Publications by authors named "Iori Sakakibara"

24 Publications

  • Page 1 of 1

MuRF1 deficiency prevents age-related fat weight gain, possibly through accumulation of PDK4 in skeletal muscle mitochondria in older mice.

J Orthop Res 2021 Jun 29. Epub 2021 Jun 29.

Department of Nutritional Physiology, Institute of Medical Nutrition, Tokushima University Graduate School, Tokushima, Japan.

Recent studies show that muscle mass and metabolic function are interlinked. Muscle RING finger 1 (MuRF1) is a critical muscle-specific ubiquitin ligase associated with muscle atrophy. Yet, the molecular target of MuRF1 in atrophy and aging remains unclear. We examined the role of MuRF1 in aging, using MuRF1-deficient (MuRF1 ) mice in vivo, and MuRF1-overexpressing cell in vitro. MuRF1 deficiency partially prevents age-induced skeletal muscle loss in mice. Interestingly, body weight and fat mass of more than 7-month-old MuRF1 mice were lower than in MuRF1 mice. Serum and muscle metabolic parameters and results of indirect calorimetry suggest significantly higher energy expenditure and enhanced lipid metabolism in 3-month-old MuRF1 mice than in MuRF1 mice, resulting in suppressed adipose tissue gain during aging. Pyruvate dehydrogenase kinase 4 (PDK4) is crucial for a switch from glucose to lipid metabolism, and the interaction between MuRF1 and PDK4 was examined. PDK4 protein levels were elevated in mitochondria from the skeletal muscle in MuRF1 mice. In vitro, MuRF1 interacted with PDK4 but did not induce degradation through ubiquitination. Instead, SUMO posttranscriptional modification (SUMOylation) of PDK4 was detected in MuRF1-overexpressing cells, in contrast to cells without the RING domain of MuRF1. MuRF1 deficiency enhances lipid metabolism possibly by upregulating PDK4 localization into mitochondrial through prevention of SUMOylation. Inhibition of MuRF1-mediated PDK4 SUMOylation is a potential therapeutic target for age-related dysfunction of lipid metabolism and muscle atrophy.
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http://dx.doi.org/10.1002/jor.25131DOI Listing
June 2021

Myofiber androgen receptor increases muscle strength mediated by a skeletal muscle splicing variant of Mylk4.

iScience 2021 Apr 13;24(4):102303. Epub 2021 Mar 13.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan.

Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT). DHT treatment increased grip strength in control mice but not in cARKO mice. Transcriptome analysis by RNA-seq, using skeletal muscles obtained from control and cARKO mice treated with or without DHT, identified a fast-type muscle-specific novel splicing variant of as a target of AR in skeletal muscles. knockout mice exhibited decreased maximum isometric torque of plantar flexion and passive stiffness of myofibers due to reduced phosphorylation of Myomesin 1 protein. This study suggests that androgen-induced skeletal muscle strength is mediated with Mylk4 and Myomesin 1 axis.
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http://dx.doi.org/10.1016/j.isci.2021.102303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041868PMC
April 2021

Morin attenuates dexamethasone-mediated oxidative stress and atrophy in mouse C2C12 skeletal myotubes.

Arch Biochem Biophys 2021 Jun 10;704:108873. Epub 2021 Apr 10.

Department of Nutritional Physiology, Institute of Medical Nutrition, Tokushima University Graduate School, Tokushima, Japan. Electronic address:

Glucocorticoids are the drugs most commonly used to manage inflammatory diseases. However, they are prone to inducing muscle atrophy by increasing muscle proteolysis and decreasing protein synthesis. Various studies have demonstrated that antioxidants can mitigate glucocorticoid-induced skeletal muscle atrophy. Here, we investigated the effect of a potent antioxidative natural flavonoid, morin, on the muscle atrophy and oxidative stress induced by dexamethasone (Dex) using mouse C2C12 skeletal myotubes. Dex (10 μM) enhanced the production of reactive oxygen species (ROS) in C2C12 myotubes via glucocorticoid receptor. Moreover, Dex administration reduced the diameter and expression levels of the myosin heavy chain protein in C2C12 myotubes, together with the upregulation of muscle atrophy-associated ubiquitin ligases, such as muscle atrophy F-box protein 1/atrogin-1, muscle ring finger protein-1, and casitas B-lineage lymphoma proto-oncogene-b. Dex also significantly decreased phosphorylated Foxo3a and increased total Foxo3a expression. Interestingly, Dex-induced ROS accumulation and Foxo3a expression were inhibited by morin (10 μM) pretreatment. Morin also prevented the Dex-induced reduction of myotube thickness, together with muscle protein degradation and suppression of the upregulation of atrophy-associated ubiquitin ligases. In conclusion, our results suggest that morin effectively prevents glucocorticoid-induced muscle atrophy by reducing oxidative stress.
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http://dx.doi.org/10.1016/j.abb.2021.108873DOI Listing
June 2021

The Prevalence and Diagnostic Ratio of Familial Hypercholesterolemia (FH) and Proportion of Acute Coronary Syndrome in Japanese FH Patients in a Healthcare Record Database Study.

Cardiovasc Ther 2020 11;2020:5936748. Epub 2020 Jun 11.

Amgen K.K., Tokyo, Japan.

Background: Familial hypercholesterolemia (FH) is a genetic disorder characterized by high levels of low-density lipoprotein cholesterol (LDL-C). Because of underdiagnosis, acute coronary syndrome (ACS) is often the first clinical manifestation of FH. In Japan, there are few reports on the prevalence and diagnostic ratios of FH and the proportion of ACS among FH patients in clinical settings.

Methods: This retrospective, observational study used anonymized data from electronic healthcare databases between April 2001 and March 2015 of patients who had ≥2 LDL-C measurements recorded after April 2009. The index date was defined as the date of the first LDL-C measurement after April 2009. The primary endpoint was the prevalence of definite or suspected FH; secondary endpoints included the proportion of FH patients hospitalized for ACS, the proportion of patients using lipid-lowering drugs (LLDs), and LDL-C levels.

Results: Of the 187,781 patients screened, 1547 had definite or suspected FH (0.8%) based on data from the entire period; 832 patients with definite ( = 299, 0.16%) or suspected FH ( = 533, 0.28%) before the index date were identified in the main analysis cohort. LLDs were used in 214 definite FH patients (71.6%) and 137 suspected FH patients (25.7%). Among definite or suspected FH patients with ACS ( = 84) and without ACS ( = 748), 32.1% and 30.1% with definite FH and 3.2% and 2.4% with suspected FH had LDL-C levels < 2.6 mmol/L (<100 mg/dL), respectively. Sixty patients (7.2%) were hospitalized due to ACS at the index date.

Conclusions: The prevalence of FH in this Japanese cohort of patients with ≥2 LDL-C measurements at hospitals was 0.8%, which is higher than that currently reported in epidemiological studies (0.2-0.5%). Patients with suspected FH, with or without ACS, had poorly controlled LDL-C levels and were undertreated. The proportion of FH patients who were hospitalized due to ACS was 7.2%.
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http://dx.doi.org/10.1155/2020/5936748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321519PMC
September 2020

Myogenesis control by SIX transcriptional complexes.

Semin Cell Dev Biol 2020 08 1;104:51-64. Epub 2020 Apr 1.

Department of Integrative Medical Biology (IMB), Umeå universitet, Sweden.

SIX homeoproteins were first described in Drosophila, where they participate in the Pax-Six-Eya-Dach (PSED) network with eyeless, eyes absent and dachsund to drive synergistically eye development through genetic and biochemical interactions. The role of the PSED network and SIX proteins in muscle formation in vertebrates was subsequently identified. Evolutionary conserved interactions with EYA and DACH proteins underlie the activity of SIX transcriptional complexes (STC) both during embryogenesis and in adult myofibers. Six genes are expressed throughout muscle development, in embryonic and adult proliferating myogenic stem cells and in fetal and adult post-mitotic myofibers, where SIX proteins regulate the expression of various categories of genes. In vivo, SIX proteins control many steps of muscle development, acting through feedforward mechanisms: in the embryo for myogenic fate acquisition through the direct control of Myogenic Regulatory Factors; in adult myofibers for their contraction/relaxation and fatigability properties through the control of genes involved in metabolism, sarcomeric organization and calcium homeostasis. Furthermore, during development and in the adult, SIX homeoproteins participate in the genesis and the maintenance of myofibers diversity.
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http://dx.doi.org/10.1016/j.semcdb.2020.03.003DOI Listing
August 2020

GPRC5A facilitates cell proliferation through cell cycle regulation and correlates with bone metastasis in prostate cancer.

Int J Cancer 2020 03 22;146(5):1369-1382. Epub 2019 Jul 22.

Department of Pathophysiology, Ehime University Graduate School of Medicine, Toon, Japan.

The prognosis of patients with progressive prostate cancers that are hormone refractory and/or have bone metastasis is poor. Multiple therapeutic targets to improve prostate cancer patient survival have been investigated, including orphan GPCRs. In our study, we identified G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5A) as a candidate therapeutic molecule using integrative gene expression analyses of registered data sets for prostate cancer cell lines. Kaplan-Meier analysis of TCGA data sets revealed that patients who have high GPRC5A expression had significantly shorter overall survival. PC3 prostate cancer cells with CRISPR/Cas9-mediated GPRC5A knockout exhibited significantly reduced cell proliferation both in vitro and in vivo. RNA-seq revealed that GPRC5A KO PC3 cells had dysregulated expression of cell cycle-related genes, leading to cell cycle arrest at the G2/M phase. Furthermore, the registered gene expression profile data set showed that the expression level of GPRC5A in original lesions of prostate cancer patients with bone metastasis was higher than that without bone metastasis. In fact, GPRC5A KO PC3 cells failed to establish bone metastasis in xenograft mice models. In addition, our clinical study revealed that GPRC5A expression levels in prostate cancer patient samples were significantly correlated with bone metastasis as well as the patient's Gleason score (GS). Combined assessment with the immunoreactivity of GPRC5A and GS displayed higher specificity for predicting the occurrence of bone metastasis. Together, our findings indicate that GPRC5A can be a possible therapeutic target and prognostic marker molecule for progressive prostate cancer.
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http://dx.doi.org/10.1002/ijc.32554DOI Listing
March 2020

Microdetermination of Sialic Acids in Blood Samples by Hydrophilic Interaction Chromatography Coupled to Post-column Derivatization and Fluorometric Detection.

Anal Sci 2019 May 28;35(5):517-520. Epub 2018 Dec 28.

Faculty of Pharmaceutical Sciences, Ritsumeikan University.

An analytical method for the determination of sialic acids in biological samples has been developed and applied to fetal bovine serum (FBS), newborn calf serum and adult bovine serum. The hydrolysis of sera was carried out and the liberated sialic acids were quantified using a rapid and sensitive HPLC. The HPLC includes the separation and detection of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) using hydrophilic interaction liquid chromatography and a fluorometric post-column reaction with 2-cyanoacetamide. The calibration graphs for Neu5Ac and Neu5Gc were linear over the range of 10 pmol - 5 nmol. The concentrations of sialic acids in FBS, newborn calf serum and adult bovine serum were 5.06, 3.79 and 1.64 mM, respectively. The ratios of Neu5Gc and Neu5Ac changed dramatically according to the development stages. The present method has a satisfactory sensitivity in the quantification of Neu5Ac and Neu5Gc in serum samples. It seems that this analytical system can therefore be applied for routine use in clinical investigations of serum sialylation changes in cancer patients.
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http://dx.doi.org/10.2116/analsci.18P500DOI Listing
May 2019

Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression.

Development 2018 01 8;145(1). Epub 2018 Jan 8.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice ( ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including , which is reported to be an IL1-related gene and to affect chondrocyte differentiation. knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation.
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http://dx.doi.org/10.1242/dev.157412DOI Listing
January 2018

The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells.

Sci Rep 2017 02 20;7:42845. Epub 2017 Feb 20.

Division of Cell Growth and Tumor Regulation, Proteo-Science Center (PROS), Ehime University, Toon, Ehime, Japan.

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.
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http://dx.doi.org/10.1038/srep42845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5317005PMC
February 2017

QRFP-Deficient Mice Are Hypophagic, Lean, Hypoactive and Exhibit Increased Anxiety-Like Behavior.

PLoS One 2016 11;11(11):e0164716. Epub 2016 Nov 11.

Department of Molecular Neuroscience and Integrative Physiology, Faculty of Medicine, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan.

How the hypothalamus transmits hunger information to other brain regions to govern whole brain function to orchestrate feeding behavior has remained largely unknown. Our present study suggests the importance of a recently found lateral hypothalamic neuropeptide, QRFP, in this signaling. Qrfp-/- mice were hypophagic and lean, and exhibited increased anxiety-like behavior, and were hypoactive in novel circumstances as compared with wild type littermates. They also showed decreased wakefulness time in the early hours of the dark period. Histological studies suggested that QRFP neurons receive rich innervations from neurons in the arcuate nucleus which is a primary region for sensing the body's metabolic state by detecting levels of leptin, ghrelin and glucose. These observations suggest that QRFP is an important mediator that acts as a downstream mediator of the arcuate nucleus and regulates feeding behavior, mood, wakefulness and activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164716PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5105951PMC
June 2017

Six1 homeoprotein drives myofiber type IIA specialization in soleus muscle.

Skelet Muscle 2016 5;6(1):30. Epub 2016 Sep 5.

INSERM U1016, Institut Cochin, Paris, 75014 France ; CNRS UMR 8104, Paris, 75014 France ; Université Paris Descartes, Sorbonne Paris Cité, Paris, 75014 France.

Background: Adult skeletal muscles are composed of slow and fast myofiber subtypes which each express selective genes required for their specific contractile and metabolic activity. Six homeoproteins are transcription factors regulating muscle cell fate through activation of myogenic regulatory factors and driving fast-type gene expression during embryogenesis.

Results: We show here that Six1 protein accumulates more robustly in the nuclei of adult fast-type muscles than in adult slow-type muscles, this specific enrichment takes place during perinatal growth. Deletion of Six1 in soleus impaired fast-type myofiber specialization during perinatal development, resulting in a slow phenotype and a complete lack of Myosin heavy chain 2A (MyHCIIA) expression. Global transcriptomic analysis of wild-type and Six1 mutant myofibers identified the gene networks controlled by Six1 in adult soleus muscle. This analysis showed that Six1 is required for the expression of numerous genes encoding fast-type sarcomeric proteins, glycolytic enzymes and controlling intracellular calcium homeostasis. Parvalbumin, a key player of calcium buffering, in particular, is a direct target of Six1 in the adult myofiber.

Conclusions: This analysis revealed that Six1 controls distinct aspects of adult muscle physiology in vivo, and acts as a main determinant of fast-fiber type acquisition and maintenance.
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http://dx.doi.org/10.1186/s13395-016-0102-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011358PMC
February 2018

MyoD reprogramming requires Six1 and Six4 homeoproteins: genome-wide cis-regulatory module analysis.

Nucleic Acids Res 2016 Oct 14;44(18):8621-8640. Epub 2016 Jun 14.

Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS), UMR 8104, Paris, France Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Paris, France

Myogenic regulatory factors of the MyoD family have the ability to reprogram differentiated cells toward a myogenic fate. In this study, we demonstrate that Six1 or Six4 are required for the reprogramming by MyoD of mouse embryonic fibroblasts (MEFs). Using microarray experiments, we found 761 genes under the control of both Six and MyoD. Using MyoD ChIPseq data and a genome-wide search for Six1/4 MEF3 binding sites, we found significant co-localization of binding sites for MyoD and Six proteins on over a thousand mouse genomic DNA regions. The combination of both datasets yielded 82 genes which are synergistically activated by Six and MyoD, with 96 associated MyoD+MEF3 putative cis-regulatory modules (CRMs). Fourteen out of 19 of the CRMs that we tested demonstrated in Luciferase assays a synergistic action also observed for their cognate gene. We searched putative binding sites on these CRMs using available databases and de novo search of conserved motifs and demonstrated that the Six/MyoD synergistic activation takes place in a feedforward way. It involves the recruitment of these two families of transcription factors to their targets, together with partner transcription factors, encoded by genes that are themselves activated by Six and MyoD, including Mef2, Pbx-Meis and EBF.
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http://dx.doi.org/10.1093/nar/gkw512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5062961PMC
October 2016

Clinical Data Interchange Standards Consortium Standardization of Biobank Data: A Feasibility Study.

Biopreserv Biobank 2016 Feb 21;14(1):45-50. Epub 2015 Dec 21.

1 Department of Clinical Study and Informatics, Center for Clinical Sciences, National Center for Global Health and Medicine , Tokyo, Japan .

The National Center Biobank Network (NCBN), consisting of six national centers (NCs) for advanced and specialized medical care, was launched in Japan in 2012 to collect biological specimens and health-related data. The common data formats of the six NCs, however, are not widely known outside the NCs. Therefore, we investigated whether the data elements collected by the NCBN could be made to conform to the international standards of the Clinical Data Interchange Standards Consortium (CDISC). We attempted to map the NCBN data elements (202 items) onto the Study Data Tabulation Model (SDTM), a set of CDISC standards on the submission format of electronic clinical data approved by the Food and Drug Administration. The results showed that all 202 items of the NCBN data could be mapped onto the SDTM and fulfilled 50%-70% of the required items of each domain specified in the SDTM. We concluded that, while the standardization of biobank data according to the CDISC standards is possible, there is a need to consider whether additional items must be included in the NCBN and to have experts familiar with the CDISC standards review the standardization needs.
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http://dx.doi.org/10.1089/bio.2015.0051DOI Listing
February 2016

Six homeoproteins and a Iinc-RNA at the fast MYH locus lock fast myofiber terminal phenotype.

PLoS Genet 2014 22;10(5):e1004386. Epub 2014 May 22.

INSERM U1016, Institut Cochin, Paris, France; CNRS UMR 8104, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Thousands of long intergenic non-coding RNAs (lincRNAs) are encoded by the mammalian genome. However, the function of most of these lincRNAs has not been identified in vivo. Here, we demonstrate a role for a novel lincRNA, linc-MYH, in adult fast-type myofiber specialization. Fast myosin heavy chain (MYH) genes and linc-MYH share a common enhancer, located in the fast MYH gene locus and regulated by Six1 homeoproteins. linc-MYH in nuclei of fast-type myofibers prevents slow-type and enhances fast-type gene expression. Functional fast-sarcomeric unit formation is achieved by the coordinate expression of fast MYHs and linc-MYH, under the control of a common Six-bound enhancer.
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http://dx.doi.org/10.1371/journal.pgen.1004386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031048PMC
December 2014

AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.

FASEB J 2014 Jul 20;28(7):3211-24. Epub 2014 Mar 20.

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité (U)1016, Institut Cochin, Paris, France; Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 8104, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France;

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle-specific AMPKα1α2 double-knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle exercise capacity, mitochondrial function, and contraction-stimulated glucose uptake. Exercise performance was significantly reduced in the mdKO mice, with a reduction in maximal force production and fatigue resistance. An increase in the proportion of myofibers with centralized nuclei was noted, as well as an elevated expression of interleukin 6 (IL-6) mRNA, possibly consistent with mild skeletal muscle injury. Notably, we found that AMPKα1 and AMPKα2 isoforms are dispensable for contraction-induced skeletal muscle glucose transport, except for male soleus muscle. However, the lack of skeletal muscle AMPK diminished maximal ADP-stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial substrate utilization but not baseline mitochondrial muscle content. Together, these results demonstrate that skeletal muscle AMPK has an unexpected role in the regulation of mitochondrial oxidative phosphorylation that contributes to the energy demands of the exercising muscle.-Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J. T., Pehmøller, C., Sanz, N., Sakakibara, I., Saint-Amand, E., Rimbaud, S., Maire, P., Marette, A., Ventura-Clapier, R., Ferry, A., Wojtaszewski, J. F. P., Foretz, M., Viollet, B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.
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http://dx.doi.org/10.1096/fj.14-250449DOI Listing
July 2014

Analysis of DNA repair and protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after exposure to UVC radiation.

PLoS One 2013 6;8(6):e64793. Epub 2013 Jun 6.

Biospheric Science Branch, NASA Ames Research Center, Moffett Field, California, United States of America.

Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m(2) of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064793PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675078PMC
January 2014

Genesis of muscle fiber-type diversity during mouse embryogenesis relies on Six1 and Six4 gene expression.

Dev Biol 2011 Nov 22;359(2):303-20. Epub 2011 Aug 22.

Institut Cochin, Université Paris Descartes, Sorbonne Paris Cité, CNRS (UMR 8104), Paris, France.

Adult skeletal muscles in vertebrates are composed of different types of myofibers endowed with distinct metabolic and contraction speed properties. Genesis of this fiber-type heterogeneity during development remains poorly known, at least in mammals. Six1 and Six4 homeoproteins of the Six/sine oculis family are expressed throughout muscle development in mice, and Six1 protein is enriched in the nuclei of adult fast-twitch myofibers. Furthermore, Six1/Six4 proteins are known to control the early activation of fast-type muscle genes in myocytes present in the mouse somitic myotome. Using double Six1:Six4 mutants (SixdKO) to dissect in vivo the genesis of muscle fiber-type heterogeneity, we analyzed here the phenotype of the dorsal/epaxial muscles remaining in SixdKO. We show by electron microscopy analysis that the absence of these homeoproteins precludes normal sarcomeric organization of the myofiber leading to a dystrophic aspect, and by immunohistochemistry experiments a deficiency in synaptogenesis. Affymetrix transcriptome analysis of the muscles remaining in E18.5 SixdKO identifies a major role for these homeoproteins in the control of genes that are specifically activated in the adult fast/glycolytic myofibers, particularly those controlling Ca(2+) homeostasis. Absence of Six1 and Six4 leads to the development of dorsal myofibers lacking expression of fast-type muscle genes, and mainly expressing a slow-type muscle program. The absence of restriction of the slow-type program during the fetal period in SixdKO back muscles is associated with a decreased HDAC4 protein level, and subcellular relocalization of the transcription repressor Sox6. Six genes thus behave as essential global regulators of muscle gene expression, as well as a central switch to drive the skeletal muscle fast phenotype during fetal development.
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http://dx.doi.org/10.1016/j.ydbio.2011.08.010DOI Listing
November 2011

The peroxisome proliferator-activated receptor gamma/retinoid X receptor alpha heterodimer targets the histone modification enzyme PR-Set7/Setd8 gene and regulates adipogenesis through a positive feedback loop.

Mol Cell Biol 2009 Jul 4;29(13):3544-55. Epub 2009 May 4.

Genome Science Division, University of Tokyo, Tokyo, Japan.

Control of cell differentiation occurs through transcriptional mechanisms and through epigenetic modification. Using a chromatin immunoprecipitation-on-chip approach, we performed a genome-wide search for target genes of peroxisome proliferator-activated receptor gamma (PPAR gamma) and its partner protein retinoid X receptor alpha during adipogenesis. We show that these two receptors target several genes that encode histone lysine methyltransferase SET domain proteins. The histone H4 Lys 20 (H4K20) monomethyltransferase PR-Set7/Setd8 gene is upregulated by PPAR gamma during adipogenesis, and the knockdown of PR-Set7/Setd8 suppressed adipogenesis. Intriguingly, monomethylated H4K20 (H4K20me1) levels are robustly increased toward the end of differentiation. PR-Set7/Setd8 positively regulates the expression of PPAR gamma and its targets through H4K20 monomethylation. Furthermore, the activation of PPAR gamma transcriptional activity leads to the induction of H4K20me1 modification of PPAR gamma and its targets and thereby promotes adipogenesis. We also show that PPAR gamma targets PPAR gamma2 and promotes its gene expression through H4K20 monomethylation. Our results connect transcriptional regulation and epigenetic chromatin modulation through H4K20 monomethylation during adipogenesis through a feedback loop.
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http://dx.doi.org/10.1128/MCB.01856-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698772PMC
July 2009

COUP-TFII acts downstream of Wnt/beta-catenin signal to silence PPARgamma gene expression and repress adipogenesis.

Proc Natl Acad Sci U S A 2009 Apr 23;106(14):5819-24. Epub 2009 Mar 23.

Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153-8904, Japan.

Wnt signaling through beta-catenin and TCF maintains preadipocytes in an un-differentiated proliferative state; however, the molecular pathway has not been completely defined. By integrating gene expression microarray, chromatin immunoprecipitation-chip, and cell-based experimental approaches, we show that Wnt/beta-catenin signaling activates the expression of COUP-TFII which recruits the SMRT corepressor complex to the first introns located downstream from the first exons of both PPARgamma1 and gamma2 mRNAs. This maintains the local chromatin in a hypoacetylated state and represses PPARgamma gene expression to inhibit adipogenesis. Our experiments define the COUP-TFII/SMRT complex as a previously unappreciated component of the linear pathway that directly links Wnt/beta-catenin signaling to repression of PPARgamma gene expression and the inhibition of adipogenesis.
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http://dx.doi.org/10.1073/pnas.0901676106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667001PMC
April 2009

Fasting-induced hypothermia and reduced energy production in mice lacking acetyl-CoA synthetase 2.

Cell Metab 2009 Feb;9(2):191-202

Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan.

Acetate is activated to acetyl-CoA by acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzyme. Here, we report that the activation of acetate by AceCS2 has a specific and unique role in thermogenesis during fasting. In the skeletal muscle of fasted AceCS2(-/-) mice, ATP levels were reduced by 50% compared to AceCS2(+/+) mice. Fasted AceCS2(-/-) mice were significantly hypothermic and had reduced exercise capacity. Furthermore, when fed a low-carbohydrate diet, 4-week-old weaned AceCS2(-/-) mice also exhibited hypothermia accompanied by sustained hypoglycemia that led to a 50% mortality. Therefore, AceCS2 plays a significant role in acetate oxidation needed to generate ATP and heat. Furthermore, AceCS2(-/-) mice exhibited increased oxygen consumption and reduced weight gain on a low-carbohydrate diet. Our findings demonstrate that activation of acetate by AceCS2 plays a pivotal role in thermogenesis, especially under low-glucose or ketogenic conditions, and is crucially required for survival.
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http://dx.doi.org/10.1016/j.cmet.2008.12.008DOI Listing
February 2009

Hepatocyte nuclear factor 4alpha contributes to thyroid hormone homeostasis by cooperatively regulating the type 1 iodothyronine deiodinase gene with GATA4 and Kruppel-like transcription factor 9.

Mol Cell Biol 2008 Jun 21;28(12):3917-31. Epub 2008 Apr 21.

Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan.

Type 1 iodothyronine deiodinase (Dio1), a selenoenzyme catalyzing the bioactivation of thyroid hormone, is highly expressed in the liver. Dio1 mRNA and enzyme activity levels are markedly reduced in the livers of hepatocyte nuclear factor 4alpha (HNF4alpha)-null mice, thus accounting for its liver-specific expression. Consistent with this deficiency, serum T4 and rT3 concentrations are elevated in these mice compared with those in HNF4alpha-floxed control littermates; however, serum T3 levels are unchanged. Promoter analysis of the mouse Dio1 gene demonstrated that HNF4alpha plays a key role in the transactivation of the mouse Dio1 gene. Deletion and substitution mutation analyses demonstrated that a proximal HNF4alpha site (direct repeat 1 [TGGACAAAGGTGC]; HNF4alpha-RE) is crucial for transactivation of the mouse Dio1 gene by HNF4alpha. Mouse Dio1 is also stimulated by thyroid hormone signaling, but a direct role for thyroid hormone receptor action has not been reported. We also showed that thyroid hormone-inducible Krüppel-like factor 9 (KLF9) stimulates the mouse Dio1 promoter very efficiently through two CACCC sequences that are located on either side of HNF4alpha-RE. Furthermore, KLF9 functions together with HNF4alpha and GATA4 to synergistically activate the mouse Dio1 promoter, suggesting that Dio1 is regulated by thyroid hormone in the mouse through an indirect mechanism requiring prior KLF9 induction. In addition, we showed that physical interactions between the C-terminal zinc finger domain (Cf) of GATA4 and activation function 2 of HNF4alpha and between the basic domain adjacent to Cf of GATA4 and a C-terminal domain of KLF9 are both required for this synergistic response. Taken together, these results suggest that HNF4alpha regulates thyroid hormone homeostasis through transcriptional regulation of the mouse Dio1 gene with GATA4 and KLF9.
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http://dx.doi.org/10.1128/MCB.02154-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423126PMC
June 2008

Cooperative interaction between hepatocyte nuclear factor 4 alpha and GATA transcription factors regulates ATP-binding cassette sterol transporters ABCG5 and ABCG8.

Mol Cell Biol 2007 Jun 2;27(12):4248-60. Epub 2007 Apr 2.

Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo, Japan.

Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4alpha (HNF4alpha) in the ABCG5/ABCG8 intergenic promoter, through which HNF4alpha strongly activated the expression of a reporter gene in both directions. The HNF4alpha-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4alpha. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4alpha leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4alpha and GATA were essential for maximal synergism. We also show that HNF4alpha, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4alpha and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4alpha acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.
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http://dx.doi.org/10.1128/MCB.01894-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1900057PMC
June 2007

A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice.

Proc Natl Acad Sci U S A 2006 May 28;103(19):7438-43. Epub 2006 Apr 28.

Yanagisawa Orphan Receptor Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Tokyo 135-0064, Japan.

Here, we report the isolation and characterization of an endogenous peptide ligand of GPR103 from rat brains. The purified peptide was found to be the 43-residue RF-amide peptide QRFP. We also describe two mouse homologues of human GPR103, termed mouse GPR103A and GPR103B. QRFP binds and activates the human GPR103, as well as mouse GPR103A and GPR103B, with nanomolar affinities in transfected cells. Systematic in situ hybridization analysis in mouse brains showed that QRFP is expressed exclusively in the periventricular and lateral hypothalamus, whereas the two receptor mRNAs are distinctly localized in various brain areas without an overlap to each other. When administered centrally in mice, QRFP induced feeding behavior, accompanied by increased general locomotor activity and metabolic rate. QRFP-induced food intake was abolished by preadministration of BIBP3226, a specific antagonist for the Y1 neuropeptide Y receptor. Hypothalamic prepro-QRFP mRNA expression was up-regulated upon fasting and in genetically obese ob/ob and db/db mice. Central QRFP administration also evoked highly sustained elevation of blood pressure and heart rate. Our findings suggest that QRFP and GPR103A/B may regulate diverse neuroendocrine and behavioral functions and implicate this neuropeptide system in metabolic syndrome.
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http://dx.doi.org/10.1073/pnas.0602371103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464357PMC
May 2006