures may be valuable and useful indicators for estimating the pathological and drug-sensitive aspects of OSCC and OASC malignancies.

3D Spheroid Configurations Are Possible Indictors for Evaluating the Pathophysiology of Melanoma Cell Lines

Hiroshi Ohguro, Megumi Watanabe, Tatsuya Sato, Fumihito Hikage, Masato Furuhashi, Masae Okura, Tokimasa Hida, Hisashi Uhara

Cells 12 ( 5 ) 2023年02月 [査読有り] [国際誌]

To study the molecular mechanisms responsible for inducing the spatial proliferation of malignant melanomas (MM), three-dimension (3D) spheroids were produced from several MM cell lines including SK-mel-24, MM418, A375, WM266-4, and SM2-1, and their 3D architectures and cellular metabolisms were evaluated by phase-contrast microscopy and Seahorse bio-analyzer, respectively. Several transformed horizontal configurations were observed within most of these 3D spheroids, and the degree of their deformity was increased in the order: WM266-4, SM2-1, A375, MM418, and SK-mel-24. An increased maximal respiration and a decreased glycolytic capacity were observed within the lesser deformed two MM cell lines, WM266-4 and SM2-1, as compared with the most deformed ones. Among these MM cell lines, two distinct cell lines, WM266-4 and SK-mel-24, whose 3D appearances were the closest and farthest, respectively, from being horizontally circular-shaped, were subjected to RNA sequence analyses. Bioinformatic analyses of the differentially expressed genes (DEGs) identified KRAS and SOX2 as potential master regulatory genes for inducing these diverse 3D configurations between WM266-4 and SK-mel-24. The knockdown of both factors altered the morphological and functional characteristics of the SK-mel-24 cells, and in fact, their horizontal deformity was significantly reduced. A qPCR analysis indicated that the levels of several oncogenic signaling related factors, including KRAS and SOX2, PCG1α, extracellular matrixes (ECMs), and ZO1 had fluctuated among the five MM cell lines. In addition, and quite interestingly, the dabrafenib and trametinib resistant A375 (A375DT) cells formed globe shaped 3D spheroids and showed different profiles in cellular metabolism while the mRNA expression of these molecules that were tested as above were different compared with A375 cells. These current findings suggest that 3D spheroid configuration has the potential for serving as an indicator of the pathophysiological activities associated with MM.

Adenosine monophosphate deaminase in the endoplasmic reticulum-mitochondria interface promotes mitochondrial Ca2+ overload in type 2 diabetes rat hearts.

Arata Osanami, Tatsuya Sato, Yuki Toda, Masaki Shimizu, Atsushi Kuno, Hidemichi Kouzu, Toshiyuki Yano, Wataru Ohwada, Toshifumi Ogawa, Tetsuji Miura, Masaya Tanno

Journal of diabetes investigation 2023年02月 [査読有り] [国内誌]

AIMS/INTRODUCTION: We previously showed that upregulation of myocardial adenosine monophosphate deaminase (AMPD) is associated with pressure overload-induced diastolic dysfunction in type 2 diabetes hearts. Here, we examined involvement of AMPD localized in the endoplasmic reticulum-mitochondria interface in mitochondrial Ca2+ overload and its pathological significance. MATERIALS AND METHODS: We used type 2 diabetes Otsuka Long-Evans Tokushima Fatty rats (OLETF) and non-diabetes Long-Evans Tokushima Otsuka Fatty rats (LETO) as well as AMPD3-overexpressing H9c2 cells and human embryonic kidney 293 cells. RESULTS: OLETF, but not LETO, showed diastolic dysfunction under the condition of phenylephrine-induced pressure overload. The levels of 90-kDa AMPD3 in outer mitochondrial membranes/endoplasmic reticulum and mitochondria-associated endoplasmic reticulum membrane (MAM) fractions were significantly higher in OLETF than in LETO. The area of the MAM quantified by electron microscopic analysis was 57% larger, mitochondrial Ca2+ level under the condition of pressure overload was 47% higher and Ca2+ retention capacity in MAM-containing crude mitochondria isolated before the pressure overloading was 21% lower in OLETF than in LETO (all P-values <0.05). Transfection of FLAG-AMPD3 in cells resulted in significant enlargement of the MAM area, and impairment in pyruvate/malate-driven adenosine triphosphate-stimulated and uncoupler-stimulated mitochondrial respiration compared with those in control cells. CONCLUSIONS: The findings suggest that 90-kDa AMPD3 localized in the endoplasmic reticulum-mitochondria interface promotes formation of the MAM, inducing mitochondrial Ca2+ overload and dysfunction in type 2 diabetes hearts.

TGF-β-3 Induces Different Effects from TGF-β-1 and -2 on Cellular Metabolism and the Spatial Properties of the Human Trabecular Meshwork Cells

Megumi Watanabe, Tatsuya Sato, Yuri Tsugeno, Megumi Higashide, Masato Furuhashi, Hiroshi Ohguro

International Journal of Molecular Sciences 2023年02月 [査読有り]

Nuclear translocation of MLKL enhances necroptosis by a RIP1/RIP3-independent mechanism in H9c2 cardiomyoblasts.

Shoya Ino, Toshiyuki Yano, Atsushi Kuno, Masaya Tanno, Hidemichi Kouzu, Tatsuya Sato, Tomohisa Yamashita, Wataru Ohwada, Arata Osanami, Toshifumi Ogawa, Yuki Toda, Masaki Shimizu, Tetsuji Miura

Journal of pharmacological sciences 151 ( 2 ) 134 - 143 2023年02月 [査読有り] [国内誌]

Accumulating evidence suggests that necroptosis of cardiomyocytes contributes to cardiovascular diseases. Lethal disruption of the plasma membrane in necroptosis is induced by oligomers of mixed lineage kinase domain-like (MLKL) that is translocated to the membrane from the cytosol. However, the role played by cytoplasmic-nuclear shuttling of MLKL is unclear. Here, we tested the hypothesis that translocation of MLKL to the nucleus promotes the necroptosis of cardiomyocytes. Activation of the canonical necroptotic signaling pathway by a combination of TNF-α and zVAD (TNF/zVAD) increased nuclear MLKL levels in a RIP1-activity-dependent manner in H9c2 cells, a rat cardiomyoblast cell line. By use of site-directed mutagenesis, we found a nuclear export signal sequence in MLKL and prepared its mutant (MLKL-L280/283/284A), though a search for a nuclear import signal was unsuccessful. MLKL-L280/283/284A localized to both the cytosol and the nucleus. Expression of MLKL-L280/283/284A induced necroptotic cell death, which was attenuated by GppNHp, an inhibitor of Ran-mediated nuclear import, but not by inhibition of RIP1 activity or knockdown of RIP3 expression. GppNHp partly suppressed H9c2 cell death induced by TNF/zVAD treatment. These results suggest that MLKL that is translocated to the nucleus via RIP1-mediated necroptotic signaling enhances the necroptosis of cardiomyocytes through a RIP1-/RIP3-independent mechanism.

Downregulation of extramitochondrial BCKDH and its uncoupling from AMP deaminase in type 2 diabetic OLETF rat hearts

Toshifumi Ogawa, Hidemichi Kouzu, Arata Osanami, Yuki Tatekoshi, Tatsuya Sato, Atsushi Kuno, Yugo Fujita, Shoya Ino, Masaki Shimizu, Yuki Toda, Wataru Ohwada, Toshiyuki Yano, Masaya Tanno, Takayuki Miki, Tetsuji Miura

Physiological Reports 11 ( 4 ) e15608 2023年02月 [査読有り] [国際誌]

Systemic branched-chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long-Evans-Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched-chain α-keto acid dehydrogenase (BCKDH), a rate-limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long-Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH-E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3-E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3-BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3-BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.

SIRT2 inhibition protects against cardiac hypertrophy and heart failure.

Xiaoyan Yang, Hsiang-Chun Chang, Yuki Tatekoshi, Maryam Balibegloo, Rongxue Wu, Chunlei Chen, Tatsuya Sato, Jason Shapiro, Hossein Ardehali

bioRxiv : the preprint server for biology 2023年01月 [国際誌]

Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of heart failure (HF) and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion of Sirt2 ( Sirt2 -/- ) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO), suggesting that SIRT2 exerts maladaptive effects in the heart in response to stress. Similar results were obtained in mice with cardiomyocyte-specific Sirt2 deletion. Mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which results in reduced expression of antioxidant proteins. Deletion of Nrf2 in the hearts of Sirt2 -/- mice reversed protection after PO. Finally, treatment of mouse hearts with a specific SIRT2 inhibitors reduces cardiac size and attenuates cardiac hypertrophy in response to PO. These data indicate that SIRT2 has detrimental effects in the heart and plays a role in the progression of HF and cardiac hypertrophy, which makes this protein a unique member of the SIRT family. Additionally, our studies provide a novel approach for treatment of cardiac hypertrophy by targeting SIRT2 pharmacologically, providing a novel avenue for the treatment of this disorder.

Unexpected Crosslinking Effects of a Human Thyroid Stimulating Monoclonal Autoantibody, M22, with IGF1 on Adipogenesis in 3T3L-1 Cells

Araya Umetsu, Tatsuya Sato, Megumi Watanabe, Yosuke Ida, Masato Furuhashi, Yuri Tsugeno, Hiroshi Ohguro

International Journal of Molecular Sciences 2023年01月 [査読有り]

G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells

Hanae Ichioka, Yoshihiko Hirohashi, Tatsuya Sato, Masato Furuhashi, Megumi Watanabe, Yosuke Ida, Fumihito Hikage, Toshihiko Torigoe, Hiroshi Ohguro

International Journal of Molecular Sciences 24 ( 1 ) 2023年01月 [査読有り] [国際誌]

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

Elevated circulating level of β-aminoisobutyric acid (BAIBA) in heart failure patients with type 2 diabetes receiving sodium-glucose cotransporter 2 inhibitors.

Satoshi Katano, Toshiyuki Yano, Hidemichi Kouzu, Ryohei Nagaoka, Ryo Numazawa, Kotaro Yamano, Yusuke Fujisawa, Katsuhiko Ohori, Nobutaka Nagano, Takefumi Fujito, Ryo Nishikawa, Wataru Ohwada, Masaki Katayose, Tatsuya Sato, Atsushi Kuno, Masato Furuhashi

Cardiovascular diabetology 21 ( 1 ) 285 - 285 2022年12月 [査読有り] [国際誌]

AIMS: The mechanism by which a sodium-glucose cotransporter inhibitor (SGLT2i) induces favorable effects on diabetes and cardiovascular diseases including heart failure (HF) remains poorly understood. Metabolomics including amino acid profiling enables detection of alterations in whole body metabolism. The aim of this study was to determine whether plasma amino acid profiles are modulated by SGLT2i use in HF patients with type 2 diabetes mellitus (T2DM). METHODS: We retrospectively examined 81 HF patients with T2DM (68 ± 11 years old; 78% male). Plasma amino acid concentrations in a fasting state after stabilization of HF were determined using ultraperformance liquid chromatography. To minimize potential selection bias in the retrospective analyses, the differences in baseline characteristics between patients receiving an SGLT2i and patients not receiving an SGLT2i were controlled by using an inverse probability of treatment weighting (IPTW)-adjusted analysis. RESULTS: Of amino acids measurable in the present assay, plasma β-aminoisobutyric acid (BAIBA), an exercise-induced myokine-like molecule also known as 3-aminoisobutyric acid or 3-amino-2-methyproponic acid, was detected in 77% of all patients and the proportion of patients in whom plasma BAIBA was detected was significantly higher in patients receiving an SGLT2i than in patients not receiving an SGLT2i (93% vs. 67%, p = 0.01). Analyses in patients in whom plasma BAIBA was detected showed that plasma BAIBA concentration was significantly higher in patients receiving an SGLT2i than in patients not receiving an SGLT2i (6.76 ± 4.72 vs. 4.56 ± 2.93 nmol/ml, p = 0.03). In multivariate logistic regression analyses that were adjusted for age and sex, SGLT2i use was independently associated with BAIBA detection. The independent association between BAIBA and SGLT2i use remained after inclusion of body mass index, HF with reduced ejection fraction, ischemic etiology, renal function, NT-proBNP, albumin, hemoglobin, and HbA1c into the Cox proportional hazards model. When the differences in baseline characteristics between patients receiving an SGLT2i and patients not receiving an SGLT2i were controlled by using an IPTW-adjusted analysis, least squares mean of plasma BAIBA concentration was significantly higher in patients receiving an SGLT2i than in patients not receiving an SGLT2i. CONCLUSION: SGLT2i use is closely associated with increased circulating BAIBA concentration in HF patients with T2DM.

The Selective α1 Antagonist Tamsulosin Alters ECM Distributions and Cellular Metabolic Functions of ARPE 19 Cells in a Concentration-Dependent Manner.

Yosuke Ida, Tatsuya Sato, Megumi Watanabe, Araya Umetsu, Yuri Tsugeno, Masato Furuhashi, Fumihito Hikage, Hiroshi Ohguro

Bioengineering (Basel, Switzerland) 9 ( 10 ) 2022年10月 [査読有り] [国際誌]

The purpose of the present study was to examine the effect of the selective α1 antagonist tamsulosin (TAM) on human retinal pigment epithelium cells, ARPE 19. Two-dimension (2D) and three-dimension (3D) cultured ARPE 19 cells were used in the following characterizations: (1) ultrastructure by scanning electron microscopy (SEM) (2D); (2) barrier functions by transepithelial electrical resistance (TEER) measurements, and FITC-dextran permeability (2D); (3) real time cellular metabolisms by Seahorse Bioanalyzer (2D); (4) physical properties, size and stiffness measurements (3D); and (5) expression of extracellular matrix (ECM) proteins, including collagen1 (COL1), COL4, COL6 and fibronectin (FN) by qPCR and immunohistochemistry (2D and 3D). TAM induced significant effects including: (1) alteration of the localization of the ECM deposits; (2) increase and decrease of the TEER values and FITC-dextran permeability, respectively; (3) energy shift from glycolysis into mitochondrial oxidative phosphorylation (OXPHOS); (4) large and stiffened 3D spheroids; and (5) down-regulations of the mRNA expressions and immune labeling of most ECM proteins in a concentration-dependent manner. However, in some ECM proteins, COL1 and COL6, their immunolabeling intensities were increased at the lowest concentration (1 μM) of TAM. Such a discrepancy between the gene expressions and immunolabeling of ECM proteins may support alterations of ECM localizations as observed by SEM. The findings reported herein indicate that the selective α1 antagonist, TAM, significantly influenced ECM production and distribution as well as cellular metabolism levels in a concentration-dependent manner.

All Trans-Retinoic Acids Facilitate the Remodeling of 2D and 3D Cultured Human Conjunctival Fibroblasts.

Yuri Tsugeno, Tatsuya Sato, Megumi Watanabe, Megumi Higashide, Masato Furuhashi, Araya Umetsu, Soma Suzuki, Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro

Bioengineering (Basel, Switzerland) 9 ( 9 ) 2022年09月 [査読有り] [国際誌]

Vitamin A derivative, all-trans-retinoic acid (ATRA), is known to be a potent regulator of the growth and differentiation of various types of cells. In the present study, the unidentified effects of ATRA on superficial and vertical spreading conjunctival scarring were examined. The study involved the use of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells in the presence or absence of TGF-β2. The effects of ATRA (1 μM) on superficial or vertical spreading conjunctival scarring were evaluated by the barrier function by trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements and real-time metabolic analysis, as well as the physical properties, namely, the size and stiffness, of 3D spheroids, respectively. In addition, the expressions of several related molecules, including extracellular matrix (ECM) molecules, ECM modulators including a tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), and ER stress-related factors, were examined. ATRA significantly induced (1) an increase in TEER values and a decrease in FITC dextran permeability, respectively, in the 2D monolayers, and (2) relatively and substantially increased the size and stiffness, respectively, of the 3D spheroids. These ATRA-induced effects were further enhanced in the TGF-β2-treated cells, whereas the TGF-β2-induced enhancement in glycolytic capacity was canceled by the presence of ATRA. Consistent with these physical and morphological effects, the mRNA expressions of several molecules were significantly but differently induced between 2D and 3D cultures by ATRA, although the presence of TGF-β2 did not substantially affect these gene expression levels. The findings reported in this study indicate that ATRA may exacerbate both superficial and vertical conjunctival fibrosis spreading independently of TGF-β2-induced changes.

Benzalkonium Chloride, Even at Low Concentrations, Deteriorates Intracellular Metabolic Capacity in Human Conjunctival Fibroblasts

Yuri Tsugeno, Tatsuya Sato, Megumi Watanabe, Masato Furuhashi, Araya Umetsu, Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro

Biomedicines 2022年09月

All-trans Retinoic Acids Synergistically and Beneficially Affect In Vitro Glaucomatous Trabecular Meshwork (TM) Models Using 2D and 3D Cell Cultures of Human TM Cells.

Megumi Watanabe, Tatsuya Sato, Yuri Tsugeno, Megumi Higashide, Masato Furuhashi, Araya Umetsu, Soma Suzuki, Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro

International journal of molecular sciences 23 ( 17 ) 2022年08月 [査読有り] [国際誌]

We report herein on the effects of all-trans retinoic acid (ATRA) on two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork (HTM) cells that were treated with transforming growth factor β2 (TGF-β2). In the presence of 5 ng/mL TGF-β2, the effects of ATRA on the following were observed: (1) the barrier function of the 2D HTM monolayers, as determined by trans-endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability measurements; (2) a Seahorse cellular bio-metabolism analysis; (3) physical properties, including the size and stiffness, of 3D spheroids; (4) the gene expression of extracellular matrix (ECM) molecules, ECM modulators including tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), tight junction (TJ)-related molecules, and endoplasmic reticulum (ER)-stress-related factors. ATRA significantly inhibited the TGF-β2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-β2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-β2-induced shift in metabolic reserve from mitochondrial oxidative phosphorylation to glycolysis in 2D HTM cells, whereas ATRA alone did not induce significant metabolic changes. In contrast, ATRA induced the formation of substantially downsized and softer 3D spheroids in the absence and presence of TGF-β2. The different effects induced by ATRA toward 2D and 3D HTM cells were also supported by the qPCR analysis of several proteins as above. The findings reported here indicate that ATRA may induce synergistic and beneficial effects on TGF-β2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.

Brimonidine Modulates the ROCK1 Signaling Effects on Adipogenic Differentiation in 2D and 3D 3T3-L1 Cells

Araya Umetsu, Yosuke Ida, Tatsuya Sato, Megumi Watanabe, Yuri Tsugeno, Masato Furuhashi, Fumihito Hikage, Hiroshi Ohguro

Bioengineering 2022年07月 [査読有り]

An α2-Adrenergic Agonist, Brimonidine, Beneficially Affects the TGF-β2-Treated Cellular Properties in an In Vitro Culture Model

Megumi Watanabe, Tatsuya Sato, Yuri Tsugeno, Megumi Higashide, Masato Furuhashi, Araya Umetsu, Soma Suzuki, Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro

Bioengineering ( {MDPI} {AG} ) 9 ( 7 ) 310 - 310 2022年07月 [査読有り]

Human Trabecular Meshwork (HTM) Cells Treated with TGF-β2 or Dexamethasone Respond to Compression Stress in Different Manners.

Megumi Watanabe, Tatsuya Sato, Yuri Tsugeno, Araya Umetsu, Soma Suzuki, Masato Furuhashi, Yosuke Ida, Fumihito Hikage, Hiroshi Ohguro

Biomedicines 10 ( 6 ) 2022年06月 [査読有り] [国際誌]

To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-β2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokines, and ER stress-related factors of those 3D HTM spheroid models. The results indicated that (1) the real-time cellular metabolism analysis indicated that TGF-β2 significantly induced an energy shift from mitochondrial oxidative phosphorylation (OXPHOS) into glycolysis, and DEX induced similar but lesser effects. In contrast, ROCK2 inhibition by KD025 caused a substantial reverse energy shift from glycolysis into OXPHOS. (2) Upon direct compression stresses toward the untreated control 3D HTM spheroids, a bimodal fluctuation of the mRNA expressions of ECM proteins was observed for 60 min, that is, initial significant upregulation (0-10 min) and subsequent downregulation (10-30 min) followed by another upregulation (30-60 min); those of inflammatory cytokines and ER stress-related factors were also bimodally changed. However, such compression stresses for 30 min toward TGF-β2- or DEX-treated 3D HTM spheroids induced downregulation of most of those of inflammatory cytokines and ER stress-related factors in addition to upregulation of COL1 and downregulation of FN. The findings presented herein indicate that (1) OXPHOS of the HTM cells was decreased or increased by TGF-β2 or DEX stimulation or ROCK2 inhibition, and (2) mechanical compression stresses toward 3D HTM spheroids may replicate acute, subacute, and chronic HTM models affected by elevated intraocular pressures.

SIRT1 in the cardiomyocyte counteracts doxorubicin-induced cardiotoxicity via regulating histone H2AX

Atsushi Kuno, Ryusuke Hosoda, Miki Tsukamoto, Tatsuya Sato, Hiromi Sakuragi, Nami Ajima, Yukika Saga, Kouhei Tada, Yoshiki Taniguchi, Naotoshi Iwahara, Yoshiyuki Horio

Cardiovascular Research ( Oxford University Press (OUP) ) 2022年03月 [査読有り] [国際誌]

Abstract Aims Cardiotoxicity by doxorubicin predicts worse prognosis of patients. Accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. SIRT1, an NAD+-dependent histone/protein deacetylase, protects cells by deacetylating target proteins. We investigated whether SIRT1 counteracts doxorubicin-induced cardiotoxicity by mediating Ser139 phosphorylation of histone H2AX, a critical signal of the DNA damage response. Methods and results Doxorubicin (5 mg/kg per week, x4) was administered to mice with intact SIRT1 (Sirt1f/f) and mice that lack SIRT1 activity in cardiomyocytes (Sirt1f/f; MHCcre/+). Reductions in left ventricular fractional shortening and ejection fraction by doxorubicin treatment were more severe in Sirt1f/f; MHCcre/+ than in Sirt1f/f. Myocardial expression level of type-B natriuretic peptide was 2.5-fold higher in Sirt1f/f; MHCcre/+ than in Sirt1f/f after doxorubicin treatment. Sirt1f/f; MHCcre/+ showed larger fibrotic areas and higher nitrotyrosine levels in the heart after doxorubicin treatment. Although doxorubicin-induced DNA damage evaluated by TUNEL staining was enhanced in Sirt1f/f; MHCcre/+, the myocardium from Sirt1f/f; MHCcre/+ showed blunted Ser139 phosphorylation of H2AX by doxorubicin treatment. In H9c2 cardiomyocytes, SIRT1 knockdown attenuated Ser139 phosphorylation of H2AX, increased DNA damage, and enhanced caspase-3 activation under doxorubicin treatment. Immunostaining revealed that acetylation level of H2AX at Lys5 was higher in hearts from Sirt1f/f; MHCcre/+. In H9c2 cells, acetyl-Lys5-H2AX level was increased by SIRT1 knockdown and reduced by SIRT1 overexpression. Ser139 phosphorylation in response to doxorubicin treatment was blunted in a mutant H2AX with substitution of Lys5 to Gln (K5Q) that mimics acetylated lysine compared with that in wild-type H2AX. Expression of K5Q-H2AX as well as S139A-H2AX, which cannot be phosphorylated at Ser139, augmented doxorubicin-induced caspase-3 activation. Treatment of mice with resveratrol, a SIRT1 activator, attenuated doxorubicin-induced cardiac dysfunction, which was associated with a reduction in acetyl-Lys5-H2AX level and a preserved phospho-Ser139-H2AX level. Conclusion These findings suggest that SIRT1 counteracts doxorubicin-induced cardiotoxicity by mediating H2AX phosphorylation through its deacetylation in cardiomyocytes. Translational perspective Doxorubicin-induced cardiotoxicity limits the further use of doxorubicin for cancer treatment and determines prognosis of patients. This work shows for the first time the protective effect of SIRT1, an NAD+-dependent deacetylase, on doxorubicin-induced cardiotoxicity using a genetically modified mouse model. We identified histone H2AX as a target of SIRT1 for proper DNA damage response. Therefore, DNA repair by SIRT1 could be a potential therapeutic target to attenuate doxorubicin cardiotoxicity. SIRT1 activity may also help to predict a risk of developing cardiotoxicity in patients treated with doxorubicin.

Role of Erythropoiesis-Stimulating Agents in Cardiovascular Protection in CKD Patients: Reappraisal of Their Impact and Mechanisms.

Tetsuji Miura, Tatsuya Sato, Toshiyuki Yano, Akira Takaguri, Takayuki Miki, Noritsugu Tohse, Keitaro Nishizawa

Cardiovascular drugs and therapy 2022年02月 [査読有り] [国際誌]

Erythropoiesis-stimulating agents (ESAs) have markedly reduced the need for blood transfusion for renal anemia and are included in standard therapies for patients with chronic kidney disease (CKD). Various protective effects of ESAs on the cardiovascular system have been discovered through basic research, and the effects have received much attention because the rates of cardiovascular events and mortality are high in CKD patients. However, randomized clinical trials did not provide strong evidence that ESAs exert cardioprotection in humans, including CKD patients. It is difficult to assess the cardioprotective effects of ESAs in CKD patients through the clinical data that has been reported to date because the relationship between hemoglobin level rather than ESA dose and cardiovascular event rates was examined in most studies. Interestingly, recent studies using a rat model of CKD showed that the infarct size-limiting effect of an ESA was lost when its dose was increased to a level that normalized blood hemoglobin levels, suggesting that the optimal dose of an ESA for myocardial protection is less than the dose required to normalize hemoglobin levels. Furthermore, animal models of traditional coronary risk factors or comorbidities were resistant to the cardioprotective effects of ESAs because of interruptions in signal-mediated mechanisms downstream of erythropoietin receptors. In this review, we briefly discuss basic and clinical data on the impact of anemia on coronary and systemic circulation, the effects of CKD on the cardiovascular system, and the multiple pharmacological actions of ESAs to examine whether the ESAs that are prescribed for renal anemia exert any cardioprotection in patients with CKD.

Successful therapeutic strategy for a patient with obese end-stage kidney disease by simultaneous laparoscopic sleeve gastrectomy and implantation of a buried peritoneal dialysis catheter: A case report.

Tomohisa Yamashita, Tatsuya Sato, Kazuyuki Yamamoto, Atsuko Abiko, Keitaro Nishizawa, Masahiro Matsuda, Yuma Ebihara, Takeshi Maehana, Toshiaki Tanaka, Toshiyuki Yano, Hironori Kobayashi

Frontiers in medicine 9 926652 - 926652 2022年 [査読有り] [国際誌]

For morbidly obese patients with end-stage kidney disease (ESKD), there are often difficulties in accessing, implementing, and maintaining kidney replacement therapy (KRT). Although recent weight-loss surgery has the potential to solve these problems, its therapeutic strategy and appropriate perioperative management for morbidly obese patients with ESKD have not been established. Here, we describe the case history of a 47-year-old man diagnosed with ESKD due to obesity-related glomerulopathy with an uncorrected estimated glomerular filtration rate (eGFR) of 16.1 ml/min. He hoped for kidney transplantation but was not eligible due to his high body mass index (BMI) (36.9 kg/m2). Therefore, a combination strategy for both attaining weight loss and preparing for KRT was needed. We performed modified laparoscopic sleeve gastrectomy (LSG) combined with a buried catheter for peritoneal dialysis (PD), which resulted in reduction of multiple surgical invasions while simultaneously preparing for PD. After these operations, his body mass dropped to below 30.0 kg/m2, making him a candidate for kidney transplantation, while maintaining PD. Finally, he was able to have kidney transplantation with success. Collectively, in this case, our novel therapeutic approach was able to avoid multiple surgeries, to assist catheter insertion by laparoscopy, and to provide optimal KRT for an obese patient with ESKD. Simultaneous LSG and implantation of a buried PD catheter may be a promising strategy for morbidly obese patients with ESKD.

Larger improvements in fatigue resistance and mitochondrial function with high- than with low-intensity contractions during interval training of mouse skeletal muscle.

Takashi Yamada, Iori Kimura, Yuki Ashida, Katsuyuki Tamai, Hiroyori Fusagawa, Noritsugu Tohse, Håkan Westerblad, Daniel C Andersson, Tatsuya Sato

FASEB journal 35 ( 11 ) e21988 2021年11月 [査読有り] [国際誌]

担当区分：最終著者

Interval training (IT) results in improved fatigue resistance in skeletal muscle mainly due to an increased aerobic capacity, which involves increased muscle mitochondrial content and/or improved mitochondrial function. We hypothesized that IT with high-intensity contractions is more effective in increasing mitochondrial function, and hence fatigue resistance, than low-intensity contractions. To study this hypothesis without interference from differences in muscle fiber recruitment obliged to occur during voluntary contractions, IT was performed with in situ supramaximal electrical stimulation where all muscle fibers are recruited. We compared the effect of IT with repeated low-intensity (20 Hz stimulation, IT20) and high-intensity (100 Hz stimulation, IT100) contractions on fatigue resistance and mitochondrial content and function in mouse plantar flexor muscles. Muscles were stimulated every other day for 4 weeks. The averaged peak torque during IT bouts was 4.2-fold higher with IT100 than with IT20. Both stimulation protocols markedly improved in situ fatigue resistance, although the improvement was larger with IT100. The citrate synthase activity, a biomarker of mitochondrial content, was similarly increased with IT20 and IT100. Conversely, increased expression of mitochondrial respiratory chain (MRC) complexes I, III, and IV was only observed with IT100 and this was accompanied by increases in MRC supercomplex formation and pyruvate-malate-driven state 3 respiration in isolated mitochondria. In conclusion, the IT-induced increase in fatigue resistance is larger with high-intensity than with low-intensity contractions and this is linked to improved mitochondrial function due to increased expression of MRC complexes and assembly of MRC supercomplexes.

Xanthine oxidoreductase-mediated injury is amplified by upregulated AMP deaminase in type 2 diabetic rat hearts under the condition of pressure overload.

Yusuke Igaki, Masaya Tanno, Tatsuya Sato, Hidemichi Kouzu, Toshifumi Ogawa, Arata Osanami, Toshiyuki Yano, Atsushi Kuno, Takayuki Miki, Takashi Nakamura, Tetsuji Miura

Journal of molecular and cellular cardiology 154 21 - 31 2021年05月 [査読有り] [国際誌]

BACKGROUND: We previously reported that upregulated AMP deaminase (AMPD) contributes to diastolic ventricular dysfunction via depletion of the adenine nucleotide pool in a rat model of type 2 diabetes (T2DM), Otsuka Long-Evans-Tokushima Fatty rats (OLETF). Meanwhile, AMPD promotes the formation of substrates of xanthine oxidoreductase (XOR), which produces ROS as a byproduct. Here, we tested the hypothesis that a functional link between upregulated AMPD and XOR is involved in ventricular dysfunction in T2DM rats. METHODS AND RESULTS: Pressure-volume loop analysis revealed that pressure overloading by phenylephrine infusion induced severer left ventricular diastolic dysfunction (tau: 14.7 ± 0.8 vs 12.5 ± 0.7 msec, left ventricular end-diastolic pressure: 18.3 ± 1.5 vs 12.2 ± 1.3 mmHg, p < 0.05) and ventricular-arterial uncoupling in OLETF than in LETO, non-diabetic rats, though the baseline parameters were comparable in the two groups. While the pressure overload did not affect AMPD activity, it increased XOR activity both in OLETF and LETO, with OLETF showing significantly higher XOR activity than that in LETO (347.2 ± 17.9 vs 243.2 ± 6.1 μg/min/mg). Under the condition of pressure overload, myocardial ATP level was lower, and levels of xanthine and uric acid were higher in OLETF than in LETO. Addition of exogenous inosine, a product of AMP deamination, to the heart homogenates augmented XOR activity. OLETF showed 68% higher tissue ROS levels and 47% reduction in mitochondrial state 3 respiration compared with those in LETO. Overexpression of AMPD3 in H9c2 cells elevated levels of hypoxanthine and ROS and reduced the level of ATP. Inhibition of XOR suppressed the production of tissue ROS and mitochondrial dysfunction and improved ventricular function under the condition of pressure overload in OLETF. CONCLUSIONS: The results suggest that increases in the activity of XOR and the formation of XOR substrates by upregulated AMPD contribute to ROS-mediated diastolic ventricular dysfunction at the time of increased cardiac workload in diabetic hearts.

Distinct intra-mitochondrial localizations of pro-survival kinases and regulation of their functions by DUSP5 and PHLPP-1.

Wataru Ohwada, Masaya Tanno, Toshiyuki Yano, Sang-Bing Ong, Koki Abe, Tatsuya Sato, Atsushi Kuno, Takayuki Miki, Hirohito Sugawara, Yusuke Igaki, Tetsuji Miura

Biochimica et biophysica acta. Molecular basis of disease 1866 ( 10 ) 165851 - 165851 2020年10月 [査読有り] [国際誌]

ERK and Akt have been shown to regulate cell sensitivity to death-inducing stress by phosphorylating GSK-3β, a major modulator of the threshold for mitochondrial permeability transition. Here we examined intra-mitochondrial localization of the pro-survival kinases and their regulation by phosphatases. Stepwise trypsin digestion of mitochondria isolated from HEK293 or H9c2 cells was performed, and immunoblotting revealed that GSK-3β and ERK localized dominantly in the outer membrane (OM), while Akt resided at comparable levels in OM, the inner membrane (IM) and the matrix. Treatment with IGF-1 increased the protein level of Akt in the matrix, while ERK and GSK-3β protein levels were increased in OM. Simultaneously, IGF-1 treatment elevated the level of Thr202/Tyr204-phospho-ERK in IM and matrix and levels of Ser473-phospho-Akt and Ser9-phospho-GSK-3β in OM, IM and matrix. Exposing cells to reactive oxygen species (ROS) by using antimycin A increased the levels of DUSP5 and PHLPP-1 mainly in OM and induced dephosphorylation of Akt, ERK and GSK-3β. The mitochondrial localization of DUSP5 was confirmed by experiments with mitochondria purified by Percoll gradient centrifugation and by transfection of cells with GFP-tagged DUSP5. Knockdown of either DUSP5 or PHLPP-1 increased the levels of both Thr202/Tyr204-phospho-ERK and Ser473-phospho-Akt in mitochondria. Cell death induced by antimycin A was suppressed by siRNA-mediated knockdown of DUSP5. The results suggest that Akt and ERK in mitochondria show distinct intra-mitochondrial localization and crosstalk in GSK-3β regulation and that recruitment of DUSP5 as well as PHLPP-1 to mitochondria contributes to ROS-induced termination of the protective signaling.

Empagliflozin attenuates acute kidney injury after myocardial infarction in diabetic rats.

Atsushi Kuno, Yukishige Kimura, Masashi Mizuno, Hiroto Oshima, Tatsuya Sato, Norihito Moniwa, Marenao Tanaka, Toshiyuki Yano, Masaya Tanno, Takayuki Miki, Tetsuji Miura

Scientific reports 10 ( 1 ) 7238 - 7238 2020年04月 [査読有り] [国際誌]

Acute kidney injury (AKI) predicts poor prognosis in patients with acute myocardial infarction (MI) and diabetes mellitus (DM) is an independent risk factor of AKI. Recent clinical studies have shown the beneficial effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors on cardiovascular and renal outcomes in patients with DM. We recently reported that canagliflozin normalized susceptibility of diabetic rats to AKI after acute MI via β-hydroxybutyrate-mediated suppression of NOX expression. Here we examined whether the same renoprotective effect is shared by empagliflozin. Serum creatinine levels were not changed by MI induced by coronary artery occlusion in LETO, non-diabetic control rats, and OLETF, obese type 2 diabetic rats. However, immunohistochemistry revealed that MI increased renal expression of NGAL and KIM-1, early markers of tubular injury, by 3.2-fold and 2.6-fold, respectively, in OLETF. These increases in injury markers were not observed in LETO. Pretreatment with empagliflozin of OLETF for 2 weeks improved hyperglycemia, increased blood β-hydroxybutyrate level, and suppressed MI-induced expression of NGAL and KIM-1. Empagliflozin suppressed upregulation of NOX2 and NOX4 in the kidney of OLETF. Taken together with the results of our previous study, it was concluded that treatment with the SGLT2 inhibitor protects the diabetic kidney from MI-induced AKI.

mTORC1 inhibition attenuates necroptosis through RIP1 inhibition-mediated TFEB activation.

Koki Abe, Toshiyuki Yano, Masaya Tanno, Takayuki Miki, Atsushi Kuno, Tatsuya Sato, Hidemichi Kouzu, Kei Nakata, Wataru Ohwada, Yukishige Kimura, Hirohito Sugawara, Satoru Shibata, Yusuke Igaki, Shoya Ino, Tetsuji Miura

Biochimica et biophysica acta. Molecular basis of disease 1865 ( 12 ) 165552 - 165552 2019年12月 [査読有り] [国際誌]

Accumulating evidence indicates that necroptosis contributes to cardiovascular diseases. We recently reported suppression of autophagy by necroptotic signals in cardiomyocytes and protective action of rapamycin. Here we examined the mechanism by which mTORC1 inhibition protects cardiomyocytes from necroptosis. Necroptosis of H9c2 cells was induced by treatment with tumor necrotic factor-α (TNF) and z-VAD-fmk (zVAD), and the extent of necroptosis was determined as the level of LDH release (as % of total). TNF/zVAD increased RIP1-RIP3 interaction and LDH release from 3.4 ± 1.3% to 46.1 ± 2.3%. The effects of TNF/zVAD were suppressed by an mTORC1 inhibitor, rapamycin, and an mTORC1/2 inhibitor, Ku-0063794, but not by a p70s6K inhibitor, PF-4708671. Protection by rapamycin was not abolished by inhibitors of TAK1, IKKα/β, and cIAP, endogenous necroptosis suppressors upstream of RIP1. Rapamycin and Ku-0063794 suppressed TNF/zVAD-induced RIP1-Ser166 phosphorylation and increased phosphorylation of RIP1-Ser320, an inhibitory phosphorylation site, though such an effect on RIP1-Ser320 was not observed for PF-4708671. Protective effects of rapamycin on TNF/zVAD-induced RIP1-RIP3 binding and necroptosis were undetected in cells transfected with RIP1-S320A. In TNF/zVAD-treated cells, rapamycin and a RIP1 inhibitor, necrostatin-1, increased nuclear localization of transcriptional factor EB (TFEB) and promoted autolysosome formation from autophagosomes in a TFEB-dependent manner. Knockdown of TFEB expression attenuated rapamycin-induced protection from necroptosis in TNF/zVAD-treated cells. The results suggest that mTORC1 inhibition promotes autophagy and protects cardiomyocytes from necroptosis by a TFEB-dependent mechanism and that inhibition of RIP1 by increased phosphorylation at Ser320 is crucial in the cardiomyocyte protection afforded by mTORC1 inhibition.

Insulin Resistance is Associated with Longitudinal Changes of Cardiac Repolarization Heterogeneity in Apparently Healthy Subjects.

Tamaki Matsumoto, Hirofumi Ohnishi, Tatsuya Sato, Takayuki Miki, Hiroshi Akasaka, Nagisa Hanawa, Masayuki Koyama, Shigeyuki Saitoh, Tetsuji Miura

Cardiology and therapy 8 ( 2 ) 239 - 251 2019年07月 [査読有り] [国際誌]

INTRODUCTION: Increased heterogeneity in ventricular repolarization is a risk factor of sudden cardiac death, but its natural history is unclear. Here we examined whether insulin resistance is associated with longitudinal change in ventricular repolarization heterogeneity in apparently healthy subjects. METHODS: The study subjects were participants in health checkups in cohort 1 and cohort 2, which were followed up for 6 years and 5 years, respectively. Subjects with diabetes, cardiovascular disease, or renal disease at baseline were excluded from the analyses. As indices of insulin resistance, the homeostasis model assessment of insulin resistance (HOMA-IR) and triglyceride to HDL-cholesterol ratio (TG/HDL-C) were used in cohort 1 and cohort 2, respectively. Heterogeneity in ventricular repolarization was assessed by heart rate-corrected Tpeak-Tend interval in V5 (cTpTe), QT interval, and QT dispersion. In regression analyses, parameters with a skewed distribution were normalized by logarithmic transformation or by Box-Cox transformation. RESULTS: In longitudinal analyses, Box-Cox-transformed cTpTe at the end of follow-up was weakly correlated with log HOMA-IR at baseline in cohort 1 (n = 153, r = - 0.207, 95% CI - 0.354 to - 0.050, p = 0.010) and with log TG/HDL-C at baseline in cohort 2 (n = 738, r = - 0.098, 95% CI - 0.169 to - 0.026, p = 0.008). Multiple regression analysis showed that indices of insulin resistance, but not glycosylated hemoglobin (HbA1c) or plasma glucose, at baseline were significant explanatory variables for cTpTe at the end of follow-up. Neither QT interval nor QT dispersion was correlated with metabolic parameters. CONCLUSION: Insulin resistance may be involved in the longitudinal increase of ventricular repolarization heterogeneity in apparently healthy subjects.

Canagliflozin, a sodium-glucose cotransporter 2 inhibitor, normalizes renal susceptibility to type 1 cardiorenal syndrome through reduction of renal oxidative stress in diabetic rats.

Yukishige Kimura, Atsushi Kuno, Masaya Tanno, Tatsuya Sato, Kouhei Ohno, Satoru Shibata, Kei Nakata, Hirohito Sugawara, Koki Abe, Yusuke Igaki, Toshiyuki Yano, Takayuki Miki, Tetsuji Miura

Journal of diabetes investigation 10 ( 4 ) 933 - 946 2019年07月 [査読有り] [国内誌]

AIMS/INTRODUCTION: Type 2 diabetes mellitus is a risk factor of acute kidney injury after myocardial infarction (MI), a form of cardiorenal syndrome. Recent clinical trials have shown that a sodium-glucose cotransporter 2 (SGLT2) inhibitor improved both cardiac and renal outcomes in patients with type 2 diabetes mellitus, but effects of an SGLT2 inhibitor on cardiorenal syndrome remain unclear. MATERIALS AND METHODS: Type 2 diabetes mellitus (Otsuka Long-Evans Tokushima Fatty rats [OLETF]) and control (Long-Evans Tokushima Otsuka rats [LETO]) were treated with canagliflozin, an SGLT2 inhibitor, for 2 weeks. Renal tissues were analyzed at 12 h after MI with or without preoperative fasting. RESULTS: Canagliflozin reduced blood glucose levels in OLETF, and blood β-hydroxybutyrate levels were increased by canagliflozin only with fasting. MI increased neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 protein levels in the kidney by 3.2- and 1.6-fold, respectively, in OLETF, but not in LETO. The renal messenger ribonucleic acid level of Toll-like receptor 4 was higher in OLETF than in LETO after MI, whereas messenger ribonucleic acid levels of cytokines/chemokines were not significantly different. Levels of lipid peroxides, nicotinamide adenine dinucleotide phosphate oxidase (NOX)2 and NOX4 proteins after MI were significantly higher in OLETF than in LETO. Canagliflozin with pre-MI fasting suppressed MI-induced renal expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in OLETF, together with reductions in lipid peroxides and NOX proteins in the kidney. Blood β-hydroxybutyrate levels before MI were inversely correlated with neutrophil gelatinase-associated lipocalin protein levels in OLETF. Pre-incubation with β-hydroxybutyrate attenuated angiotensin II-induced upregulation of NOX4 in NRK-52E cells. CONCLUSIONS: The findings suggest that SGLT2 inhibitor treatment with a fasting period protects kidneys from MI-induced cardiorenal syndrome, possibly by β-hydroxybutyrate-mediated reduction of NOXs and oxidative stress, in type 2 diabetic rats.

Empagliflozin, an SGLT2 Inhibitor, Reduced the Mortality Rate after Acute Myocardial Infarction with Modification of Cardiac Metabolomes and Antioxidants in Diabetic Rats.

Hiroto Oshima, Takayuki Miki, Atsushi Kuno, Masashi Mizuno, Tatsuya Sato, Masaya Tanno, Toshiyuki Yano, Kei Nakata, Yukishige Kimura, Koki Abe, Wataru Ohwada, Tetsuji Miura

The Journal of pharmacology and experimental therapeutics 368 ( 3 ) 524 - 534 2019年03月 [査読有り] [国際誌]

The mechanism by which SGLT2 inhibitors reduce cardiac events in diabetic patients remains unclear. Here, we examined the effects of an SGLT2 inhibitor on the acute survival rate after myocardial infarction (MI) in an animal model of type 2 diabetes mellitus (DM) and the possible involvement of modification of cardiac metabolomes and antioxidative proteins. MI was induced in DM Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) control rats. Treatment with empagliflozin (10 mg/kg per day, 14 days) before MI reduced blood glucose and increased blood and myocardial β-hydroxybutyrate (βOHB) levels in OLETF. Survival rate at 48 hours after MI was significantly lower in OLETF rats than in LETO rats (40% vs. 84%), and empagliflozin significantly improved the survival rate in OLETF rats to 70%, although the sizes of MI were comparable. Patterns of metabolomes and gene expression in the noninfarcted myocardium of OLETF rats were consistent with increased fatty acid oxidation and decreased glucose oxidation. The patterns were modified by empagliflozin, suggesting both increased glucose oxidation and ketone utilization in OLETF rats. Empagliflozin prevented reduction of ATP level in the noninfarcted myocardium after MI and significantly increased myocardial levels of Sirt3 and superoxide dismutase 2 in OLETF rats. Administration of βOHB partially mimicked the effects of empagliflozin in OLETF rats. The results suggest that empagliflozin prevents DM-induced increase in post-MI mortality, possibly by protective modification of cardiac energy metabolism and antioxidant proteins.

Antinociceptive effects of hyaluronic acid on monoiodoacetate-induced ankle osteoarthritis in rats.

Shunsuke Jimbo, Yoshinori Terashima, Atsushi Teramoto, Tsuneo Takebayashi, Izaya Ogon, Kota Watanabe, Tatsuya Sato, Nobutoshi Ichise, Noritsugu Tohse, Toshihiko Yamashita

Journal of pain research 12 191 - 200 2019年 [査読有り] [国際誌]

Purpose: Ankle osteoarthritis (OA) causes significant pain and debilitation; yet, its underlying mechanisms remain unclear. Clinically, hyaluronic acid (HA) is widely used to treat OA. The present study aimed to investigate the roles of HA in pain-related behavior, joint function, swelling, and pathological changes in cartilage in a rat model of monoiodoacetate (MIA)-induced ankle OA. Materials and methods: Male Sprague Dawley rats were assigned to three experimental groups as follows: 1) MIA rats injected with 1 mg MIA in the right tibiotarsal joint for two consecutive days; 2) sham rats injected with saline instead of MIA; and 3) MIA-HA rats injected with HA in the tibiotarsal joint at 7, 14, and 21 days after MIA injection. Joint swelling, range of motion (ROM), and pain-related behavior were evaluated 1 day before and on the 7th, 14th, 21st, and 28th day after MIA or saline injection. Pathological changes in the ankle joint were assessed 28 days after MIA or saline injection. Results: No significant difference in the degree of ankle swelling or ROM reduction was observed between MIA rats and MIA-HA rats. However, compared with those in MIA rats, mechanical and thermal hypersensitivity was significantly reduced and stride length significantly improved in MIA-HA rats. Histologic analysis revealed that cartilage degeneration was significantly suppressed in MIA-HA rats compared with that in MIA rats, reflecting the chondroprotective effects of HA. Conclusion: HA improved pain-related behavior and stride length and suppressed MIA-induced cartilage degeneration. HA may thus inhibit OA progression and suppress peripheral and/or central sensitization.

Analgesic effects of calcitonin on radicular pain in male rats.

Yoshinori Terashima, Tsuneo Takebayashi, Shunsuke Jimbo, Izaya Ogon, Tatsuya Sato, Nobutoshi Ichise, Noritsugu Tohse, Toshihiko Yamashita

Journal of pain research 12 223 - 230 2019年 [査読有り] [国際誌]

Purpose: Radicular pain is a frequently observed symptom of lumbar disk herniation or lumbar spinal canal stenosis. Achieving radicular pain relief is difficult. This type of pain may progress to chronic neuropathic pain. Calcitonin (elcatonin [eCT]) has been used mainly for hypercalcemia and pain associated with osteoporosis. The purpose of this study was to investigate analgesic effects of repeated eCT administration on radicular pain in male rats and changes in mRNA-expression levels of voltage-dependent sodium channels in the dorsal root ganglion (DRG). Methods: Seventy male Sprague-Dawley rats were used. A right L5 hemilaminectomy and an L5-L6 partial facetectomy were performed to expose the right L5 nerve root. Under a microscope, the right L5 spinal nerve root was tightly ligated extradurally with 8-0 nylon suture proximally to the DRG to cause radicular pain in rats. Mechanical hyperalgesia, thermal hyperalgesia, and analgesic effects of eCT were compared among rats with radicular pain that received eCT, those that received the vehicle, and sham rats that received the vehicle. Real-time reverse-transcription PCR was performed to measure mRNA-expression levels of tetrodotoxin-sensitive (NaV1.3 and NaV1.6) and tetrodotoxin-resistant (NaV1.8 and NaV1.9) sodium channels in the DRG. Results: Mechanical and thermal hyperalgesic reactions occurring in rats with radicular pain significantly improved on days 5 and 9 of eCT administration, respectively. In rats with radicular pain, mRNA-expression levels of NaV1.3, NaV1.8, and NaV1.9 increased. After repeated eCT administration, mRNA-expression levels of these sodium channels in rats with radicular pain improved to the same levels as in sham rats. Conclusion: The present study demonstrated that repeated systemic eCT administration was effective for radicular pain. No serious side effects of eCT have been reported thus far. Therefore, calcitonin may be a preferred therapeutic option for patients with radicular pain or for those requiring long-term treatment.

Translational regulation by miR-301b upregulates AMP deaminase in diabetic hearts.

Yuki Tatekoshi, Masaya Tanno, Hidemichi Kouzu, Koki Abe, Takayuki Miki, Atsushi Kuno, Toshiyuki Yano, Satoko Ishikawa, Wataru Ohwada, Tatsuya Sato, Takeshi Niinuma, Hiromu Suzuki, Tetsuji Miura

Journal of molecular and cellular cardiology 119 138 - 146 2018年06月 [査読有り] [国際誌]

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism. Recently we found that upregulated AMPD activity is associated with ATP depletion and contractile dysfunction under the condition of pressure overloading in the heart of a rat model of type 2 diabetes mellitus (T2DM), OLETF. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates by 55% in OLETF compared to those in LETO, a non-diabetic control. In contrast, the mRNA levels of AMPD3 in the myocardium were similar in OLETF and LETO. AMPD3 was comparably ubiquitinated in OLETF and LETO, and its degradation ex vivo was more sensitive to MG-132, a proteasome inhibitor, in OLETF than in LETO. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3'UTR of AMPD3 mRNA. AMPD3 protein level was significantly increased by a miR-301b inhibitor and was decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay confirmed binding of miR-301b to the 3'UTR of AMPD3 mRNA. Transfection of neonatal rat cardiomyocytes with a miR-301b inhibitor increased 90-kDa AMPD3 and reduced ATP level. The results indicate that translational regulation by miR-301b mediates upregulated expression of cardiac AMPD3 protein in OLETF, which potentially reduces the adenine nucleotide pool at the time of increased work load. The miR-301b-AMPD3 axis may be a novel therapeutic target for intervening enegy metabolism in diabetic hearts.

Empagliflozin normalizes the size and number of mitochondria and prevents reduction in mitochondrial size after myocardial infarction in diabetic hearts.

Masashi Mizuno, Atsushi Kuno, Toshiyuki Yano, Takayuki Miki, Hiroto Oshima, Tatsuya Sato, Kei Nakata, Yukishige Kimura, Masaya Tanno, Tetsuji Miura

Physiological reports 6 ( 12 ) e13741 2018年06月 [査読有り] [国際誌]

To explore mechanisms by which SGLT2 inhibitors protect diabetic hearts from heart failure, we examined the effect of empagliflozin (Empa) on the ultrastructure of cardiomyocytes in the noninfarcted region of the diabetic heart after myocardial infarction (MI). OLETF, a rat model of type 2 diabetes, and its nondiabetic control, LETO, received a sham operation or left coronary artery ligation 12 h before tissue sampling. Tissues were sampled from the posterior ventricle (i.e., the remote noninfarcted region in rats with MI). The number of mitochondria was larger and small mitochondria were more prevalent in OLETF than in LETO. Fis1 expression level was higher in OLETF than in LETO, while phospho-Ser637-Drp1, total Drp1, Mfn1/2, and OPA1 levels were comparable. MI further reduced the size of mitochondria with increased Drp1-Ser616 phosphorylation in OLETF. The number of autophagic vacuoles was unchanged after MI in LETO but was decreased in OLETF. Lipid droplets in cardiomyocytes and tissue triglycerides were increased in OLETF. Empa administration (10 mg/kg per day) reduced blood glucose and triglycerides and paradoxically increased lipid droplets in cardiomyocytes in OLETF. Empa suppressed Fis1 upregulation, increased Bnip3 expression, and prevented reduction in both mitochondrial size and autophagic vacuole number after MI in OLETF. Together with the results of our parallel study showing upregulation of SOD2 and catalase by Empa, the results indicate that Empa normalizes the size and number of mitochondria in diabetic hearts and that diabetes-induced excessive reduction in mitochondrial size after MI was prevented by Empa via suppression of ROS and restoration of autophagy.

Hippocampal GABAA antagonism reverses the novel object recognition deficit in sub-chronic phencyclidine-treated rats.

Nichole M Neugebauer, Masanori Miyauchi, Tatsuya Sato, Jun Tadano, Hanife Akal, Hossein Ardehali, Herbert Y Meltzer

Behavioural brain research 342 11 - 18 2018年04月 [査読有り] [国際誌]

BACKGROUND: Abnormalities in prefrontal cortical and hippocampal GABAergic function are postulated to be major causes of the cognitive impairment associated with schizophrenia (CIAS). There are conflicting views on whether diminished or enhanced GABAergic activity contributes to the deficit in short-term novel object recognition (NOR) in the sub-chronic phencyclidine (scPCP) rodent model of CIAS. This study assessed the role of GABAA signaling in the medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC) in NOR in saline (scSAL)- and scPCP-treated rats. METHODS: The effects of local administration of a GABAA agonist (muscimol) into the vHPC or mPFC and an antagonist (bicuculline) or a GABAA/benzodiazepine partial agonist (bretazenil) into the vHPC on NOR in scSAL and scPCP-treated rats were determined. RESULTS: In scSAL-treated rats, injection of muscimol into the vHPC, but not mPFC, induced a deficit in NOR. The scPCP-induced NOR deficit was significantly reversed by intra-vHPC bicuculline, while intra-vHPC bretazenil produced a non-significant trend for reversal (p = .06). scPCP treatment increased mRNA expression of GABAA γ2 in PFC and GABAA α5 and GABAA β1 in the HPC. However, GABA concentration in the PFC or HPC was not altered. CONCLUSIONS: These findings indicate that the scPCP-induced NOR deficit can be rescued by reducing GABAA receptor stimulation in vHPC, indicating that increased vHPC GABAA inhibition may contribute to the scPCP-induced NOR deficit in rats. These results also indicate that excessive GABAA receptor signalling in the vHPC has a deleterious effect on NOR in normal rats.

Muscarinic receptor signaling contributes to atypical antipsychotic drug reversal of the phencyclidine-induced deficit in novel object recognition in rats.

Masanori Miyauchi, Nichole M Neugebauer, Tatsuya Sato, Hossein Ardehali, Herbert Y Meltzer

Journal of psychopharmacology (Oxford, England) 31 ( 12 ) 1588 - 1604 2017年12月 [査読有り] [国際誌]

Enhancement of cholinergic function via muscarinic acetylcholine receptor M1 agonism improves cognition in some schizophrenia patients. Most atypical antipsychotic drugs, including clozapine and its active metabolite, N-desmethylclozapine, and lurasidone, enhance the release of acetylcholine in key brain regions involved in cognition (e.g. hippocampus). We determined the effect of muscarinic acetylcholine receptor M1 stimulation on novel object recognition and its contribution to the ability of atypical antipsychotic drugs to reverse the novel object recognition deficit in rats withdrawn from subchronic phencyclidine, a rodent model of cognitive impairment in schizophrenia. In control rats, the non-specific muscarinic acetylcholine receptor antagonist, scopolamine, and the M1 selective antagonist, VU0255035, induced a novel object recognition deficit, which was reversed by the M1 agonist, AC260584. Scopolamine fully blocked the effect of clozapine and N-desmethylclozapine, but not lurasidone, to restore novel object recognition in subchronic phencyclidine-treated rats. VU0255035 also blocked these effects of clozapine and N-desmethylclozapine, but not lurasidone; however, the blockade was not as complete as that achieved with scopolamine. Furthermore, subchronic phencyclidine increased hippocampal M1 mRNA expression. These data suggest that M1 agonism is required for clozapine and N-desmethylclozapine to ameliorate the phencyclidine-induced deficit in novel object recognition, additional evidence that M1 agonism is a potential target for treating cognitive impairment in schizophrenia.

Snf1-related kinase improves cardiac mitochondrial efficiency and decreases mitochondrial uncoupling.

Amy K Rines, Hsiang-Chun Chang, Rongxue Wu, Tatsuya Sato, Arineh Khechaduri, Hidemichi Kouzu, Jason Shapiro, Meng Shang, Michael A Burke, Eltyeb Abdelwahid, Xinghang Jiang, Chunlei Chen, Tenley A Rawlings, Gary D Lopaschuk, Paul T Schumacker, E Dale Abel, Hossein Ardehali

Nature communications 8 14095 - 14095 2017年01月 [査読有り] [国際誌]

Ischaemic heart disease limits oxygen and metabolic substrate availability to the heart, resulting in tissue death. Here, we demonstrate that the AMP-activated protein kinase (AMPK)-related protein Snf1-related kinase (SNRK) decreases cardiac metabolic substrate usage and mitochondrial uncoupling, and protects against ischaemia/reperfusion. Hearts from transgenic mice overexpressing SNRK have decreased glucose and palmitate metabolism and oxygen consumption, but maintained power and function. They also exhibit decreased uncoupling protein 3 (UCP3) and mitochondrial uncoupling. Conversely, Snrk knockout mouse hearts have increased glucose and palmitate oxidation and UCP3. SNRK knockdown in cardiac cells decreases mitochondrial efficiency, which is abolished with UCP3 knockdown. We show that Tribbles homologue 3 (Trib3) binds to SNRK, and downregulates UCP3 through PPARα. Finally, SNRK is increased in cardiomyopathy patients, and SNRK reduces infarct size after ischaemia/reperfusion. SNRK also decreases cardiac cell death in a UCP3-dependent manner. Our results suggest that SNRK improves cardiac mitochondrial efficiency and ischaemic protection.

Distinct impacts of sleep-disordered breathing on glycemic variability in patients with and without diabetes mellitus.

Kei Nakata, Takayuki Miki, Masaya Tanno, Hirofumi Ohnishi, Toshiyuki Yano, Atsuko Muranaka, Tatsuya Sato, Hiroto Oshima, Yuki Tatekoshi, Masashi Mizuno, Koki Abe, Tetsuji Miura

PloS one 12 ( 12 ) e0188689 2017年 [査読有り] [国際誌]

BACKGROUND: Sleep-disordered breathing (SDB) is highly prevalent in patients with diabetes mellitus (DM) and heart failure (HF) and contributes to poor cardiovascular outcomes. Enlarged glycemic variability (GV) is a risk factor of cardiac events independently of average blood glucose level, but the influence of SDB on GV is uncertain. In this study, we examined whether the impact of SDB on GV is modified by the presence of DM with or without HF. METHODS AND RESULTS: Two hundred three patients (67.5±14.1 [SD] years old, 132 males) who were admitted to our institute for examination or treatment of DM and/or HF underwent continuous glucose monitoring and polysomnography. Both HbA1c (8.0±2.0 vs. 5.7±0.4%) and mean amplitude of glycemic excursion (MAGE, median: 95.5 vs. 63.5 mg/dl) were significantly higher in a DM group (n = 100) than in a non-DM group (n = 103), but apnea-hypopnea index (AHI: 29.0±22.7 vs. 29.3±21.5) was similar in the two groups. AHI was correlated with log MAGE in the non-DM group but not in the DM group, and multivariate regression analysis revealed that AHI was an independent variable for log MAGE in the non-DM group but not in the DM group. We then divided the non-DM patients into two subgroups according to BNP level (100 pg/ml). AHI was positively correlated with log MAGE (r = 0.74, p<0.001) in the non-DM low-BNP subgroup, but such a correlation was not found in the non-DM high-BNP subgroup. Continuous positive airway pressure (CPAP) reduced MAGE from 75.3 to 53.0 mg/dl in the non-DM group but did not reduce MAGE in the DM group. CONCLUSION: Severity of SDB was associated with higher GV, but DM as well as HF diminished the contribution of SDB to GV. Treatment with CPAP was effective for reduction of GV only in patients without DM.

Reduction in mitochondrial iron alleviates cardiac damage during injury.

Hsiang-Chun Chang, Rongxue Wu, Meng Shang, Tatsuya Sato, Chunlei Chen, Jason S Shapiro, Ting Liu, Anita Thakur, Konrad T Sawicki, Sathyamangla V N Prasad, Hossein Ardehali

EMBO molecular medicine 8 ( 3 ) 247 - 67 2016年03月 [査読有り] [国際誌]

Excess cellular iron increases reactive oxygen species (ROS) production and causes cellular damage. Mitochondria are the major site of iron metabolism and ROS production; however, few studies have investigated the role of mitochondrial iron in the development of cardiac disorders, such as ischemic heart disease or cardiomyopathy (CM). We observe increased mitochondrial iron in mice after ischemia/reperfusion (I/R) and in human hearts with ischemic CM, and hypothesize that decreasing mitochondrial iron protects against I/R damage and the development of CM. Reducing mitochondrial iron genetically through cardiac-specific overexpression of a mitochondrial iron export protein or pharmacologically using a mitochondria-permeable iron chelator protects mice against I/R injury. Furthermore, decreasing mitochondrial iron protects the murine hearts in a model of spontaneous CM with mitochondrial iron accumulation. Reduced mitochondrial ROS that is independent of alterations in the electron transport chain's ROS producing capacity contributes to the protective effects. Overall, our findings suggest that mitochondrial iron contributes to cardiac ischemic damage, and may be a novel therapeutic target against ischemic heart disease.

Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice.

Yuzhi Jia, Hsiang-Chun Chang, Matthew J Schipma, Jing Liu, Varsha Shete, Ning Liu, Tatsuya Sato, Edward B Thorp, Philip M Barger, Yi-Jun Zhu, Navin Viswakarma, Yashpal S Kanwar, Hossein Ardehali, Bayar Thimmapaya, Janardan K Reddy

PloS one 11 ( 8 ) e0160755 2016年 [査読有り] [国際誌]

Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart. Collectively, these observations suggest that Med1 plays a critical role in the maintenance of heart function impacting on multiple metabolic, compensatory and reparative pathways with a likely therapeutic potential in the management of heart failure.

Increased Heme Levels in the Heart Lead to Exacerbated Ischemic Injury.

Konrad Teodor Sawicki, Meng Shang, Rongxue Wu, Hsiang-Chun Chang, Arineh Khechaduri, Tatsuya Sato, Christine Kamide, Ting Liu, Sathyamangla V Naga Prasad, Hossein Ardehali

Journal of the American Heart Association 4 ( 8 ) e002272 2015年07月 [査読有り] [国際誌]

BACKGROUND: Heme is an essential iron-containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid synthase 2 (ALAS2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart. METHODS AND RESULTS: We first showed that ALAS2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac-specific overexpression of ALAS2. While ALAS2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with ALAS2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS2-overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to nonischemic cardiomyopathy. CONCLUSIONS: Heme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a novel approach for protection against the development of heart failure.

Excessive degradation of adenine nucleotides by up-regulated AMP deaminase underlies afterload-induced diastolic dysfunction in the type 2 diabetic heart.

Hidemichi Kouzu, Takayuki Miki, Masaya Tanno, Atsushi Kuno, Toshiyuki Yano, Takahito Itoh, Tatsuya Sato, Daisuke Sunaga, Hiromichi Murase, Toshiyuki Tobisawa, Makoto Ogasawara, Satoko Ishikawa, Tetsuji Miura

Journal of molecular and cellular cardiology 80 136 - 45 2015年03月 [査読有り] [国際誌]

Type 2 diabetes mellitus (T2DM) is often complicated with diastolic heart failure, which decompensates under increased afterload. Focusing on cardiac metabolomes, we examined mechanisms by which T2DM augments ventricular diastolic stiffness in response to pressure overloading. Pressure-volume relationships (PVRs) and myocardial metabolomes were determined at baseline and during elevation of aortic pressure by phenylephrine infusion in a model of T2DM, OLETF, and its non-diabetic control, LETO. Pressure overloading augmented diastolic stiffness without change in systolic reserve in OLETF as indicated by a left-upward shift of end-diastolic PVR. In contrast, PVRs under cardioplegic arrest in buffer-perfused isolated hearts were similar in OLETF and LETO, indicating that extracellular matrix or titin remodeling does not contribute to the afterload-induced increase in stiffness of the beating ventricle of OLETF. Metabolome analyses revealed impaired glycolysis and facilitation of the pentose phosphate pathway in OLETF. Pressure overloading significantly reduced ATP and total adenine nucleotides by 34% and 40%, respectively, in OLETF but not in LETO, while NADH-to-NAD(+) ratios were similar in the two groups. The decline in ATP by pressure overloading in OLETF was associated with increased inosine 5-monophosphate and decreased adenosine levels, being consistent with the 2.5-times higher activity of cardiac AMP deaminase in OLETF. Tissue ATP level was negatively correlated with tau of LV pressure and LVEDP. These results suggest that ATP depletion due to excessive degradation of adenine nucleotides by up-regulated AMP deaminase underlies ventricular stiffening during acute pressure overloading in T2DM hearts.

Does glycemic control reverse dispersion of ventricular repolarization in type 2 diabetes?

Takayuki Miki, Toshiyuki Tobisawa, Tatsuya Sato, Masaya Tanno, Toshiyuki Yano, Hiroshi Akasaka, Atsushi Kuno, Makoto Ogasawara, Hiromichi Murase, Shigeyuki Saitoh, Tetsuji Miura

Cardiovascular diabetology 13 125 - 125 2014年08月 [査読有り] [国際誌]

BACKGROUND: Abnormal ventricular repolarization is a predictor of cardiovascular mortality. In this study, we tested the hypothesis that glycemic control reverses abnormal ventricular repolarization in patients with type 2 diabetes. METHODS: We analyzed longitudinal changes in repolarization indices of electrocardiograms in retrospectively enrolled 44 patients with type 2 diabetes and 44 age-matched healthy subjects. RESULTS: In the diabetic group, BMI was greater, levels of HbA1c (10.0 ± 1.6 vs. 5.6 ± 0.3%) and triglyceride were higher and level of HDL cholesterol was lower than those in the control group. Although mean QTc intervals were similar (413.6 ± 18.5 vs. 408.3 ± 22.7 ms), QT dispersion (41.8 ± 15.4 vs. 28.7 ± 7.7 ms) and Tpeak-Tend in lead V5 (83.6 ± 13.6 vs. 71.3 ± 10.3 ms) were significantly longer in the diabetic group than in the control group, indicating increased heterogeneity of ventricular repolarization in type 2 diabetes. During follow-up of 36 patients in the diabetic group for 787 ± 301 days, HbA1c level decreased to 7.3 ± 1.6%, while BMI did not significantly change. In contrast to HbA1c, QT dispersion (45.8 ± 15.0 ms) and Tpeak-Tend in lead V5 (83.6 ± 10.6 ms) were not significantly reduced during the follow-up period. There was no correlation between the change in HbA1c and the change in QT dispersion or Tpeak-Tend. CONCLUSIONS: Increased heterogeneity of ventricular repolarization in type 2 diabetic patients was not reduced during the relatively short follow-up period despite significantly improved glycemic control.

Cytoprotective regulation of the mitochondrial permeability transition pore is impaired in type 2 diabetic Goto-Kakizaki rat hearts.

Takahito Itoh, Hidemichi Kouzu, Takayuki Miki, Masaya Tanno, Atsushi Kuno, Tatsuya Sato, Daisuke Sunaga, Hiromichi Murase, Tetsuji Miura

Journal of molecular and cellular cardiology 53 ( 6 ) 870 - 9 2012年12月 [査読有り] [国際誌]

Our recent studies indicated that up-regulation of calcineurin activity and unfolded protein responses (UPRs) disrupt cytoprotective Akt- and ERK-signaling in OLETF, a model of obese type 2 diabetes (T2DM). To determine whether the mechanisms can be generalized, we used Goto-Kakizaki rats (GK), a model of non-obese T2DM, in this study. Infarct sizes after 20-min ischemia/2-h reperfusion were similar in GK and non-diabetic controls, Wistar rats (Wistar). However, erythropoietin (EPO) limited infarct size in Wistar (64.0±5.3% vs. 45.7±4.4%, p<0.05) but not in GK (56.2±2.2% vs. 52.6±2.3%). Levels of calcineurin activity and EPO-induced phosphorylation of Akt and ERK were similar in GK and Wistar, though cytosolic HSP70 level was 50% lower and mitochondrial HSP60 level was 60% higher in GK. EPO preserved mitochondrial calcium retention capacity (CRC), an index of the threshold for opening of the mitochondrial permeability transition pore (mPTP), after ischemia/reperfusion in Wistar but not in GK. Interaction of cyclophilin D (CypD) with mitochondrial inorganic phosphate carrier (PiC), which sensitizes the mPTP, was enhanced in GK. There was a negative exponential relationship between CypD-PiC interaction and CRC upon reperfusion, indicating that increase in CRC by reduction of CypD-PiC interaction is smaller when CypD-PiC interaction level is at a higher range. A chemical chaperone, 4-phenylbutyric acid, attenuated the changes in HSPs and CypD-PiC interaction and restored responses of CRC and infarct size to EPO in GK. These results suggest that cytoprotective regulation of the mPTP is impaired in GK by enhanced CypD-PiC interaction in which UPRs are involved.

Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes.

Satoko Ishikawa, Atsushi Kuno, Masaya Tanno, Takayuki Miki, Hidemichi Kouzu, Takahito Itoh, Tatsuya Sato, Daisuke Sunaga, Hiromichi Murase, Tetsuji Miura

American journal of physiology. Heart and circulatory physiology 302 ( 12 ) H2536-44 2012年06月 [査読有り] [国際誌]

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.

Role of ER stress in ventricular contractile dysfunction in type 2 diabetes.

Akifumi Takada, Takayuki Miki, Atsushi Kuno, Hidemichi Kouzu, Daisuke Sunaga, Takahito Itoh, Masaya Tanno, Toshiyuki Yano, Tatsuya Sato, Satoko Ishikawa, Tetsuji Miura

PloS one 7 ( 6 ) e39893 2012年 [査読有り] [国際誌]

BACKGROUND: Diabetes mellitus (DM) is associated with an increased risk of ischemic heart disease and of adverse outcomes following myocardial infarction (MI). Here we assessed the role of endoplasmic reticulum (ER) stress in ventricular dysfunction and outcomes after MI in type 2 DM (T2DM). METHODOLOGY AND PRINCIPAL FINDINGS: In hearts of OLETF, a rat model of T2DM, at 25∼30 weeks of age, GRP78 and GRP94, markers of ER stress, were increased and sarcoplasmic reticulum calcium ATPase (SERCA)2a protein was reduced by 35% compared with those in LETO, a non-diabetic control. SERCA2a mRNA levels were similar, but SERCA2a protein was more ubiquitinated in OLETF than in LETO. Left ventricular (LV) end-diastolic elastance (Eed) was higher in OLETF than in LETO (53.9±5.2 vs. 20.2±5.6 mmHg/µl), whereas LV end-systolic elastance and positive inotropic responses to β-adrenergic stimulation were similar in OLETF and LETO. 4-Phenylbutyric acid (4-PBA), an ER stress modulator, suppressed both GRP up-regulation and SERCA2a ubiquitination and normalized SERCA2a protein level and Eed in OLETF. Sodium tauroursodeoxycholic acid, a structurally different ER stress modulator, also restored SERCA2a protein level in OLETF. Though LV dysfunction was modest, mortality within 48 h after coronary occlusion was markedly higher in OLETF than in LETO (61.3% vs. 7.7%). Telemetric recording showed that rapid progression of heart failure was responsible for the high mortality rate in OLETF. ER stress modulators failed to reduce the mortality rate after MI in OLETF. CONCLUSIONS: ER stress reduces SERCA2a protein via its augmented ubiquitination and degradation, leading to LV diastolic dysfunction in T2DM. Even at a stage without systolic LV dysfunction, susceptibility to lethal heart failure after infarction is markedly increased, which cannot be explained by ER stress or change in myocardial response to sympathetic nerve activation.

The beginning of the calcium transient in rat embryonic heart.

Takeshi Kobayashi, Sachiko Maeda, Nobutoshi Ichise, Tatsuya Sato, Takehito Iwase, Sumihiko Seki, Yoichi Yamada, Noritsugu Tohse

The journal of physiological sciences : JPS 61 ( 2 ) 141 - 9 2011年03月 [査読有り] [国内誌]

Although many researchers have tried to observe the beginning of the heartbeat, no study has shown the beginning of the calcium transient. Here, we evaluate the beginning of the calcium transient in the Wistar rat heart. We first tried to reveal when the heart of the Wistar rat begins to contract because no previous study has evaluated the beginning of the heartbeat in Wistar rats. Observation of embryos transferred to a small incubator mounted on a microscope revealed that the heart primordium, the so-called cardiac crescent, began to contract at embryonic day 9.99-10.13. Observation of embryos loaded with fluo-3 AM revealed that the beginning of the calcium transient precedes the initiation of contraction which precedes the appearance of the linear heart tube. Nifedipine (1 μM), but not ryanodine (1 μM), abolished the calcium transients. These results indicate that calcium transients in the early embryonic period involve exclusively calcium entry through L-type calcium channels in contrast to the situation in mature hearts. This study provides the first demonstration of the relationship between morphological changes in the heart primordium and the beginning of the calcium transient and contraction.

Hypertensive hypertrophied myocardium is vulnerable to infarction and refractory to erythropoietin-induced protection.

Toshiyuki Yano, Takayuki Miki, Masaya Tanno, Atsushi Kuno, Takahito Itoh, Akifumi Takada, Tatsuya Sato, Hidemichi Kouzu, Kazuaki Shimamoto, Tetsuji Miura

Hypertension (Dallas, Tex. : 1979) 57 ( 1 ) 110 - 5 2011年01月 [査読有り] [国際誌]

The objective of this study was to examine the hypothesis that hypertensive hypertrophy is vulnerable to infarction and defective in cytoprotective mechanisms by modification of intracellular signaling and mitochondrial proteins. Myocardial infarction was induced by 20-minute coronary occlusion/reperfusion in spontaneously hypertensive stroke-prone rats (SHR-SPs) and their controls (Wistar-Kyoto rats [WKYs]). Infarct size expressed as a percentage of area-at-risk was larger by 29% in SHR-SPs than in WKYs. Pretreatment with erythropoietin (EPO) significantly limited infarct size in WKYs but not in SHR-SPs. Ca(2+) retention capacity of mitochondria, an index of the threshold for opening of the mitochondrial permeability transition pore, on reperfusion was reduced in SHR-SPs compared with that in WKYs. Suppression of reactive oxygen species by N-(2-mercaptopropionyl)-glycine increased Ca(2+) retention capacity after reperfusion and limited infarct size in SHR-SPs to levels in WKYs. EPO induced phosphorylation of Akt, extracellular signal-related kinase, and glycogen synthase kinase-3β in the myocardium in both WKYs and SHR-SPs. EPO enhanced interaction of phospho-glycogen synthase kinase-3β and adenine nucleotide translocase on reperfusion in WKYs, although such an effect of EPO was not detected in SHR-SPs. The results suggest that enhanced opening of mitochondrial permeability transition pores by reactive oxygen species and modification of the signal downstream of phospho-glycogen synthase kinase-3β in the mitochondria underlie the increased vulnerability to infarction and the lack of anti-infarct tolerance by EPO, respectively, in hypertensive hypertrophied hearts.

Erythropoietin (EPO) affords more potent cardioprotection by activation of distinct signaling to mitochondrial kinases compared with carbamylated EPO.

Yoshiaki Terashima, Tatsuya Sato, Toshiyuki Yano, Ole Maas, Takahito Itoh, Takayuki Miki, Masaya Tanno, Atsushi Kuno, Kazuaki Shimamoto, Tetsuji Miura

Cardiovascular drugs and therapy 24 ( 5-6 ) 401 - 8 2010年12月 [査読有り] [国際誌]

PURPOSE: Erythropoietin (EPO) and its non-erythrogenic derivative, carbarmylated EPO (CEPO), have been reported to activate different receptors (homomeric EPO receptor vs. heteromeric receptor consisting of EPO receptor monomer and common β-subunit). The aim of this study was to examine differences between EPO and CEPO in efficacy of cardioprotection against infarction and in activation of pro-survival kinases. METHODS: In isolated rat hearts, infarction was induced by global ischemia followed by reperfusion. Infarct size was determined 2 h after reperfusion, and ventricular tissues for immunoblotting were sampled at 5 min after reperfusion. RESULTS: Pretreatment with EPO (10 units/ml) before ischemia reduced infarct size (% of risk area; %IS/AR) from 47.0 ± 2.1% of the control after 20-min ischemia to 24.7 ± 4.3% and from 62.0 ± 3.0% after 25-min ischemia to 45.5 ± 4.1%. Desialylated EPO (asialoEPO, 100 ng/ml) mimicked the protection by EPO. However, CEPO (100 ng/ml) failed to reduce infarct size after 20-min ischemia (%IS/AR = 47.5 ± 5.9%) and that after 25-min ischemia (%IS/AR = 56.1 ± 4.2%). The infarct size-limiting effect of CEPO was not shown either by increasing CEPO dose to 500 ng/ml or by shortening ischemia to 15 min. Both EPO and CEPO enhanced phosphorylation of cytosolic GSK-3β upon reperfusion. In contrast, phosphorylation of GSK-3β, Akt, and PKC-ε in mitochondria upon reperfusion was significantly enhanced by EPO but not by CEPO. CONCLUSION: EPO affords more potent protection against infarction than does CEPO by distinct activation of signaling leading to phosphorylation of pro-survival protein kinases in mitochondria upon reperfusion.

Mitochondrial kinase signalling pathways in myocardial protection from ischaemia/reperfusion-induced necrosis.

Tetsuji Miura, Masaya Tanno, Tatsuya Sato

Cardiovascular research 88 ( 1 ) 7 - 15 2010年10月 [査読有り] [国際誌]

Multiple cardioprotective signal pathways that are activated by ischaemic preconditioning (IPC) and those by IPC mimetics converge on mitochondria. Recent studies have shown that pools of Akt, protein kinase C-ε, extracellular-regulated kinases, glycogen synthase kinase-3beta (GSK-3beta), and hexokinases (HK) I and II, are localized in mitochondria in addition to their pools in the cytosol. Accumulating evidence indicates that such 'mitochondrial protein kinases' receive signals from cytosolic molecules and enhance tolerance of myocytes to injury. Proteomic analyses suggest that these kinases form complexes with each other and with subunit proteins of the mitochondrial permeability transition pore (mPTP). Functional relationships between the protein kinases in mitochondria have not been fully clarified, but GSK-3beta and HKs appear to be at the end of the signal pathways and directly responsible for inhibition of opening of the mPTP and, thus, for myocyte protection from necrosis. In this review, recent findings supporting roles of mitochondrial protein kinases in protection from myocardial necrosis after ischaemia/reperfusion are summarized and discussed.

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