Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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Effect of storage conditions on salivary polyamines quantified via liquid chromatography-mass spectrometry

Tomita, A., Mori, M., Hiwatari, K., Yamaguchi, E., Itoi, T., Sunamura, M., Soga, T., Tomita, M., Sugimoto, M.

Sci. Rep. 8 ( 1 ) 12075 2018.08

Research paper (scientific journal), Joint Work, Accepted

© 2018, The Author(s). Salivary polyamines are potential non-invasive tools for screening various types of cancers. For clinical use, the reproducibility of these metabolites should be evaluated under various storage conditions, including duration and temperature, to establish standard operating protocols. Polyamines and amino acids in unstimulated whole saliva were quantified via liquid chromatography-mass spectrometry. Concentrations of time course samples were analysed after short-term storage for up to 240 min and long-term storage for up to 8 days under various storage conditions. As expected, storage at the lowest temperature (−18 °C) exerted the least pronounced effects on the quantified values in both tests. At a higher temperature, polyamines were more stable than amino acids, as evident from polyamine profiling. Addition of ethanol significantly stabilized polyamine profiles even at a higher temperature. Comparative processing of saliva revealed a minor effect of the solvent, whereas drying had a more prominent effect on polyamine profiles. Computational analyses evaluated the ability of polyamines to discriminate pancreatic cancer from controls. Repeated noise added tests were designed on the basis of the results of the storage tests; these analyses confirmed that the discriminative abilities were robust. These data contribute to the standardization of salivary storage conditions, thereby highlighting the clinical utility of saliva.

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Lacking ketohexokinase-A exacerbates renal injury in streptozotocin-induced diabetic mice

Doke, T., Ishimoto, T., Hayasaki, T., Ikeda, S., Hasebe, M., Hirayama, A., Soga, T., Kato, N., Kosugi, T., Tsuboi, N., Lanaspa, MA., Johnson, RJ., Kadomatsu, K., Maruyama, S.

Metabolism (Metabolism: Clinical and Experimental) 85 161 - 170 2018.08

Research paper (scientific journal), Joint Work, ISSN 00260495

© 2018 Elsevier Inc. Objective: Ketohexokinase (KHK), a primary enzyme in fructose metabolism, has two isoforms, namely, KHK-A and KHK-C. Previously, we reported that renal injury was reduced in streptozotocin-induced diabetic mice which lacked both isoforms. Although both isoforms express in kidney, it has not been elucidated whether each isoform plays distinct roles in the development of diabetic kidney disease (DKD). The aim of the study is to elucidate the role of KHK-A for DKD progression. Materials and Methods: Diabetes was induced by five consecutive daily intraperitoneal injections of streptozotocin (50 mg/kg) in C57BL/6J wild-type mice, mice lacking KHK-A alone (KHK-A KO), and mice lacking both KHK-A and KHK-C (KHK-A/C KO). At 35 weeks, renal injury, inflammation, hypoxia, and oxidative stress were examined. Metabolomic analysis including polyol pathway, fructose metabolism, glycolysis, TCA (tricarboxylic acid) cycle, and NAD (nicotinamide adenine dinucleotide) metabolism in kidney and urine was done. Results: Diabetic KHK-A KO mice developed severe renal injury compared to diabetic wild-type mice, and this was associated with further increases of intrarenal fructose, dihydroxyacetone phosphate (DHAP), TCA cycle intermediate levels, and severe inflammation. In contrast, renal injury was prevented in diabetic KHK-A/C KO mice compared to both wild-type and KHK-A KO diabetic mice. Further, diabetic KHK-A KO mice contained decreased renal NAD + level with the increase of renal hypoxia-inducible factor 1-alpha expression despite having increased renal nicotinamide (NAM) level. Conclusion: These results suggest that KHK-C might play a deleterious role in DKD progression through endogenous fructose metabolism, and that KHK-A plays a unique protective role against the development of DKD.

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Low tumor glutathione level as a sensitivity marker for glutamate-cysteine ligase inhibitors

Nishizawa S., Araki H., Ishikawa Y., Kitazawa S., Hata A., Soga T., Hara T.

Oncol. Lett. 15 ( 6 ) 8735 - 8743 2018.06

Research paper (scientific journal), Joint Work, ISSN 17921074

© 2018, Spandidos Publications. All rights reserved. Previous metabolomic analyses of cancer have revealed elevated glutathione levels in tumors. An inhibitor of cystine uptake was identified to suppress glutathione biosynthesis, leading to ferroptosis, a novel iron-dependent form of cell death that differs from apoptosis and necrosis. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione biosynthesis pathway. Buthionine sulfoximine (BSO), a GCL inhibitor, has previously demonstrated limited clinical benefits. Therefore, selecting patients who respond well to the inhibitor is a key approach for successful future drug development. Ferroptosis induction by BSO has not been fully examined in prior studies. Therefore, the present study investigated the pharmacological effects of BSO and the association between basal intracellular glutathione levels and sensitivity to BSO in cultured cell lines derived from various types of cancer, including those of the kidney [769P, 786-O, A-498, A704, ACHN, Caki-1, Caki-2, G401, G402, RCC4 VHL(-/-), RCC4 VHL(+/+), SK-NEP-1 and SW156] and ovaries (A2780 and A2780/CDDP). BSO was demonstrated to suppress glutathione levels and induce lipid peroxidation, thereby inhibiting cell viability. The viability-reducing effects of BSO were attenuated by ferroptosis inhibition and enhanced by iron, indicating that BSO induced ferroptosis in cancer cells. The cell lines sensitive to BSO, including G402, tended to exhibit non-significantly lower levels of glutathione compared with the BSO-insensitive cell lines, including Caki-2 (P=0.08). Patient sample data indicated the existence of a population of colorectal tumors with lower glutathione levels compared with those of matched normal tissues that might be suitable for the clinical testing of sensitivity to GCLC inhibitors. Collectively, these data suggest that GCL inhibition leads to ferroptosis in cancer cells, and that low glutathione tumor levels may be a patient selection marker for the use of GCL inhibitors in the treatment of tumors.

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Development of a sheathless CE‐ESI‐MS interface

Hirayama, A., Abe, H., Yamaguchi, N., Tabata, S., Tomita, T., Soga, T.

Electrophoresis 39 ( 11 ) 1382 - 1389 2018.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 01730835

© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
A sheath-flow interface is the most common ionization technique in CE-ESI-MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE-MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath-flow CE-MS. Because the interface is easy to construct, it is cost-effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE-MS-based metabolomic analysis.

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Changes of Liver Metabolites following Hepatectomy with Ischemia Reperfusion towards Liver Regeneration

Saito, Y., Morine Y., Iwahashi, S., Ikemoto, T., Imura, S., Yamanaka-Okumura, H., Hirayama, A., Soga, T., Tomita, M., Shimada, M.

Ann. Gastroenterol. Surg. 2 ( 3 ) 204 - 211 2018.05

Research paper (scientific journal), Joint Work, Accepted

Background: Metabolome analysis is one of the omics which investigates the final product of a central dogma. Changes of liver metabolites during liver regeneration following hepatectomy (Hx) continue to remain unclear. The aim of the present study was to investigate the changes of liver metabolites following Hx with ischemia reperfusion (I/R) towards liver regeneration. Methods: Twenty-three patients who underwent Hx were enrolled in this study. Non-tumor tissues were sampled immediately before and after Hx and a comparison was made between the liver samples taken before and after Hx using capillary electrophoresis (CE)-time-of-flight mass spectrometry (TOFMS) as metabolome analysis. Results: The metabolic pathway showed that there was a significant increase in "lactate" following Hx. There was a significant decrease in metabolites only in the first half of the tricarboxylic acid cycle (TCA) cycle, and adenosine triphosphate (ATP) by anaerobiotic glycolysis did not occur in time for energy consumption of the Hx. Principal component analysis revealed remarkably different component profiles between the samples taken before and after Hx. One hundred and three metabolites were selected as critical metabolites for separating components. Valine and tryptophan increased significantly after Hx and they were regulated by resected liver volume, ischemic time and liver function. Conclusion: The liver metabolites changed remarkably between before and after Hx. Especially, liver valine and tryptophan were increased.

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Thymidine catabolism promotes NADPH oxidase-derived reactive oxygen species (ROS) signalling in KB and yumoto cells

Tabata, S., Yamamoto, M., Goto, H., Hirayama, A., Ohishi, M., Kuramoto, T., Mitsuhashi, A., Ikeda, R., Haraguchi, M., Kawahara, K., Shinsato, Y., Minami, K., Saijo, A., Toyoda, Y., Hanibuchi, M., Nishioka, Y., Sone, S., Esumi, H., Tomita, M., Soga, T., Furukawa, T., Akiyama, S.

Sci. Rep. 8 ( 1 ) 6760 2018.04

Research paper (scientific journal), Joint Work, Accepted

Thymidine phosphorylase (TP) is a rate-limiting enzyme in the thymidine catabolic pathway. TP is identical to platelet-derived endothelial cell growth factor and contributes to tumour angiogenesis. TP induces the generation of reactive oxygen species (ROS) and enhances the expression of oxidative stress-responsive genes, such as interleukin (IL)-8. However, the mechanism underlying ROS induction by TP remains unclear. In the present study, we demonstrated that TP promotes NADPH oxidase-derived ROS signalling in cancer cells. NADPH oxidase inhibition using apocynin or small interfering RNAs (siRNAs) abrogated the induction of IL-8 and ROS in TP-expressing cancer cells. Meanwhile, thymidine catabolism induced by TP increased the levels of NADPH and intermediates of the pentose phosphate pathway (PPP). Both siRNA knockdown of glucose 6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme in PPP, and a G6PD inhibitor, dihydroepiandrosterone, reduced TP-induced ROS production. siRNA downregulation of 2-deoxy-D-ribose 5-phosphate (DR5P) aldolase, which is needed for DR5P to enter glycolysis, also suppressed the induction of NADPH and IL-8 in TP-expressing cells. These results suggested that TP-mediated thymidine catabolism increases the intracellular NADPH level via the PPP, which enhances the production of ROS by NADPH oxidase and activates its downstream signalling.

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Purine nucleotide metabolism regulates expression of the human immune ligand MICA

McCarthy, MT., Moncayo, G., Hiron, TK., Jakobsen, NA., Valli, A., Soga, T., Adam, J., O'Callaghan, CA.

J. Biol. Chem. 293 ( 11 ) 3913-3924 - 3924 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219258

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the cell-surface glycoprotein MHC class I polypeptide- related sequence A (MICA) is induced in dangerous, abnormal, or "stressed" cells, including cancer cells, virus-infected cells, and rapidly proliferating cells. MICA is recognized by the activating immune cell receptor natural killer group 2D (NKG2D), providing a mechanism by which immune cells can identify and potentially eliminate pathological cells. Immune recognition through NKG2D is implicated in cancer, atherosclerosis, transplant rejection, and inflammatory diseases, such as rheumatoid arthritis. Despite the wide range of potential therapeutic applications ofMICAmanipulation, the factors that control MICA expression are unclear. Here we use metabolic interventions and metabolomic analyses to show that the transition from quiescent cellular metabolism to a "Warburg" or biosynthetic metabolic state induces MICA expression. Specifically, we show that glucose transport into the cell and active glycolytic metabolism are necessary to up-regulate MICA expression. Active purine synthesis is necessary to support this effect of glucose, and increases in purine nucleotide levels are sufficient to induce MICA expression. Metabolic induction of MICA expression directly influences NKG2D-dependent cytotoxicity by immune cells. These findings support a model of MICA regulation whereby the purine metabolic activity of individual cells is reflected by cell-surface MICA expression and is the subject of surveillance by NKG2D receptor-expressing immune cells.

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PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth

Morita, M., Sato, T., Nomura, M., Sakamoto, Y., Inoue, Y., Tanaka, R., Ito, S., Kurosawa, K., Yamaguchi, K., Sugiura, Y., Takizaki, H., Yamashita, Y., Katakura, R., Sato, I., Kawai, M., Okada, Y., Watanabe, H., Kondoh, G., Matsumoto, S., Kishimoto, A., Obata, M., Matsumoto, M., Fukuhara, T., Motohashi, H., Suematsu, M., Komatsu, M., Nakayama, KI., Watanabe, T., Soga, T., Shima, H., Maemondo, M., Tanuma. N.

Cancer Cell (Cancer Cell) 33 ( 3 ) 355 - 367.e7 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 15356108

Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including small-cell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs.

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Urinary Polyamine Biomarker Panels with Machine-Learning Differentiated Colorectal Cancers, Benign Disease, and Healthy Controls

Nakajima, T., Katsumata, K., Kuwabara, H., Soya, R., Enomoto, M., Ishizaki, T., Tsuchida, A., Mori, M., Hiwatari, K., Soga, T., Tomita, M., Sugimoto, M.

Int. J. Mol. Sci. (Int. J. Mol. Sci.) 19 ( 3 ) 756 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 16616596

Colorectal cancer (CRC) is one of the most daunting diseases due to its increasing worldwide prevalence, which requires imperative development of minimally or non-invasive screening tests. Urinary polyamines have been reported as potential markers to detect CRC, and an accurate pattern recognition to differentiate CRC with early stage cases from healthy controls are needed. Here, we utilized liquid chromatography triple quadrupole mass spectrometry to profile seven kinds of polyamines, such as spermine and spermidine with their acetylated forms. Urinary samples from 201 CRCs and 31 non-CRCs revealed the N₁,N12-diacetylspermine showing the highest area under the receiver operating characteristic curve (AUC), 0.794 (the 95% confidence interval (CI): 0.704-0.885, p < 0.0001), to differentiate CRC from the benign and healthy controls. Overall, 59 samples were analyzed to evaluate the reproducibility of quantified concentrations, acquired by collecting three times on three days each from each healthy control. We confirmed the stability of the observed quantified values. A machine learning method using combinations of polyamines showed a higher AUC value of 0.961 (95% CI: 0.937-0.984, p < 0.0001). Computational validations confirmed the generalization ability of the models. Taken together, polyamines and a machine-learning method showed potential as a screening tool of CRC.

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Microhomology-assisted scarless genome editing in human iPSCs

Kim, S., Matsumoto, T., Kagawa, H., Nakamura, M., Hirohata, R., Ueno, A., Ohishi, M., Sakuma, T., Soga, T., Yamamoto, T., Woltjen, K.

Nat. Commun. 9 ( 1 ) 939 2018.03

Research paper (scientific journal), Joint Work, Accepted

© 2018 The Author(s). Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.

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Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors

Oshima, K., Saiki, N., Tanaka, M., Imamura, H. Niwa, A., Tanimura, A., Nagahashi;, A., Hirayama, A., Okita, K., Hotta, A., Kitayama, S., Osawa, M., Kaneko, S., Watanabe, A., Asaka, I., Fujibuchi W., Imai, K., Yabe, H., Kamachi, Y., Hara J., Kojima, S., Tomita, M., Soga, T., Noma, T., Nonoyama, S., Nakahata, T., Saito, M.K.

Biochem. Biophys. Res. Commun. 497 ( 2 ) 719-725 - 725 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells.

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Elevated Polyamines in Saliva of Pancreatic Cancer

Asai, Y., Itoi, T., Sugimoto, M., Sofuni, A., Tsuchiya, T, Tanaka, R., Tonozuka, R., Honjo, M., Mukai, S., Fujita, M., Yamamoto, K., Matsunami, Y., Kurosawa, T., Nagakawa, Y., Kaneko, M., Ota, S., Kawachi, S., Shimazu, M., Soga, T., Tomita, M., Sunamura., M.

Cancers 10 ( 2 ) pii: E43 2018.02

Research paper (scientific journal), Joint Work, Accepted

Detection of pancreatic cancer (PC) at a resectable stage is still difficult because of the lack of accurate detection tests. The development of accurate biomarkers in low or non-invasive biofluids is essential to enable frequent tests, which would help increase the opportunity of PC detection in early stages. Polyamines have been reported as possible biomarkers in urine and saliva samples in various cancers. Here, we analyzed salivary metabolites, including polyamines, using capillary electrophoresis-mass spectrometry. Salivary samples were collected from patients with PC (n = 39), those with chronic pancreatitis (CP, n = 14), and controls (C, n = 26). Polyamines, such as spermine, N₁-acetylspermidine, and N₁-acetylspermine, showed a significant difference between patients with PC and those with C, and the combination of four metabolites including N₁-acetylspermidine showed high accuracy in discriminating PC from the other two groups. These data show the potential of saliva as a source for tests screening for PC.

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A Transient Rise in Free Mg2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation

Maeshima, K., Matsuda, T., Shindo, Y., Imamura, H., Tamura, S., Imai, R., Kawakami, S., Nagashima, R., Soga, T., Noji, H., Oka, K., Nagai, T.

Curr. Biol. 28 ( 3 ) 444 - 451 2018.02

Research paper (scientific journal), Joint Work, Accepted

For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg2+, have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ increase is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell [33]. ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance.

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Ketone body 3-hydroxybutyrate mimics calorie restriction via the Nrf2 activator, fumarate, in the retina

Izuta, Y., Imada, T., Hisamura, R., Oonishi, E., Nakamura. S., Inagaki, E., Ito, M., Soga, T., Tsubota, K.

Aging Cell 17 ( 1 ) e12699 2018.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 14749718

Calorie restriction (CR) being the most robust dietary intervention provides various health benefits. D-3-hydroxybutyrate (3HB), a major physiological ketone, has been proposed as an important endogenous molecule for CR. To investigate the role of 3HB in CR, we investigated potential shared mechanisms underlying increased retinal 3HB induced by CR and exogenously applied 3HB without CR to protect against ischemic retinal degeneration. The repeated elevation of retinal 3HB, with or without CR, suppressed retinal degeneration. Metabolomic analysis showed that the antioxidant pentose phosphate pathway and its limiting enzyme, glucose-6-phosphate dehydrogenase (G6PD), were concomitantly preserved. Importantly, the upregulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a regulator of G6PD, and elevation of the tricarboxylic acid cycle's Nrf2 activator, fumarate, were also shared. Together, our findings suggest that CR provides retinal antioxidative defense by 3HB through the antioxidant Nrf2 pathway via modification of a tricarboxylic acid cycle intermediate during 3HB metabolism.

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Comparative analysis of cerebrospinal fluid metabolites in Alzheimer’s disease and idiopathic normal pressure hydrocephalus in a Japanese cohort

Nagata, Y., Hirayama, A., Ikeda, S., Shirahata, A., Shoji, F., Maruyama, M., Kayano, M., Bundo, M., Hattori, K., Yoshida, S., Goto, Y., Urakami, K., Soga, T., Ozaki, K., Niida, S.

Biomark. Res. 6 ( 1 ) 5 2018.01

Research paper (scientific journal), Joint Work, Accepted

© 2018 The Author(s).
Background: Alzheimer's disease (AD) is a most common dementia in elderly people. Since AD symptoms resemble those of other neurodegenerative diseases, including idiopathic normal pressure hydrocephalus (iNPH), it is difficult to distinguish AD from iNPH for a precise and early diagnosis. iNPH is caused by the accumulation of cerebrospinal fluid (CSF) and involves gait disturbance, urinary incontinence, and dementia. iNPH is treatable with shunt operation which removes accumulated CSF from the brain ventricles. Methods: We performed metabolomic analysis in the CSF of patients with AD and iNPH with capillary electrophoresis-mass spectrometry. We assessed metabolites to discriminate between AD and iNPH with Welch's t-test, receiver operating characteristic (ROC) curve analysis, and multiple logistic regression analysis. Results: We found significant increased levels of glycerate and N-acetylneuraminate and significant decreased levels of serine and 2-hydroxybutyrate in the CSF of patients with AD compared to the CSF of patients with iNPH. The ROC curve analysis with these four metabolites showed that the area under the ROC curve was 0.90, indicating good discrimination between AD and iNPH. Conclusions: This study identified four metabolites that could possibly discriminate between AD and iNPH, which previous research has shown are closely related to the risk factors, pathogenesis, and symptoms of AD. Analyzing pathway-specific metabolites in the CSF of patients with AD may further elucidate the mechanism and pathogenesis of AD.

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Reliability of plasma polar metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry

Harada, S., Hirayama, A., Chan, Q., Kurihara, A., Fukai, K., Iida, M., Kato, S., Sugiyama, D., Kuwabara, K., Takeuchi, A., Akiyama, M., Okamura, T., Ebbels, TMD., Elliott, P., Tomita, M., Sato, A., Suzuki, C., Sugimoto, M., Soga, T., Takebayashi, T.

PLoS One 13 ( 1 ) e0191230 2018.01

Research paper (scientific journal), Single Work, Accepted

Background: Cohort studies with metabolomics data are becoming more widespread, however, large-scale studies involving 10,000s of participants are still limited, especially in Asian populations. Therefore, we started the Tsuruoka Metabolomics Cohort Study enrolling 11,002 community-dwelling adults in Japan, and using capillary electrophoresis-mass spectrometry (CE-MS) and liquid chromatography-mass spectrometry. The CE-MS method is highly amenable to absolute quantification of polar metabolites, however, its reliability for large-scale measurement is unclear. The aim of this study is to examine reproducibility and validity of large-scale CE-MS measurements. In addition, the study presents absolute concentrations of polar metabolites in human plasma, which can be used in future as reference ranges in a Japanese population.

Methods: Metabolomic profiling of 8,413 fasting plasma samples were completed using CE-MS, and 94 polar metabolites were structurally identified and quantified. Quality control (QC) samples were injected every ten samples and assessed throughout the analysis. Inter- and intra-batch coefficients of variation of QC and participant samples, and technical intraclass correlation coefficients were estimated. Passing-Bablok regression of plasma concentrations by CE-MS on serum concentrations by standard clinical chemistry assays was conducted for creatinine and uric acid.

Results and conclusions: In QC samples, coefficient of variation was less than 20% for 64 metabolites, and less than 30% for 80 metabolites out of the 94 metabolites. Inter-batch coefficient of variation was less than 20% for 81 metabolites. Estimated technical intraclass correlation coefficient was above 0.75 for 67 metabolites. The slope of Passing-Bablok regression was estimated as 0.97 (95% confidence interval: 0.95, 0.98) for creatinine and 0.95 (0.92, 0.96) for uric acid. Compared to published data from other large cohort measurement platforms, reproducibility of metabolites common to the platforms was similar to or better than in the other studies. These results show that our CE-MS platform is suitable for conducting large-scale epidemiological studies.

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Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

Matsuda, J., Namba, T., Takabatake, Y., Kimura, T., Takahashi, A., Yamamoto, T., Minami, S., Sakai, S., Fujimura, R., Kaimori, J.Y., Matsui, I., Hamano, T., Fukushima, Y., Matsui, K., Soga, T., Isaka, Y.

Autophagy 14 ( 1 ) 53 - 65 2018.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 15548627

Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific atg5-deficient mice (atg5-conditional KO [cKO] mice). Glomeruli of 4-wk-old atg5-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old atg5-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The atg5-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk atg5-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the atg5-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to atg5-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.

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Dynamic metabolomics reveals that insulin primes the adipocyte for glucose metabolism

Krycer, JR., Yugi, K., Hirayama, A., Fazakerley, DJ., Quek, LE., Scalzo, R., Ohno, S., Hodson, MP. Ikeda, S., Shoji, F., Suzuki, K., Domanova, W., Parker, BL.,. Nelson, ME ., Humphrey, SJ., Turner, N., Hoehn, KL., Cooney, GJ., Soga, T., Kuroda, S., James, DE.

Cell Rep. 21 ( 12 ) 3536 - 3547 2017.12

Research paper (scientific journal), Joint Work, Accepted

Insulin triggers an extensive signaling cascade to coordinate adipocyte glucose metabolism. It is considered that the major role of insulin is to provide anabolic substrates by activating GLUT4-dependent glucose uptake. However, insulin stimulates phosphorylation of many metabolic proteins. To examine the implications of this on glucose metabolism, we performed dynamic tracer metabolomics in cultured adipocytes treated with insulin. Temporal analysis of metabolite concentrations and tracer labeling revealed rapid and distinct changes in glucose metabolism, favoring specific glycolytic branch points and pyruvate anaplerosis. Integrating dynamic metabolomics and phosphoproteomics data revealed that insulin-dependent phosphorylation of anabolic enzymes occurred prior to substrate accumulation. Indeed, glycogen synthesis was activated independently of glucose supply. We refer to this phenomenon as metabolic priming, whereby insulin signaling creates a demand-driven system to "pull" glucose into specific anabolic pathways. This complements the supply-driven regulation of anabolism by substrate accumulation and highlights an additional role for insulin action in adipocyte glucose metabolism.

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Cancer-Specific Energy Metabolism in Rhabdomyosarcoma Cells Is Regulated by MicroRNA

Sugito, N., Taniguchi, K., Kuranaga, Y., Ohishi, M., Soga, T., Ito, Y., Miyachi, M., Kikuchi, K., Hosoi, H., Akao, Y.

Nucleic Acid Ther. 27 ( 6 ) 365 - 377 2017.12

Research paper (scientific journal), Accepted

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma and is most frequently found in children. In RMS, there are two major subtypes, that is, embryonal RMS and alveolar RMS (ARMS). ARMS has exclusively the worse prognosis and is caused by formation of the chimeric PAX3-FOXO1 gene. Regarding cancer, the Warburg effect is known as a feature of cancer-specific metabolism. Polypyrimidine tract-binding protein 1 (PTBP1), a splicer of pyruvate kinase muscle (PKM) mRNA, is a positive regulator of cancer-specific energy metabolism. We investigated the expression and effects of muscle-specific miR-1 and miR-133b on RMS cells (RD, KYM-1, Rh30, and Rh41) from the view of energy metabolism and regulation of the chimeric gene. As a result, downregulated miR-1 and miR-133b/upregulated PTBP1 were found in RMS cell lines as well as in RMS clinical cases. Ectopic expression of either miR in both types of RMS cells induced autophagic cell death through silencing of PTBP1. Interestingly, we validated that miR-133b also knock downed PAX3-FOXO1. Moreover, we found that PAX3-FOXO1 positively regulated the PKM2-dominant expression through enhanced expression of PTBP1. These findings suggest that the miR-1 and miR-133b/PTBP1 axis and miR-133b/PAX3-FOXO1/PTBP1 axis contributed to the maintenance of cancer-specific energy metabolism.

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UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homestasis

Ikeda, K., Kang, Q., Yoneshiro, T., Camporez, J.P., Maki, H., Homma, M., Shinoda, K., Chen, Y., Lu, X., Maretich, P., Tajima, K., Ajuwon, K.M., Soga, T., Kajimura, S.

Nat. Med. 23 ( 12 ) 1454 - 1465 2017.12

Research paper (scientific journal), Joint Work, Accepted

Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.

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