Publications by authors named "Tomoyoshi Soga"

299 Publications

SI-MOIRAI: A new method to identify and quantify the metabolic fate of nucleotides.

J Biochem 2021 Jul 9. Epub 2021 Jul 9.

Division of Hematology and Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, 45267, USA.

Since the discovery of nucleotides over 100 years ago, extensive studies have revealed the importance of nucleotides for homeostasis, health, and disease. However, there remains no established method to investigate quantitively and accurately intact nucleotide incorporation into RNA and DNA. Herein, we report a new method, Stable-Isotope Measure Of Influxed Ribonucleic Acid Index (SI-MOIRAI), for the identification and quantification of the metabolic fate of ribonucleotides and their precursors. SI-MOIRAI, named after Greek goddesses of fate, combines a stable isotope-labeling flux assay with mass spectrometry to enable quantification of the newly synthesized ribonucleotides into r/m/tRNA under a metabolic stationary state. Using glioblastoma U87MG cells and a patient-derived xenograft (PDX) glioblastoma mouse model, SI-MOIRAI analyses showed that newly synthesized GTP was particularly and disproportionally highly utilized for rRNA and tRNA synthesis but not for mRNA synthesis in glioblastoma (GBM) in vitro and in vivo. Furthermore, newly synthesized pyrimidine nucleotides exhibited a significantly lower utilization rate for RNA synthesis than newly synthesized purine nucleotides. The results reveal the existence of discrete pathways and compartmentalization of purine and pyrimidine metabolism designated for RNA synthesis, demonstrating the capacity of SI-MOIRAI to reveal previously unknown aspects of nucleotide biology.
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http://dx.doi.org/10.1093/jb/mvab077DOI Listing
July 2021

High-throughput screening of salivary polyamine markers for discrimination of colorectal cancer by multisegment injection capillary electrophoresis tandem mass spectrometry.

J Chromatogr A 2021 Jun 20;1652:462355. Epub 2021 Jun 20.

Institute for Advanced Biosciences, Keio University, 246-2 Mizukami, Kakuganji, Tsuruoka 997-0052, Japan; Faculty of Environmental Information Studies, Keio University, 5322 Endo, Fujisawa 252-0882, Japan. Electronic address:

Polyamine metabolites provide pathophysiological information on disease or therapeutic efficacy, yet rapid screening methods for these biomarkers are lacking. Here, we developed high-throughput polyamine metabolite profiling based on multisegment injection capillary electrophoresis triple quadrupole tandem mass spectrometry (MSI-CE-MS/MS), which allows sequential 40-sample injection followed by electrophoretic separation and specific mass detection. To achieve consecutive analysis of polyamine samples, 1 M formic acid was used as the background electrolyte (BGE). The BGE spacer volume had an apparent effect on peak resolution among samples, and 20 nL was selected as the optimal volume. The use of polyamine isotopomers as the internal standard enabled the correction of matrix effects in MS detection. This method is sensitive, selective and quantitative, and its utility was demonstrated by screening polyamines in 359 salivary samples within 360 min, resulting in discrimination of colorectal cancer patients from noncancer controls.
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http://dx.doi.org/10.1016/j.chroma.2021.462355DOI Listing
June 2021

De novo deoxyribonucleotide biosynthesis regulates cell growth and tumor progression in small-cell lung carcinoma.

Sci Rep 2021 Jun 29;11(1):13474. Epub 2021 Jun 29.

Shonai Regional Industry Promotion Center, Tsuruoka, Yamagata, 997-0052, Japan.

Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.
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http://dx.doi.org/10.1038/s41598-021-92948-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8242079PMC
June 2021

TGF-β-dependent reprogramming of amino acid metabolism induces epithelial-mesenchymal transition in non-small cell lung cancers.

Commun Biol 2021 Jun 24;4(1):782. Epub 2021 Jun 24.

Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.

Epithelial-mesenchymal transition (EMT)-a fundamental process in embryogenesis and wound healing-promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer.
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http://dx.doi.org/10.1038/s42003-021-02323-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225889PMC
June 2021

Amino acid transporters as emerging therapeutic targets in cancer.

Cancer Sci 2021 Jun 6. Epub 2021 Jun 6.

Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.

Amino acids are indispensable nutrients for both normal and cancer cells. Cancer cells are unable to synthesize essential amino acids as well as some non-essential amino acids adequately to support rapid proliferation, and must take up amino acids from the surroundings. To meet the increased demand for the amino acid needed for proliferation, high levels of amino acid transporters are expressed on the surface of cancer cells. Cancer cells utilize amino acids to synthesize proteins and nucleotides, as well as to obtain energy. In addition, amino acids are known to play pathological roles in cancer cells. Interestingly, breast cancer cells limit the use of amino acids for cell proliferation based on amino acid availability, which depends on estrogen receptor status. Here, we present a summarized literature review of novel amino acid functions in cancer cells. This review organizes the available knowledge on 2 amino acid transporters, SLC7A5 and SLC7A11, which are considered essential for breast cancer cell growth in a cell-dependent manner. In particular, we propose the glutamine recycling model to clarify the mechanism underlying aberrant SLC7A5 activation. Finally, we overview the pathological significances of SLC7A5 and SLC7A11 in cancer tissues.
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http://dx.doi.org/10.1111/cas.15006DOI Listing
June 2021

The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia.

Curr Biol 2021 Jul 3;31(14):3086-3097.e7. Epub 2021 Jun 3.

Department of Breast and Endocrine Surgery (Surgery II), Nagoya University Graduate School of Medicine, Nagoya, Japan.

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.
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http://dx.doi.org/10.1016/j.cub.2021.04.078DOI Listing
July 2021

CE-MS-Based Identification of Uremic Solutes Specific to Hemodialysis Patients.

Toxins (Basel) 2021 04 30;13(5). Epub 2021 Apr 30.

Division of Nephrology, Endocrinology and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.

Uremic toxins are suggested to be involved in the pathophysiology of hemodialysis (HD) patients. However, the profile of uremic solutes in HD patients has not been fully elucidated. In this study using capillary electrophoresis mass spectrometry (CE-MS), we comprehensively quantified the serum concentrations of 122 ionic solutes before and after HD in 11 patients. In addition, we compared the results with those in non-HD patients with chronic kidney disease (CKD) to identify HD patient-specific solutes. We identified 38 solutes whose concentrations were higher in pre-HD than in CKD stage G5. Ten solutes among them did not significantly accumulate in non-HD CKD patients, suggesting that these solutes accumulate specifically in HD patients. We also identified 23 solutes whose concentrations were lower in both pre- and post-HD than in CKD stage G5. The serum levels of 14 solutes among them were not affected by renal function in non-HD patients, suggesting that these solutes tend to be lost specifically in HD patients. Our data demonstrate that HD patients have a markedly different profile of serum uremic solute levels compared to that in non-HD CKD patients. The solutes identified in our study may contribute to the pathophysiology of HD patients.
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http://dx.doi.org/10.3390/toxins13050324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147146PMC
April 2021

A Metabolomic Profile Predictive of New Osteoporosis or Sarcopenia Development.

Metabolites 2021 Apr 28;11(5). Epub 2021 Apr 28.

Department of Orthopedic Surgery, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan.

The increasing number of patients with osteoporosis and sarcopenia is a global concern among countries with progressively aging societies. The high medical costs of treating those patients suggest that prevention rather than treatment is preferable. We enrolled 729 subjects who attended both the second and third surveys of the Research on Osteoarthritis/Osteoporosis Against Disability (ROAD) study. Blood samples were collected from subjects at the second survey, and then a comprehensive metabolomic analysis was performed. It was found that 35 had newly developed osteoporosis at the third survey performed four years later, and 39 were newly diagnosed with sarcopenia at the third survey. In the second survey, we found that serum Gly levels were significantly higher even after adjustment for age, sex, and BMI in subjects with newly developed osteoporosis relative to those who remained osteoporosis-negative during the four-year follow-up. We also show that serum taurine levels were significantly lower at the second survey, even after adjustment for age, sex, and BMI in subjects with newly developed sarcopenia during the four-year follow-up compared with those not diagnosed with sarcopenia at the second or third surveys. Though our sample size and odds ratios were small, increased Gly and decreased taurine levels were found to be predictive of new development of osteoporosis and sarcopenia, respectively, within four years.
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http://dx.doi.org/10.3390/metabo11050278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8145554PMC
April 2021

Quality Assessment of Untargeted Analytical Data in a Large-Scale Metabolomic Study.

J Clin Med 2021 Apr 22;10(9). Epub 2021 Apr 22.

Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan.

Large-scale metabolomic studies have become common, and the reliability of the peak data produced by the various instruments is an important issue. However, less attention has been paid to the large number of uncharacterized peaks in untargeted metabolomics data. In this study, we tested various criteria to assess the reliability of 276 and 202 uncharacterized peaks that were detected in a gathered set of 30 plasma and urine quality control samples, respectively, using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). The linear relationship between the amounts of pooled samples and the corresponding peak areas was one of the criteria used to select reliable peaks. We used samples from approximately 3000 participants in the Tsuruoka Metabolome Cohort Study to investigate patterns of the areas of these uncharacterized peaks among the samples and clustered the peaks by combining the patterns and differences in the migration times. Our assessment pipeline removed substantial numbers of unreliable or redundant peaks and detected 35 and 74 reliable uncharacterized peaks in plasma and urine, respectively, some of which may correspond to metabolites involved in important physiological processes such as disease progression. We propose that our assessment pipeline can be used to help establish large-scale untargeted clinical metabolomic studies.
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http://dx.doi.org/10.3390/jcm10091826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122759PMC
April 2021

Metabolomic Analysis of Small Extracellular Vesicles Derived from Pancreatic Cancer Cells Cultured under Normoxia and Hypoxia.

Metabolites 2021 Apr 1;11(4). Epub 2021 Apr 1.

Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan.

Extracellular vesicles (EVs) released from cancer cells contribute to various malignant phenotypes of cancer, including metastasis, cachexia, and angiogenesis. Although DNA, mRNAs, miRNAs, and proteins contained in EVs have been extensively studied, the function of metabolites in EVs remains unclear. In this study, we performed a comprehensive metabolomic analysis of pancreatic cancer cells, PANC-1, cultured under different oxygen concentrations, and small EVs (sEVs) released from them, considering the fact that hypoxia contributes to the malignant behavior of cells in pancreatic cancer, which is a poorly diagnosed cancer. sEVs were collected by ultracentrifugation, and hydrophilic metabolites were analyzed using capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercritical fluid chromatography-tandem mass spectrometry. A total of 140 hydrophilic metabolites and 494 lipids were detected in sEVs, and their profiles were different from those in cells. In addition, the metabolomic profile of sEVs was observed to change under hypoxic stress, and an increase in metabolites involved in angiogenesis was also detected. We reveal the hallmark of the metabolites contained in sEVs and the effect of tumor hypoxia on their profiles, which may help in understanding EV-mediated cancer malignancy.
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http://dx.doi.org/10.3390/metabo11040215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066639PMC
April 2021

Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in .

Elife 2021 04 27;10. Epub 2021 Apr 27.

RIKEN CPR, Kobe, Japan.

Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.
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http://dx.doi.org/10.7554/eLife.59809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079150PMC
April 2021

Reliability of urinary charged metabolite concentrations in a large-scale cohort study using capillary electrophoresis-mass spectrometry.

Sci Rep 2021 Apr 1;11(1):7407. Epub 2021 Apr 1.

Department of Preventive Medicine and Public Health, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, Japan.

Currently, large-scale cohort studies for metabolome analysis have been launched globally. However, only a few studies have evaluated the reliability of urinary metabolome analysis. This study aimed to establish the reliability of urinary metabolomic profiling in cohort studies. In the Tsuruoka Metabolomics Cohort Study, 123 charged metabolites were identified and routinely quantified using capillary electrophoresis-mass spectrometry (CE-MS). We evaluated approximately 750 quality control (QC) samples and 6,720 participants' spot urine samples. We calculated inter- and intra-batch coefficients of variation in the QC and participant samples and technical intraclass correlation coefficients (ICC). A correlation of metabolite concentrations between spot and 24-h urine samples obtained from 32 sub-cohort participants was also evaluated. The coefficient of variation (CV) was less than 20% for 87 metabolites (70.7%) and 20-30% for 19 metabolites (15.4%) in the QC samples. There was less than 20% inter-batch CV for 106 metabolites (86.2%). Most urinary metabolites would have reliability for measurement. The 96 metabolites (78.0%) was above 0.75 for the estimated ICC, and those might be useful for epidemiological analysis. Among individuals, the Pearson correlation coefficient of 24-h and spot urine was more than 70% for 59 of the 99 metabolites. These results show that the profiling of charged metabolites using CE-MS in morning spot human urine is suitable for epidemiological metabolomics studies.
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http://dx.doi.org/10.1038/s41598-021-86600-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016858PMC
April 2021

Cardiac mitofusin-1 is reduced in non-responding patients with idiopathic dilated cardiomyopathy.

Sci Rep 2021 Mar 24;11(1):6722. Epub 2021 Mar 24.

Department of Cardiovascular Biology and Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 951-8510, Japan.

Prognosis of severe heart failure remains poor. Urgent new therapies are required. Some heart failure patients do not respond to established multidisciplinary treatment and are classified as "non-responders". The outcome is especially poor for non-responders, and underlying mechanisms are largely unknown. Mitofusin-1 (Mfn1), a mitochondrial fusion protein, is significantly reduced in non-responding patients. This study aimed to elucidate the role of Mfn1 in the failing heart. Twenty-two idiopathic dilated cardiomyopathy (IDCM) patients who underwent endomyocardial biopsy of intraventricular septum were included. Of the 22 patients, 8 were non-responders (left ventricular (LV) ejection fraction (LVEF) of < 10% improvement at late phase follow-up). Electron microscopy (EM), quantitative PCR, and immunofluorescence studies were performed to explore the biological processes and molecules involved in failure to respond. Studies in cardiac specific Mfn1 knockout mice (c-Mfn1 KO), and in vitro studies with neonatal rat ventricular myocytes (NRVMs) were also conducted. A significant reduction in mitochondrial size in cardiomyocytes, and Mfn1, was observed in non-responders. A LV pressure overload with thoracic aortic constriction (TAC) c-Mfn1 KO mouse model was generated. Systolic function was reduced in c-Mfn1 KO mice, while mitochondria alteration in TAC c-Mfn1 KO mice increased. In vitro studies in NRVMs indicated negative regulation of Mfn1 by the β-AR/cAMP/PKA/miR-140-5p pathway resulting in significant reduction in mitochondrial respiration of NRVMs. The level of miR140-5p was increased in cardiac tissues of non-responders. Mfn1 is a biomarker of heart failure in non-responders. Therapies targeting mitochondrial dynamics and homeostasis are next generation therapy for non-responding heart failure patients.
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http://dx.doi.org/10.1038/s41598-021-86209-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990924PMC
March 2021

MEK inhibition preferentially suppresses anchorage-independent growth in osteosarcoma cells and decreases tumors in vivo.

J Orthop Res 2021 Mar 10. Epub 2021 Mar 10.

Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Osteosarcoma is the most common high-grade malignancy of bone, and novel therapeutic options are urgently required. Previously, we developed mouse osteosarcoma AXT cells that can proliferate both under adherent and nonadherent conditions. Based on metabolite levels, nonadherent conditions were more similar to the in vivo environment than adherent conditions. A drug screen identified MEK inhibitors, including trametinib, that preferentially decreased the viability of nonadherent AXT cells. Trametinib inhibited the cell cycle and induced apoptosis in AXT cells, and both effects were stronger under nonadherent conditions. Trametinib also potently decreased viability in U2OS cells, but its effects were less prominent in MG63 or Saos2 cells. By contrast, MG63 and Saos2 cells were more sensitive to PI3K inhibition than AXT or U2OS cells. Notably, the combination of MAPK/ERK kinase (MEK) and PI3K inhibition synergistically decreased viability in U2OS and AXT cells, but this effect was less pronounced in MG63 or Saos2 cells. Therefore, signal dependence for cell survival and crosstalk between MEK-ERK and PI3K-AKT pathways in osteosarcoma are cell context-dependent. The activation status of other kinases including CREB varied in a cell context-dependent manner, which might determine the response to MEK inhibition. A single dose of trametinib was sufficient to decrease the size of the primary tumor and circulating tumor cells in vivo. Moreover, combined administration of trametinib and rapamycin or conventional anticancer drugs further increased antitumor activity. Thus, given optimal biomarkers for predicting its effects, trametinib holds therapeutic potential for the treatment of osteosarcoma.
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http://dx.doi.org/10.1002/jor.25023DOI Listing
March 2021

Trans-omic analysis reveals obesity-associated dysregulation of inter-organ metabolic cycles between the liver and skeletal muscle.

iScience 2021 Mar 20;24(3):102217. Epub 2021 Feb 20.

Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.

Systemic metabolic homeostasis is regulated by inter-organ metabolic cycles involving multiple organs. Obesity impairs inter-organ metabolic cycles, resulting in metabolic diseases. The systemic landscape of dysregulated inter-organ metabolic cycles in obesity has yet to be explored. Here, we measured the transcriptome, proteome, and metabolome in the liver and skeletal muscle and the metabolome in blood of fasted wild-type and leptin-deficient obese (/) mice, identifying components with differential abundance and differential regulation in / mice. By constructing and evaluating the trans-omic network controlling the differences in metabolic reactions between fasted wild-type and / mice, we provided potential mechanisms of the obesity-associated dysfunctions of metabolic cycles between liver and skeletal muscle involving glucose-alanine, glucose-lactate, and ketone bodies. Our study revealed obesity-associated systemic pathological mechanisms of dysfunction of inter-organ metabolic cycles.
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http://dx.doi.org/10.1016/j.isci.2021.102217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961104PMC
March 2021

Quantitative and Molecular Similarity Analyses of the Metabolites of Cold- and Hot-Natured Chinese Herbs.

Evid Based Complement Alternat Med 2021 5;2021:6646507. Epub 2021 Mar 5.

Institute for Advanced Biosciences, Keio University, 246-2 Mizukami, Kakuganji, Tsuruoka 997-0052, Japan.

Background: Based on the theory of traditional Chinese medicine, Chinese herbs possess four different medicinal properties: hot, warm, cold, and cool. These serve as a reference guide for these herbal medicines. However, the molecular mechanisms supporting their relevance remain unclear.

Methods: We performed metabolomics based on capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) and multivariate data analysis for the structural identification of compounds of cold- and hot-natured Chinese herbs.

Results: To this end, 30 selected herbs were analyzed and a total of 416 metabolites were identified via CE-TOF/MS, of which 193 compounds were detected in most herbs. The observed profiles offered the potential to understand the mechanism of association between the compounds and nature of the Chinese herbs. Comparison of the similarity in terms of chemical and molecular structures and content revealed that hot-natured herbs contained more nucleotides. In contrast, principal component analysis revealed the presence of more amino acid compounds in cold-natured herbs.

Conclusion: Comparing the structural similarities between the samples using the Tanimoto coefficient revealed that a general non-specific structure was observed between cold- and hot-natured herbs; however, the distribution of the molecular groups seemed to contribute more toward the energy properties.
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http://dx.doi.org/10.1155/2021/6646507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954617PMC
March 2021

Quantification of Salivary Charged Metabolites using Capillary Electrophoresis Time-of-flight-mass Spectrometry.

Bio Protoc 2020 Oct 20;10(20):e3797. Epub 2020 Oct 20.

Institute for Advanced Biosciences, Keio University, Yamagata, Japan.

Salivary metabolomics have provided the potentials to detect both oral and systemic diseases. Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) enables the identification and quantification of various charged metabolites. This method has been employed to biomarker discoveries using human saliva samples, especially for various types of cancers. The untargeted analysis contributes to finding new biomarkers. , the analysis of all detectable signals including both known and unknown metabolites extends the coverage of metabolite to be observed. However, the observed data includes thousands of peaks. Besides, non-linear migration time fluctuation and skewed peaks are caused by the sample condition. The presented pretreatment protocols of saliva samples enhance the reproducibility of migration time drift, which facilitates the matching peaks across the samples and also results in reproducible absolute concentrations of the detected metabolites. The described protocols are utilized not only for saliva but for any liquid samples with slight modifications.
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http://dx.doi.org/10.21769/BioProtoc.3797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842545PMC
October 2020

Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells.

BMC Cancer 2021 Feb 17;21(1):174. Epub 2021 Feb 17.

Institute for Advanced Biosciences, Keio University, 246-2 Mizukami, Kakuganji, Tsuruoka, 997-0052, Japan.

Background: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance.

Methods: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Results: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2'-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance.

Conclusions: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.
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http://dx.doi.org/10.1186/s12885-021-07879-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891143PMC
February 2021

Charged metabolite biomarkers of food intake assessed via plasma metabolomics in a population-based observational study in Japan.

PLoS One 2021 10;16(2):e0246456. Epub 2021 Feb 10.

Graduate School of Health Management, Keio University, Fujisawa, Kanagawa, Japan.

Food intake biomarkers can be critical tools that can be used to objectively assess dietary exposure for both epidemiological and clinical nutrition studies. While an accurate estimation of food intake is essential to unravel associations between the intake and specific health conditions, random and systematic errors affect self-reported assessments. This study aimed to clarify how habitual food intake influences the circulating plasma metabolome in a free-living Japanese regional population and to identify potential food intake biomarkers. To achieve this aim, we conducted a cross-sectional analysis as part of a large cohort study. From a baseline survey of the Tsuruoka Metabolome Cohort Study, 7,012 eligible male and female participants aged 40-69 years were chosen for this study. All data on patients' health status and dietary intake were assessed via a food frequency questionnaire, and plasma samples were obtained during an annual physical examination. Ninety-four charged plasma metabolites were measured using capillary electrophoresis mass spectrometry, by a non-targeted approach. Statistical analysis was performed using partial-least-square regression. A total of 21 plasma metabolites were likely to be associated with long-term food intake of nine food groups. In particular, the influential compounds in each food group were hydroxyproline for meat, trimethylamine-N-oxide for fish, choline for eggs, galactarate for dairy, cystine and betaine for soy products, threonate and galactarate for carotenoid-rich vegetables, proline betaine for fruits, quinate and trigonelline for coffee, and pipecolate for alcohol, and these were considered as prominent food intake markers in Japanese eating habits. A set of circulating plasma metabolites was identified as potential food intake biomarkers in the Japanese community-dwelling population. These results will open the way for the application of new reliable dietary assessment tools not by self-reported measurements but through objective quantification of biofluids.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0246456PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875413PMC
February 2021

Artificial hibernation/life-protective state induced by thiazoline-related innate fear odors.

Commun Biol 2021 01 22;4(1):101. Epub 2021 Jan 22.

Institute of Biomedical Science, Kansai Medical University, Osaka, 573-1010, Japan.

Innate fear intimately connects to the life preservation in crises, although this relationships is not fully understood. Here, we report that presentation of a supernormal innate fear inducer 2-methyl-2-thiazoline (2MT), but not learned fear stimuli, induced robust systemic hypothermia/hypometabolism and suppressed aerobic metabolism via phosphorylation of pyruvate dehydrogenase, thereby enabling long-term survival in a lethal hypoxic environment. These responses exerted potent therapeutic effects in cutaneous and cerebral ischemia/reperfusion injury models. In contrast to hibernation, 2MT stimulation accelerated glucose uptake in the brain and suppressed oxygen saturation in the blood. Whole-brain mapping and chemogenetic activation revealed that the sensory representation of 2MT orchestrates physiological responses via brain stem Sp5/NST to midbrain PBN pathway. 2MT, as a supernormal stimulus of innate fear, induced exaggerated, latent life-protective effects in mice. If this system is preserved in humans, it may be utilized to give rise to a new field: "sensory medicine."
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http://dx.doi.org/10.1038/s42003-020-01629-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822961PMC
January 2021

L-type amino acid transporter 1 is associated with chemoresistance in breast cancer via the promotion of amino acid metabolism.

Sci Rep 2021 01 12;11(1):589. Epub 2021 Jan 12.

Department of Pathology, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan.

F-FDG PET/CT has been used as an indicator of chemotherapy effects, but cancer cells can remain even when no FDG uptake is detected, indicating the importance of exploring other metabolomic pathways. Therefore, we explored the amino acid metabolism, including L-type amino acid transporter-1 (LAT1), in breast cancer tissues and clarified the role of LAT1 in therapeutic resistance and clinical outcomes of patients. We evaluated LAT1 expression before and after neoadjuvant chemotherapy and examined the correlation of glucose uptake using FDG-PET with the pathological response of patients. It revealed that LAT1 levels correlated with proliferation after chemotherapy, and amino acid and glucose metabolism were closely correlated. In addition, LAT1 was considered to be involved in treatment resistance and sensitivity only in luminal type breast cancer. Results of in vitro analyses revealed that LAT1 promoted amino acid uptake, which contributed to energy production by supplying amino acids to the TCA cycle. However, in MCF-7 cells treated with chemotherapeutic agents, oncometabolites and branched-chain amino acids also played a pivotal role in energy production and drug resistance, despite decreased glucose metabolism. In conclusion, LAT1 was involved in drug resistance and could be a novel therapeutic target against chemotherapy resistance in luminal type breast cancer.
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http://dx.doi.org/10.1038/s41598-020-80668-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803739PMC
January 2021

Relationship between Standard Uptake Values of Positron Emission Tomography/Computed Tomography and Salivary Metabolites in Oral Cancer: A Pilot Study.

J Clin Med 2020 Dec 7;9(12). Epub 2020 Dec 7.

Department of Dentistry, Oral and Maxillofacial Plastic and Reconstructive Surgery, Faculty of Medicine, Yamagata University, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan.

F-fluorodeoxyglucose (F-FDG) positron emission tomography (PET)/computed tomography (CT) is usually used for staging or evaluation of treatment response rather than for cancer screening. However, F-FDG PET/CT has also been used in Japan for cancer screening in people with no cancer symptoms, and accumulating evidence supports this application of F-FDG PET/CT. Previously, we have observed a correlation between the saliva and tumor metabolomic profiles in patients with oral cancer. Hence, if salivary metabolites demonstrate a significant correlation with PET parameters such as the maximum standardized uptake value (SUV), they may have the potential to be used as a screening tool before PET/CT to identify patients with high SUV. Hence, in this study, we aimed to explore the relationship between salivary metabolites and SUV of F-FDG PET/CT using previously collected data. F-FDG PET/CT was performed for staging 26 patients with oral cancer. The collected data were integrated and analyzed along with quantified salivary hydrophilic metabolites obtained from the same patients with oral cancer and controls ( = 44). In total, 11 metabolites showed significant correlations with SUV in the delayed phases. A multiple logistic regression model of the two metabolites showed the ability to discriminate between patients with oral cancer and controls, with an area under the receiver operating characteristic curve of 0.738 ( = 0.001). This study uniquely confirmed a relationship between salivary metabolites and SUV of PET/CT in patients with oral cancer; salivary metabolites were significantly correlated with SUV. These salivary metabolites can be used as a screening tool before PET/CT to identify patients with high SUV, i.e., to detect the presence of oral cancer.
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http://dx.doi.org/10.3390/jcm9123958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762245PMC
December 2020

Transomics analysis reveals allosteric and gene regulation axes for altered hepatic glucose-responsive metabolism in obesity.

Sci Signal 2020 12 1;13(660). Epub 2020 Dec 1.

Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Impaired glucose tolerance associated with obesity causes postprandial hyperglycemia and can lead to type 2 diabetes. To study the differences in liver metabolism in healthy and obese states, we constructed and analyzed transomics glucose-responsive metabolic networks with layers for metabolites, expression data for metabolic enzyme genes, transcription factors, and insulin signaling proteins from the livers of healthy and obese mice. We integrated multiomics time course data from wild-type and leptin-deficient obese (/) mice after orally administered glucose. In wild-type mice, metabolic reactions were rapidly regulated within 10 min of oral glucose administration by glucose-responsive metabolites, which functioned as allosteric regulators and substrates of metabolic enzymes, and by Akt-induced changes in the expression of glucose-responsive genes encoding metabolic enzymes. In / mice, the majority of rapid regulation by glucose-responsive metabolites was absent. Instead, glucose administration produced slow changes in the expression of carbohydrate, lipid, and amino acid metabolic enzyme-encoding genes to alter metabolic reactions on a time scale of hours. Few regulatory events occurred in both healthy and obese mice. Thus, our transomics network analysis revealed that regulation of glucose-responsive liver metabolism is mediated through different mechanisms in healthy and obese states. Rapid changes in allosteric regulators and substrates and in gene expression dominate the healthy state, whereas slow changes in gene expression dominate the obese state.
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http://dx.doi.org/10.1126/scisignal.aaz1236DOI Listing
December 2020

Gut microbiota depletion by chronic antibiotic treatment alters the sleep/wake architecture and sleep EEG power spectra in mice.

Sci Rep 2020 11 11;10(1):19554. Epub 2020 Nov 11.

International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.

Dysbiosis of the gut microbiota affects physiological processes, including brain functions, by altering the intestinal metabolism. Here we examined the effects of the gut microbiota on sleep/wake regulation. C57BL/6 male mice were treated with broad-spectrum antibiotics for 4 weeks to deplete their gut microbiota. Metabolome profiling of cecal contents in antibiotic-induced microbiota-depleted (AIMD) and control mice showed significant variations in the metabolism of amino acids and vitamins related to neurotransmission, including depletion of serotonin and vitamin B6, in the AIMD mice. Sleep analysis based on electroencephalogram and electromyogram recordings revealed that AIMD mice spent significantly less time in non-rapid eye movement sleep (NREMS) during the light phase while spending more time in NREMS and rapid eye movement sleep (REMS) during the dark phase. The number of REMS episodes seen in AIMD mice increased during both light and dark phases, and this was accompanied by frequent transitions from NREMS to REMS. In addition, the theta power density during REMS was lower in AIMD mice during the light phase compared with that in the controls. Consequently, the gut microbiota is suggested to affect the sleep/wake architecture by altering the intestinal balance of neurotransmitters.
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http://dx.doi.org/10.1038/s41598-020-76562-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659342PMC
November 2020

Comprehensive Dipeptide Analysis Revealed Cancer-Specific Profile in the Liver of Patients with Hepatocellular Carcinoma and Hepatitis.

Metabolites 2020 Nov 1;10(11). Epub 2020 Nov 1.

Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan.

As the physical properties and functionality of dipeptides differ from those of amino acids, they have attracted attention in metabolomics; however, their functions in vivo have not been clarified in detail. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, and its major cause is chronic hepatitis. This study was conducted to explore tumor-specific dipeptide characteristics by performing comprehensive dipeptide analysis in the tumor and surrounding nontumor tissue of patients with HCC. Dipeptides were analyzed by liquid chromatography tandem mass spectrometry and capillary electrophoresis tandem mass spectrometry. Principal component analysis using 236 detected dipeptides showed differences in the dipeptide profiles between nontumor and tumor tissues; however, no clear difference was observed in etiological comparison. In addition, the N- and C-terminal amino acid compositions of the detected dipeptides significantly differed, suggesting the substrate specificity of enzyme proteins, such as peptidase. Furthermore, hepatitis-derived HCC may show a characteristic dipeptide profile even before tumor formation. These results provide insight into HCC pathogenesis and may help identify novel biomarkers for diagnosis.
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http://dx.doi.org/10.3390/metabo10110442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692321PMC
November 2020

Trans-omic Analysis Reveals ROS-Dependent Pentose Phosphate Pathway Activation after High-Frequency Electrical Stimulation in C2C12 Myotubes.

iScience 2020 Oct 12;23(10):101558. Epub 2020 Sep 12.

Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Skeletal muscle adaptation is mediated by cooperative regulation of metabolism, signal transduction, and gene expression. However, the global regulatory mechanism remains unclear. To address this issue, we performed electrical pulse stimulation (EPS) in differentiated C2C12 myotubes at low and high frequency, carried out metabolome and transcriptome analyses, and investigated phosphorylation status of signaling molecules. EPS triggered extensive and specific changes in metabolites, signaling phosphorylation, and gene expression during and after EPS in a frequency-dependent manner. We constructed trans-omic network by integrating these data and found selective activation of the pentose phosphate pathway including metabolites, upstream signaling molecules, and gene expression of metabolic enzymes after high-frequency EPS. We experimentally validated that activation of these molecules after high-frequency EPS was dependent on reactive oxygen species (ROS). Thus, the trans-omic analysis revealed ROS-dependent activation in signal transduction, metabolome, and transcriptome after high-frequency EPS in C2C12 myotubes, shedding light on possible mechanisms of muscle adaptation.
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http://dx.doi.org/10.1016/j.isci.2020.101558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522805PMC
October 2020

Kinetic Trans-omic Analysis Reveals Key Regulatory Mechanisms for Insulin-Regulated Glucose Metabolism in Adipocytes.

iScience 2020 Aug 20;23(9):101479. Epub 2020 Aug 20.

Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

Insulin regulates glucose metabolism through thousands of regulatory mechanisms; however, which regulatory mechanisms are keys to control glucose metabolism remains unknown. Here, we performed kinetic trans-omic analysis by integrating isotope-tracing glucose flux and phosphoproteomic data from insulin-stimulated adipocytes and built a kinetic mathematical model to identify key allosteric regulatory and phosphorylation events for enzymes. We identified nine reactions regulated by allosteric effectors and one by enzyme phosphorylation and determined the regulatory mechanisms for three of these reactions. Insulin stimulated glycolysis by promoting Glut4 activity by enhancing phosphorylation of AS160 at S595, stimulated fatty acid synthesis by promoting Acly activity through allosteric activation by glucose 6-phosphate or fructose 6-phosphate, and stimulated glutamate synthesis by alleviating allosteric inhibition of Gls by glutamate. Most of glycolytic reactions were regulated by amounts of substrates and products. Thus, phosphorylation or allosteric modulator-based regulation of only a few key enzymes was sufficient to change insulin-induced metabolism.
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http://dx.doi.org/10.1016/j.isci.2020.101479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479629PMC
August 2020

Comparison of the ischemic and non-ischemic lung cancer metabolome reveals hyper activity of the TCA cycle and autophagy.

Biochem Biophys Res Commun 2020 09 13;530(1):285-291. Epub 2020 Aug 13.

Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, Natori, 981-1293, Japan; Division of Cancer Molecular Biology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan. Electronic address:

Recent advances in cancer biology reveal the importance of metabolic changes in cancer; however, less is known about how metabolic pathways in tumors are regulated in vivo. Here, we report analysis of the lung cancer metabolism based on different surgical procedures, namely lobectomy and partial resection. In lobectomy, but not in partial resection, pulmonary arteries and veins are ligated prior to removal of tissues, rendering tissues ischemic. We show that tumors indeed undergo ischemia upon lobectomy and that the tumor metabolome differs markedly from that of tumors removed by partial resection. Comparison of the responses to ischemia in tumor and normal lung tissues revealed that lung cancer tissue exhibits greater TCA cycle and autophagic activity than do normal lung tissues in vivo in patients. Finally, we report that deleting ATG7, which encodes a protein essential for autophagy, antagonizes growth of tumors derived from lung cancer cell lines, suggesting that autophagy confers metabolic advantages to lung cancer. Our findings shed light on divergent metabolic responses to ischemia seen in tumors and normal tissues.
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http://dx.doi.org/10.1016/j.bbrc.2020.07.082DOI Listing
September 2020
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