Takako Takai-Igarashi

The Tohoku Medical Megabank Project: Design and Mission

The Great East Japan Earthquake (GEJE) and resulting tsunami of March 11, 2011 gave rise to devastating damage on the Pacific coast of the Tohoku region. The Tohoku Medical Megabank Project (TMM), which is being conducted by Tohoku University Tohoku Medical Megabank Organization (ToMMo) and Iwate Medical University Iwate Tohoku Medical Megabank Organization (IMM), has been launched to realize creative reconstruction and to solve medical problems in the aftermath of this disaster. We started two prospective cohort studies in Miyagi and Iwate Prefectures: a population-based adult cohort study, the TMM Community-Based Cohort Study (TMM CommCohort Study), which will recruit 80 000 participants, and a birth and three-generation cohort study, the TMM Birth and Three-Generation Cohort Study (TMM BirThree Cohort Study), which will recruit 70 000 participants, including fetuses and their parents, siblings, grandparents, and extended family members. The TMM CommCohort Study will recruit participants from 2013 to 2016 and follow them for at least 5 years. The TMM BirThree Cohort Study will recruit participants from 2013 to 2017 and follow them for at least 4 years. For children, the ToMMo Child Health Study, which adopted a cross-sectional design, was also started in November 2012 in Miyagi Prefecture. An integrated biobank will be constructed based on the two prospective cohort studies, and ToMMo and IMM will investigate the chronic medical impacts of the GEJE. The integrated biobank of TMM consists of health and clinical information, biospecimens, and genome and omics data. The biobank aims to establish a firm basis for personalized healthcare and medicine, mainly for diseases aggravated by the GEJE in the two prefectures. Biospecimens and related information in the biobank will be distributed to the research community. TMM itself will also undertake genomic and omics research. The aims of the genomic studies are: 1) to construct an integrated biobank; 2) to return genomic research results to the participants of the cohort studies, which will lead to the implementation of personalized healthcare and medicine in the affected areas in the near future; and 3) to contribute the development of personalized healthcare and medicine worldwide. Through the activities of TMM, we will clarify how to approach prolonged healthcare problems in areas damaged by large-scale disasters and how useful genomic information is for disease prevention.

Activating Transcription Factor 4 Links Metabolic Stress to Interleukin-6 Expression in Macrophages

Chronic inflammation is a molecular element of the metabolic syndrome and type 2 diabetes. Saturated fatty acids (SFAs) are considered to be an important proinflammatory factor. However, it is still incompletely understood how SFAs induce proinflammatory cytokine expression. Hereby we report that activating transcription factor (ATF) 4, a transcription factor that is induced downstream of metabolic stresses including endoplasmic reticulum (ER) stress, plays critical roles in SFA-induced interleukin-6 (Il6) expression. DNA microarray analysis using primary macrophages revealed that the ATF4 pathway is activated by SFAs. Haploinsufficiency and short hairpin RNA–based knockdown of ATF4 in macrophages markedly inhibited SFA- and metabolic stress–induced Il6 expression. Conversely, pharmacological activation of the ATF4 pathway and overexpression of ATF4 resulted in enhanced Il6 expression. Moreover, ATF4 acts in synergy with the Toll-like receptor-4 signaling pathway, which is known to be activated by SFAs. At a molecular level, we found that ATF4 exerts its proinflammatory effects through at least two different mechanisms: ATF4 is involved in SFA-induced nuclear factor-κB activation; and ATF4 directly activates the Il6 promoter. These findings provide evidence suggesting that ATF4 links metabolic stress and Il6 expression in macrophages.

PGC-1α-Mediated Branched-Chain Amino Acid Metabolism in the Skeletal Muscle

Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain α-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism.

Ligand-Activated PPARα-Dependent DNA Demethylation Regulates the Fatty Acid β-Oxidation Genes in the Postnatal Liver

The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator–activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and life-stage–specific DNA demethylation of a particular metabolic pathway.

CELLPEDIA: a repository for human cell information for cell studies and differentiation analyses

CELLPEDIA is a repository database for current knowledge about human cells. It contains various types of information, such as cell morphologies, gene expression and literature references. The major role of CELLPEDIA is to provide a digital dictionary of human cells for the biomedical field, including support for the characterization of artificially generated cells in regenerative medicine. CELLPEDIA features (i) its own cell classification scheme, in which whole human cells are classified by their physical locations in addition to conventional taxonomy; and (ii) cell differentiation pathways compiled from biomedical textbooks and journal papers. Currently, human differentiated cells and stem cells are classified into 2260 and 66 cell taxonomy keys, respectively, from which 934 parent–child relationships reported in cell differentiation or transdifferentiation pathways are retrievable. As far as we know, this is the first attempt to develop a digital cell bank to function as a public resource for the accumulation of current knowledge about human cells. The CELLPEDIA homepage is freely accessible except for the data submission pages that require authentication (please send a password request to cell-info@cbrc.jp). Database URL: http://cellpedia.cbrc.jp/

Increased Systemic Glucose Tolerance with Increased Muscle Glucose Uptake in Transgenic Mice Overexpressing RXRγ in Skeletal Muscle

Background Retinoid X receptor (RXR) γ is a nuclear receptor-type transcription factor expressed mostly in skeletal muscle, and regulated by nutritional conditions. Previously, we established transgenic mice overexpressing RXRγ in skeletal muscle (RXRγ mice), which showed lower blood glucose than the control mice. Here we investigated their glucose metabolism. Methodology/Principal Findings RXRγ mice were subjected to glucose and insulin tolerance tests, and glucose transporter expression levels, hyperinsulinemic-euglycemic clamp and glucose uptake were analyzed. Microarray and bioinformatics analyses were done. The glucose tolerance test revealed higher glucose disposal in RXRγ mice than in control mice, but insulin tolerance test revealed no difference in the insulin-induced hypoglycemic response. In the hyperinsulinemic-euglycemic clamp study, the basal glucose disposal rate was higher in RXRγ mice than in control mice, indicating an insulin-independent increase in glucose uptake. There was no difference in the rate of glucose infusion needed to maintain euglycemia (glucose infusion rate) between the RXRγ and control mice, which is consistent with the result of the insulin tolerance test. Skeletal muscle from RXRγ mice showed increased Glut1 expression, with increased glucose uptake, in an insulin-independent manner. Moreover, we performed in vivo luciferase reporter analysis using Glut1 promoter (Glut1-Luc). Combination of RXRγ and PPARδ resulted in an increase in Glut1-Luc activity in skeletal muscle in vivo. Microarray data showed that RXRγ overexpression increased a diverse set of genes, including glucose metabolism genes, whose promoter contained putative PPAR-binding motifs. Conclusions/Significance Systemic glucose metabolism was increased in transgenic mice overexpressing RXRγ. The enhanced glucose tolerance in RXRγ mice may be mediated at least in part by increased Glut1 in skeletal muscle. These results show the importance of skeletal muscle gene regulation in systemic glucose metabolism. Increasing RXRγ expression may be a novel therapeutic strategy against type 2 diabetes.

Analysis of DNA methylation change induced by Dnmt3b in mouse hepatocytes

DNA methylation is a key epigenetic contributor to gene regulation in mammals. We have recently found that in the mouse liver, the promoter region of glycerol-3-phosphate acyltransferase 1, a rate-limiting enzyme of de novo lipogenesis, is regulated by DNA methylation, which is mediated by Dnmt3b, an enzyme required for the initiation of de novo methylation. In this study, using primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3b, we characterized Dnmt3b-dependent DNA methylation on a genome-wide basis. A genome-wide DNA methylation analysis, called microarray-based integrated analysis of methylation by isoschizomers, identified 108 genes with Dnmt3b dependent DNA methylation. In DNA expression array analysis, expression of some genes with Dnmt3b-dependent DNA methylation was suppressed. Studies with primary mouse hepatocytes overexpressing Dnmt3b or Dnmt3a revealed that many genes with Dnmt3b-dependent methylation are not methylated by Dnmt3a, whereas those methylated by Dnmt3a are mostly methylated by Dnmt3b. Bioinformatic analysis showed that the CANAGCTG and CCGGWNCSC (N denotes A, T, G, or C; W denotes A or T; and S denotes C or G) sequences are enriched in genes methylated by overexpression of Dnmt3b and Dnmt3a, respectively. We also observed a large number of genes with Dnmt3b-dependent DNA methylation in primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3, suggesting that Dnmt3b is an important DNA methyltransferase in primary mouse hepatocytes, targets specific genes, and potentially plays a role in vivo.

Accurate mass comparison coupled with two endopeptidases enables identification of protein termini

Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys‐C and peptidyl‐Lys metalloendopeptidase (Lys‐N), to digest proteins, followed by LC‐MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys‐C digest is one lysine residue mass heavier than that in Lys‐N digest; (ii) the carboxyl terminal peptide in Lys‐N digest is one lysine residue mass heavier than that in Lys‐C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys‐C and the Lys‐N digest, although amino acid sequences of Lys‐C and Lys‐N peptides are different (Lys‐C peptides end with lysine, whereas Lys‐N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N‐terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures.

Epigenetic modulation of Fgf21 in the perinatal mouse liver ameliorates diet-induced obesity in adulthood

The nutritional environment to which animals are exposed in early life can lead to epigenetic changes in the genome that influence the risk of obesity in later life. Here, we demonstrate that the fibroblast growth factor-21 gene (Fgf21) is subject to peroxisome proliferator-activated receptor (PPAR) α–dependent DNA demethylation in the liver during the postnatal period. Reductions in Fgf21 methylation can be enhanced via pharmacologic activation of PPARα during the suckling period. We also reveal that the DNA methylation status of Fgf21, once established in early life, is relatively stable and persists into adulthood. Reduced DNA methylation is associated with enhanced induction of hepatic FGF21 expression after PPARα activation, which may partly explain the attenuation of diet-induced obesity in adulthood. We propose that Fgf21 methylation represents a form of epigenetic memory that persists into adulthood, and it may have a role in the developmental programming of obesity.FGF21 exerts beneficial metabolic effects on multiple tissues. Here the authors show that the Fgf21 gene is demethylated during the postnatal suckling period, creating an epigenetic memory that determines the responsiveness of the Fgf21 gene to inducers such as PPARα activators or fasting in adulthood.

Evaluation of reported pathogenic variants and their frequencies in a Japanese population based on a whole-genome reference panel of 2049 individuals

Clarifying allele frequencies of disease-related genetic variants in a population is important in genomic medicine; however, such data is not yet available for the Japanese population. To estimate frequencies of actionable pathogenic variants in the Japanese population, we examined the reported pathological variants in genes recommended by the American College of Medical Genetics and Genomics (ACMG) in our reference panel of genomic variations, 2KJPN, which was created by whole-genome sequencing of 2049 individuals of the resident cohort of the Tohoku Medical Megabank Project. We searched for pathogenic variants in 2KJPN for 57 autosomal ACMG-recommended genes responsible for 26 diseases and then examined their frequencies. By referring to public databases of pathogenic variations, we identified 143 reported pathogenic variants in 2KJPN for the 57 ACMG recommended genes based on a classification system. At the individual level, 21% of the individuals were found to have at least one reported pathogenic allele. We then conducted a literature survey to review the variants and to check for evidence of pathogenicity. Our results suggest that a substantial number of people have reported pathogenic alleles for the ACMG genes, and reviewing variants is indispensable for constructing the information infrastructure of genomic medicine for the Japanese population.