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Supplemental Material, Amoasii et al.A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolismLeonela Amoasii1,3,4, William Holland2, Efrain Sanchez-Ortiz1,3,4, Kedryn K. Baskin1,3,4, Mackenzie Pearson2, Shawn C. Burgess5, Benjamin R. Nelson1,3,4, Rhonda Bassel-Duby1,3,4 and Eric N. Olson1,3,4*Figure S1. (Characterization of MED13-mKO mouse)Figure S2. (Related to Figure 1)Figure S3. (Related to Figure 2)Figure S4. (Related to Figure 2)Figure S5. (Related to Figure 2)Figure S6. (Related to Figure 2)Figure S7. (Related to Figure 3)Figure S8. (Related to Figure 3)Figure S9. (Related to Figure 3)Figure S10. (Related to Figure 4)Figure S11. (Related to Figure 4)Figure S12. (Related to Figure 4)Figure S13. (Related to Figure 4)Figure S14. (Related to Figure 5)Supplemental Materials and Methods centertop00Supplemental Figure S1. Skeletal muscle deletion of exons 7 and 8 of the Med13 gene does not affect Mediator complex subunit expression. (A) RNA-seq data of control (CTL) and MED13-mKO adult gastrocnemius muscle show the deletion of exon 7 and exon 8 (in red) of the Med13 gene. (B) RNA expression level of Mediator complex subunits in CTL and MED13-mKO gastrocnemius muscle. The data are presented as FPKM (fragments per kilobase of transcript per million fragments mapped) of the gene expression measurements. The graphs represent 3 biological replicates of each genotype. NC, normal chow; HFD, high fat dietcentertop00Supplemental Figure S2. MED13-mKO and CTL mice on normal chow (NC) diet display similar body weight, body composition, muscle fiber type composition, glucose tolerance and comparable exercise performance. (A) Growth curves of body weight. (B) Body composition measured by NMR to determine fat mass and lean tissue mass. (C) Ratio of muscle weight to total body weight (soleus (SOL), extensor digitorum longus (EDL), gastrocnemius (GA), quadriceps (Q) and tibialis anterior (TA)). (D) Immunohistochemistry of soleus cryosections using antibody (Noq7) to slow type I myofibers. Scale bar, 50 ?m. (E) Real-time qRT-PCR of myosin heavy chain genes in gastrocnemius muscle. (F) Real-time qRT-PCR of myosin heavy chain genes in tibialis anterior muscle. (G) Average distance run by 13-week-old male MED13-mKO and CTL mice on NC diet during 6 weeks of voluntary wheel running (Exercise (Exc)). (H) Body weight of MED13-mKO and CTL upon sedentary condition (Sed) and after 6 weeks of voluntary wheel running (Exc). (I) Running time of CTL and MED13-mKO mice at exhaustion. (J) Blood lactate concentration at exhaustion after treadmill exercise. (K) Glucose tolerance test (GTT). All data are represented as mean ± SEM. (n=10) *P< 0.05, ***P<0.0005. centertop00Supplemental Figure S3. Similar liver glycogen content in MED13-mKO mice as CTL mice on NC and HFD. Data are represented as mean ± SEM.centertop00Supplemental Figure S4. MED13-mKO mice present similar energy expenditure and activity as CTL mice on HFD. Thirteen-week-old male MED13-mKO and CTL mice on HFD were analyzed in metabolic cages over 4.5 days. (A) Physical activity and number of beam breaks in the x axis over a 12 hr light/dark cycle. (B) Physical activity and number of beam breaks in the y axis over a 12 hr light/dark cycle. (C) Food consumption. (D) Average heat production per hour during the light/dark cycle normalized to lean mass. (E) Average oxygen consumption per hour during the light/dark cycle normalized to lean mass. (F) Average carbon dioxide production per hour during the light/dark cycle normalized to lean mass. Data are represented as mean ± SEM. (n=8) for HFD metabolic cage experiment. centertop00Supplemental Figure S5. MED13-mKO mice display similar adiposity as CTL mice on HFD. (A) Hematoxylin and eosin (H&E) of white adipose tissue (WAT). Scale bar, 50 ?m. (B) Real-time qRT-PCR of browning adipose marker genes (uncoupling protein 1, Ucp1; cell death-inducing DNA fragmentation factor α subunit-like effector A, Cidea; PR domain containing 16, Prdm16) and adipogenesis gene expression (Leptin, adaptor-related protein complex 2, aP2; CCAAT/Enhancer Binding Protein (C/EBP), cEBP1c; elongation of very long chain fatty acids, Elovl3) in WAT. (C) H&E of brown adipose tissue (BAT). Scale bar, 50 ?m. (D) Real-time qRT-PCR of metabolic genes expression (citrate synthase, CS; ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit 5, Atp5b; cytochrome C oxidase subunit 4i, Cox4i; carnitine palmitoyltransferase 1B, Cpt1b) in BAT. Data are represented as mean ± SEM. (n=8) *P< 0.05-800100-114300Supplemental Figure S6. Liver mitochondrial gene expression and bioenergetics analysis show moderate changes in gene expression and respiration rates. (A) MED13-mKO mice on HFD display decreased expression in lipid oxidation genes (acyl-CoA dehydrogenase, C-4 to C-12 straight chain, Acadm; acetyl-CoA carboxylase alpha, Acca2; NADH dehydrogenase (ubiquinone) Fe-S protein 2, Nduf2; NADH dehydrogenase (ubiquinone) Fe-S protein 8, Nduf8; cytochrome C oxidase subunit 1, Cox1; cytochrome C oxidase subunit 7, Cox7; ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, Atp5a1; 3-hydroxy-3-methylglutaryl-CoA synthase 2, Hmgcs2; diacylglycerol O-acyltransferase, Dgat; enoyl CoA hydratase 1, Ech1) compared to CTL mice. Real-time qRT-PCR of genes involved in lipid oxidation in the liver. (n=8). (B) Liver mitochondrial bioenergetics analysis shows a moderate increase in respiration and response to drug treatments. OCR of isolated mitochondria from liver of CTL and MED13-mKO mice on HFD in basal assay medium (basal respiration), in the presence of rotenone (complex I inhibitor), succinate (uncoupling agent), Antimycin A (complex III inhibitor) and ascorbate TMPD ((uncoupling agent for complex IV)). (n=5). Data are represented as mean ± SEM. *P < 0.05.centertopSupplemental Figure S7. MED13-mKO mice display similar muscle triglyceride as CTL mice on HFD. (A) H&E of tibialis anterior (TA) muscle. Scale bar, 50 ?m. (B) TA and gastrocnemius (GA) muscle triglyceride level. n=10. Data are represented as mean ± SEM. centertop00Supplemental Figure S8. MED13-mKO mice display similar muscle lipid content as CTL mice on NC or HFD. Un-targeted lipidomics analysis was performed using tibialis anterior muscle from MED13-mKO and CTL mice on HFD or NC diet for 12 weeks. Analysis of muscle (A) phospholipid (PL) species, (phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) (B) neutral lipid species (cholesterol esters (CE), diacylglycerols (DAG) and triacylglycerols (TAG)). (C) polar lipid species (hexosyl ceramides (HexCer), sphingomyelins (SM), ceramides (Cer) and free fatty acids (FFA)). (D) lysophospholipid species (lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC)). (E) fatty acid ester of a hydroxyl fatty acid (FAHFA). Graphs display total lipid counts normalized to muscle weight. Data are represented as mean ± SEM. (n=4).centertopSupplemental Figure S9. MED13-mKO mice display similar glucose oxidation rates as CTL mice on NC or HFD. U-13C6-glucose tracing analysis was performed in vivo in MED13-mKO and CTL mice on HFD or NC diet for 12 weeks. Isotopomer distribution enrichment of M3 lactate, M3 pyruvate, total succinate, total oxaloacetate (OAA), total alpha ketoglutarate (AKG) and total citrate. (A) Gastrocnemius muscle, (B) Quadriceps muscle, (C) Liver tissue. Graphs display isotopomer distribution enrichment. Data are represented as mean ± SEM.centertopSupplemental Figure S10. MED13-mKO mice display similar expression of secreted muscle factors as CTL mice. (A) Serum IL-6 level in MED13-mKO and CTL mice on HFD, NC diet and after 6 weeks of voluntary wheel running exercise. (B) Real-time qRT-PCR of secreted muscle factor genes expression in gastrocnemius muscle (follistatin, Fst; myostatin, Mstn; fibroblast growth factor 21, Fgf21; fibroblast growth factor 15, Fgf15; fibronectin Type III Domain Containing 5, Fdnc5). (n=8). Data are represented as mean ± SEM. centertopSupplemental Figure S11. Overall changes in skeletal muscle gene expression in MED13-mKO mice. Differentially expressed genes from the Illumina RNA-seq analysis comparing RNA isolated from gastrocnemius muscle of 18-week old MED13-mKO and CTL mice after 12 weeks on respective diets. (A) Venn diagram showing overlap of genes differentially regulated in skeletal muscle from MED13-mKO mice compared to CTL on HFD and NC. (B) Heat map of hierarchical clustering of 31 genes that only changed in muscle of MED13-mKO mice in response to HFD (red upper panel), 21 genes that changed in muscle of MED13-mKO mice, independent of dietary conditions (gray middle panel), 22 genes that changed in muscle in both MED13-mKO and CTL in response to HFD (blue lower panel). The data are presented as a log2 FPKM (fragments per kilobase of transcript per million fragments mapped) and Z-scores of the gene expression measurements are used for the clustering. The heat maps represent 3 biological replicates of each genotype.Supplemental Figure S12. Similar Nurr1, Sik1, Glut4 and Glut1 gene expression in muscle of MED13-mKO compared to CTL mice on NC diet. (n=8). Data are represented as mean ± SEM. centertop00Supplemental Figure S13. NURR1 protein expression is increased in MED13-mKO muscle. (A) Western blot analysis of NURR1 expression in MED13-mKO and CTL mice on HFD for 12 weeks. GAPDH is a loading control. (B) Quantification of NURR1 expression after normalization to GAPDH. Data are represented as mean ± SEM. **P<0.005centertopSupplemental Figure S14. MED13 does not bind MEF2. Co-IP experiments were performed by co-transfecting Myc-tagged MEF2 and GFP-tagged MED13 in HEK293 cells. Antibodies against the Myc epitope were used for co-IP. The extracts (Input) from the HEK293 cells and the proteins from the immunoprecipitation were analyzed by immunoblotting (IB). Representative results for co-IP (repeated three times) are shown. The expected position of GFP-MED13 is indicated by an asterisk (*).Supplemental Materials and Methods Animals Animals were housed in a pathogen free barrier facility with a 12 hour light/dark cycle and maintained on standard chow (2916 Teklad Global). Med13flox/flox mice were generated through homologous recombination ADDIN PAPERS2_CITATIONS <citation><uuid>D4CF5680-16EE-47EE-89E7-4CF8F53AD718</uuid><priority>0</priority><publications><publication><uuid>67ACF7E3-5A5F-46B5-9696-9E5CA3E23956</uuid><volume>149</volume><doi>10.1016/j.cell.2012.03.029</doi><startpage>671</startpage><publication_date>99201204271200000000222000</publication_date><url> cardiac microRNA governs systemic energy homeostasis by regulation of MED13</title><publisher>Elsevier Inc.</publisher><number>3</number><subtype>400</subtype><endpage>683</endpage><bundle><publication><publisher>Elsevier Inc.</publisher><title>Cell</title><type>-100</type><subtype>-100</subtype><uuid>1C861806-8B46-4F2A-AA75-59E45C2A9DA4</uuid></publication></bundle><authors><author><firstName>Chad</firstName><middleNames>E</middleNames><lastName>Grueter</lastName></author><author><nonDroppingParticle>van</nonDroppingParticle><firstName>Eva</firstName><lastName>Rooij</lastName></author><author><firstName>Brett</firstName><middleNames>A</middleNames><lastName>Johnson</lastName></author><author><firstName>Susan</firstName><middleNames>M</middleNames><lastName>DeLeon</lastName></author><author><firstName>Lillian</firstName><middleNames>B</middleNames><lastName>Sutherland</lastName></author><author><firstName>Xiaoxia</firstName><lastName>Qi</lastName></author><author><firstName>Laurent</firstName><lastName>Gautron</lastName></author><author><firstName>Joel</firstName><middleNames>K</middleNames><lastName>Elmquist</lastName></author><author><firstName>Rhonda</firstName><lastName>Bassel-Duby</lastName></author><author><firstName>Eric</firstName><middleNames>N</middleNames><lastName>Olson</lastName></author></authors></publication></publications><cites></cites></citation>(Grueter et al. 2012). Exons 7 and 8 of the Med13 gene were flanked by two loxP sites. The Med13flox/flox mice were backcrossed with C57/BL6J mice for more than three generations. To inactivate MED13 in skeletal muscle, we crossed Med13flox/flox mice with Myo-Cre transgenic mice in the C57BL/6J genetic background ADDIN PAPERS2_CITATIONS <citation><uuid>A3C2ADC6-E583-4456-98B7-B4DB95A12832</uuid><priority>1</priority><publications><publication><uuid>C1D4659C-3E20-494A-B4FB-6944FDC29F7B</uuid><volume>102</volume><doi>10.1073/pnas.0409103102</doi><startpage>1082</startpage><publication_date>99200501251200000000222000</publication_date><url> for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice.</title><location><html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"/><title>Sorry...</title><style> body { font-family: verdana, arial, sans-serif; background-color: #fff; color: #000; }</style></head><body><div><table><tr><td><b><font face=times color=#0039b6 size=10>G</font><font face=times color=#c41200 size=10>o</font><font face=times color=#f3c518 size=10>o</font><font face=times color=#0039b6 size=10>g</font><font face=times color=#30a72f size=10>l</font><font face=times color=#c41200 size=10>e</font></b></td><td style="text-align: left; vertical-align: bottom; padding-bottom: 15px; width: 50%"><div style="border-bottom: 1px solid #dfdfdf;">Sorry...</div></td></tr></table></div><div style="margin-left: 4em;"><h1>We're sorry...</h1><p>... but your computer or network may be sending automated queries. To protect our users, we can't process your request right now.</p></div><div style="margin-left: 4em;">See <a href="">Google Help</a> for more information.<br/><br/></div><div style="text-align: center; border-top: 1px solid #dfdfdf;"><a href="">Google Home</a></div></body></html></location><institution>Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.</institution><number>4</number><subtype>400</subtype><endpage>1087</endpage><bundle><publication><title>Proceedings of the National Academy of Sciences of the United States of America</title><type>-100</type><subtype>-100</subtype><uuid>645C70DA-4E67-4CAD-8C59-D984178D986B</uuid></publication></bundle><authors><author><firstName>Shijie</firstName><lastName>Li</lastName></author><author><firstName>Michael</firstName><middleNames>P</middleNames><lastName>Czubryt</lastName></author><author><firstName>John</firstName><lastName>McAnally</lastName></author><author><firstName>Rhonda</firstName><lastName>Bassel-Duby</lastName></author><author><firstName>James</firstName><middleNames>A</middleNames><lastName>Richardson</lastName></author><author><firstName>Franziska</firstName><middleNames>F</middleNames><lastName>Wiebel</lastName></author><author><firstName>Alfred</firstName><lastName>Nordheim</lastName></author><author><firstName>Eric</firstName><middleNames>N</middleNames><lastName>Olson</lastName></author></authors></publication></publications><cites></cites></citation>(Li et al. 2005). The littermates were screened by genotyping, and mice with two copies of loxP sites and Cre recombinase were characterized as MED13-mKO (Med13flox/flox ;Myo-Cre). Male mice were used in all experiments. For HFD (60% fat calories; D12492, Research Diet), mice were fed from the age of 5 weeks to the indicated times. Tissues were taken in the fed state except when otherwise mentioned. Study approvalAll experimental procedures involving animals in this study were reviewed and approved by the University of Texas Southwestern Medical Center’s Institutional Animal Care and Use Committee.Plasmids DNA fragments from the promoter region of Glut4 and Nurr1 were isolated by PCR using mouse genomic DNA as a template and cloned into the luciferase reporter pGL3 (Promega). Mutagenesis of MEF2 and NRE sites was performed using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s instructions. The pcDNA3.1 Myc-based MEF2 expression vectors were previously reported ADDIN PAPERS2_CITATIONS <citation><uuid>BEEEA399-4A23-4ABB-879C-CAA152A6C5C8</uuid><priority>2</priority><publications><publication><uuid>EDFB1632-1F5F-47A7-A9DE-525CD0C14FBE</uuid><volume>97</volume><doi>10.1073/pnas.260501497</doi><startpage>14400</startpage><publication_date>99200012191200000000222000</publication_date><url> of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5.</title><location><html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"/><title>Sorry...</title><style> body { font-family: verdana, arial, sans-serif; background-color: #fff; color: #000; }</style></head><body><div><table><tr><td><b><font face=times color=#0039b6 size=10>G</font><font face=times color=#c41200 size=10>o</font><font face=times color=#f3c518 size=10>o</font><font face=times color=#0039b6 size=10>g</font><font face=times color=#30a72f size=10>l</font><font face=times color=#c41200 size=10>e</font></b></td><td style="text-align: left; vertical-align: bottom; padding-bottom: 15px; width: 50%"><div style="border-bottom: 1px solid #dfdfdf;">Sorry...</div></td></tr></table></div><div style="margin-left: 4em;"><h1>We're sorry...</h1><p>... but your computer or network may be sending automated queries. To protect our users, we can't process your request right now.</p></div><div style="margin-left: 4em;">See <a href="">Google Help</a> for more information.<br/><br/></div><div style="text-align: center; border-top: 1px solid #dfdfdf;"><a href="">Google Home</a></div></body></html></location><institution>Department of Molecular Biology, The University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.</institution><number>26</number><subtype>400</subtype><endpage>14405</endpage><bundle><publication><title>Proceedings of the National Academy of Sciences of the United States of America</title><type>-100</type><subtype>-100</subtype><uuid>645C70DA-4E67-4CAD-8C59-D984178D986B</uuid></publication></bundle><authors><author><firstName>T</firstName><middleNames>A</middleNames><lastName>McKinsey</lastName></author><author><firstName>C</firstName><middleNames>L</middleNames><lastName>Zhang</lastName></author><author><firstName>E</firstName><middleNames>N</middleNames><lastName>Olson</lastName></author></authors></publication></publications><cites></cites></citation>(McKinsey et al. 2000). Primer sequences and plasmid construct designs are available upon request. Plasmids containing Nurr1, Nur77 were obtained from Invitrogen library.Antibodies Antibodies to MYH1 (1:3000, Noq7, M8421, Sigma-Aldrich), NURR1 (1:1000, Ab41917, Abcam), MEF2 (1:1000, sc-313, Santa Cruz Biotechnology), GFP (1:1000, A11122, Life Technology), FLAG (1:1000, Clone M2, Sigma-Aldrich), MYC (1:3000, clone 9E10, Sigma-Aldrich), GAPDH (1:8000, MAB374, Millipore), goat anti-mouse and goat-anti rabbit HRP-conjugated secondary antibodies (1:3000, Bio-Rad) were used for described experiments. Retrovirus production and C2C12 infectionRetrovirus production and C2C12 myotubes infection were performed as previously described ADDIN PAPERS2_CITATIONS <citation><uuid>E88AACC7-F378-485E-B3C0-70328C7A242D</uuid><priority>3</priority><publications><publication><volume>499</volume><publication_date>99201307171200000000222000</publication_date><number>7458</number><doi>10.1038/nature12343</doi><startpage>301</startpage><title>Myomaker is a membrane activator of myoblast fusion and muscle formation</title><uuid>9F57FDE3-DBE5-418C-99E7-19662911EC61</uuid><subtype>400</subtype><endpage>305</endpage><type>400</type><url> Publishing Group</publisher><title>Nature</title><type>-100</type><subtype>-100</subtype><uuid>FF6BABE6-876B-4677-AAF5-641E714438AB</uuid></publication></bundle><authors><author><firstName>Douglas</firstName><middleNames>P</middleNames><lastName>Millay</lastName></author><author><firstName>Jason</firstName><middleNames>R</middleNames><lastName>O’Rourke</lastName></author><author><firstName>Lillian</firstName><middleNames>B</middleNames><lastName>Sutherland</lastName></author><author><firstName>Svetlana</firstName><lastName>Bezprozvannaya</lastName></author><author><firstName>John</firstName><middleNames>M</middleNames><lastName>Shelton</lastName></author><author><firstName>Rhonda</firstName><lastName>Bassel-Duby</lastName></author><author><firstName>Eric</firstName><middleNames>N</middleNames><lastName>Olson</lastName></author></authors></publication></publications><cites></cites></citation>(Millay et al. 2013). Briefly, ten micrograms of retroviral plasmid DNA were transfected with FuGENE 6 (Roche) into Platinum E cells (Cell Biolabs), which were plated on a 10?cm culture dish at a density of 3?×?106 cells per dish, 24?hours before transfection. Forty-eight hours after transfection, viral media was collected, filtered through a 0.45?μm cellulose syringe filter and mixed with polybrene (Sigma) at a final concentration of 6?μg?ml?1. C2C12 myoblasts (obtained from the ATCC) were plated 24?hours before infection with viral media. Eighteen hours after infection, virus was removed, cells were washed with PBS and replaced with differentiation media. Cells were harvested with TRIzol (Invitrogen) after 5 days of differentiation.RNA analysisRNA was isolated from mouse tissues using TRIzol reagent (Invitrogen). Reverse Transcription-PCR (RT-PCR) was performed to generate cDNA. Primers for ribosomal 18S RNA served as internal controls for the quality of RNA. The sequence of primers is available upon request. Illumina RNA-seq analysis was performed by the University of Texas Southwestern Microarray Core Facility using RNA extracted from tissues of 12-week-old CTL or MED13-mKO on HFD or NC diet. Data analysis was performed using the TopHat and Cufflink software suite. Statistical tests, correlation analyses and plots were implemented in R () with default parameters, unless stated otherwise. Heatmaps were produced using the heatmap.2 function of the gplots package (). The canonical pathways, affected biological network and functional analyses were generated through the use of Ingenuity Pathways Analysis (IPA) from Illumina gene expression data. IPA application () was used to identify relationships among genes and place them in categories.Chromatin immunoprecipitation assaysChIP assays were performed as previously described by Tuteja et al ADDIN PAPERS2_CITATIONS <citation><uuid>9F0E272D-3DB1-481F-B2D5-BCE9D2751275</uuid><priority>4</priority><publications><publication><uuid>E5A6C8D0-807B-4781-B49F-AA2086A66F58</uuid><volume>36</volume><doi>10.1093/nar/gkn366</doi><startpage>4149</startpage><publication_date>99200807001200000000220000</publication_date><url> modules in the mammalian liver: composition depends on strength of Foxa2 consensus site.</title><institution>Department of Genetics, Genomics and Computational Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.</institution><number>12</number><subtype>400</subtype><endpage>4157</endpage><bundle><publication><title>Nucleic Acids Research</title><type>-100</type><subtype>-100</subtype><uuid>6D89FA9B-4DE2-44ED-8E41-4B60B0F2DB51</uuid></publication></bundle><authors><author><firstName>Geetu</firstName><lastName>Tuteja</lastName></author><author><firstName>Shane</firstName><middleNames>T</middleNames><lastName>Jensen</lastName></author><author><firstName>Peter</firstName><lastName>White</lastName></author><author><firstName>Klaus</firstName><middleNames>H</middleNames><lastName>Kaestner</lastName></author></authors></publication></publications><cites></cites></citation>(Tuteja et al. 2008). Briefly, quadriceps muscle powder was cross-linked with 1% formaldehyde for 15 min at room temperature (RT). Chromatin fragmentation was performed by sonication using a Diagnode BioRupter (7 min with 30 sec on/off). Proteins were immuno-precipitated by using three micrograms anti-MEF2, anti-Nurr1 antibody or IgG control. The antibody/chromatin complexes were left to rotate end to end overnight at 4°C. Antibody/chromatin complexes were pulled down using Dynabeads protein G-conjugated magnetic beads (Life Technologies). Chromatin was washed, eluted, and reverse-cross-linked, followed by protease treatment. Chromatin fragments were then analyzed by quantitative PCR using SYBR Green fluorescence. Primers are available upon request. All values are expressed as mean ± SEM. Statistical analysis was performed using a two-tailed Student's unpaired t-test. Results were considered significant when P< 0.05.Luciferase reporter assaysLuciferase assays were performed as previously described ADDIN PAPERS2_CITATIONS <citation><uuid>AD6B6876-0014-4BB1-9BD9-45B5EDCDC84B</uuid><priority>5</priority><publications><publication><uuid>67ACF7E3-5A5F-46B5-9696-9E5CA3E23956</uuid><volume>149</volume><doi>10.1016/j.cell.2012.03.029</doi><startpage>671</startpage><publication_date>99201204271200000000222000</publication_date><url> cardiac microRNA governs systemic energy homeostasis by regulation of MED13</title><publisher>Elsevier Inc.</publisher><number>3</number><subtype>400</subtype><endpage>683</endpage><bundle><publication><publisher>Elsevier Inc.</publisher><title>Cell</title><type>-100</type><subtype>-100</subtype><uuid>1C861806-8B46-4F2A-AA75-59E45C2A9DA4</uuid></publication></bundle><authors><author><firstName>Chad</firstName><middleNames>E</middleNames><lastName>Grueter</lastName></author><author><nonDroppingParticle>van</nonDroppingParticle><firstName>Eva</firstName><lastName>Rooij</lastName></author><author><firstName>Brett</firstName><middleNames>A</middleNames><lastName>Johnson</lastName></author><author><firstName>Susan</firstName><middleNames>M</middleNames><lastName>DeLeon</lastName></author><author><firstName>Lillian</firstName><middleNames>B</middleNames><lastName>Sutherland</lastName></author><author><firstName>Xiaoxia</firstName><lastName>Qi</lastName></author><author><firstName>Laurent</firstName><lastName>Gautron</lastName></author><author><firstName>Joel</firstName><middleNames>K</middleNames><lastName>Elmquist</lastName></author><author><firstName>Rhonda</firstName><lastName>Bassel-Duby</lastName></author><author><firstName>Eric</firstName><middleNames>N</middleNames><lastName>Olson</lastName></author></authors></publication></publications><cites></cites></citation>(Grueter et al. 2012). Briefly, COS-7 cells (CRL-1651; ATCC) were grown in DMEM containing 10% fetal bovine serum (FBS). Transfections were performed with FuGENE 6 Transfection Reagent (Promega) according to the manufacturer’s instructions. For luciferase assays, cells were plated into 12-well dishes. After approximately 12 hours of incubation, transfection reagent was added. Unless indicated otherwise, 700 ng total plasmid DNA was used. A CMV promoter–driven LacZ expression plasmid was included for all transfections as an internal control (100 ng). The total amount of each plasmid DNA per condition was kept constant by adding expression vectors without a cDNA insert if necessary. Forty-eight hours later, cells were lysed, and luciferase activity (Promega) and β-galactosidase activity (Invitrogen) were assessed according to the manufacturer’s instructions. All experiments were performed in technical triplicate and were repeated at least three times.ImmunoprecipitationHEK293 cells were grown in DMEM containing 10% FBS. Transfections were performed with FuGENE 6 Transfection Reagent (Promega) according to the manufacturer’s instructions. Twenty-four hours after transfection cells were lysed with a syringe and with a Fischer Scientific 550 sonicator in lysis buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 1% TritonX-100, complete protease inhibitors cocktail (Roche), 1 mM PMSF, 10 mM NaF). For the Co-IP analysis of NURR1 and MEF2 (using FLAG-tagged NURR1 and Myc-tagged-MEF2), anti-MYC agarose beads (Sigma-Aldrich) were incubated with extract. The immunoprecipitated proteins were analyzed by immunoblotting. For the Co-IP analysis of NURR1 and MED13 (using FLAG-tagged NURR1 and GFP-tagged-MED13), one microgram of anti-GFP antibody was incubated with extract and Dynabeads protein G-conjugated magnetic beads (Life Technologies), and the immunoprecipitated proteins were analyzed by immunoblotting. All experiments were repeated at least two times.HistologyWAT, BAT and liver were isolated and fixed in 4% paraformaldehyde (PFA) and processed for H&E staining. For oil red O staining, liver tissues were fixed in 4% PFA overnight, incubated in 12% sucrose for 12 hours then in 18% sucrose overnight before being cryoembedded and sectioned by the UT Southwestern Histology Core Facility. For skeletal muscle fiber analysis, tissues were frozen in liquid-nitrogen precooled isopentane, and 8 ?m sections were used for H&E and fiber type staining.Metabolic chambers and whole-body composition analysis Metabolic phenotyping of CTL and MED13-mKO mice on HFD was performed using TSE metabolic chamber analysis by the Mouse Metabolic Phenotyping Core Facility at University of Texas Southwestern Medical Center. Thirteen week old mice on HFD were placed in TSE metabolic chambers for an initial 5 days acclimation period, followed by a 4.5 days experimental period with data collection. Whole-body composition parameters were measured by magnetic resonance imaging (MRI) using a Bruker Minispec mq10 system.Plasma and tissue chemistryBlood was collected using a 1 ml syringe coated in 0.5 M K2EDTA and serum collected by centrifugation for 20 min at 1000xg. Insulin and leptin levels were measured by ELISA. Serum triglycerides levels were measured using the Ortho Vitros 250 chemistry system. To measure triglyceride in the liver and skeletal muscle, tissue specimens were frozen immediately after isolation and pulverized in liquid nitrogen with a cell crusher. Serum and tissue triglyceride levels were measured by Mouse Metabolic Phenotyping Core Facility at University of Texas Southwestern Medical Center.Muscle lipidomics analysisLipids were quantified by shotgun lipidomics: Using an ABI 5600+ (AB Sciex, Gramingham, MA). Lipids were quantified by shotgun lipidomics: Using an ABI 5600+ (AB Sciex, Gramingham, MA), we simultaneously identified changes in hundreds of distinct lipid species via a nonbiased approach following direct infusion of extracted lipids containing 18 mM ammonium fluoride to aid in ionization of neutral lipids and to reduce salt adducts. Data from the AB Sciex 5600+ was collected and calibrated with Analyst and PeakView Software (AB Sciex, Framingham, MA). The in-house-developed Lipid Explorer software assists with simplifying the data by identifying lipid species based on exact mass and fragmentation patterns.13C-glucose isotope metabolic tracing analysisAfter a 6 h fast, mice underwent a GTT where 10% of [U-13C]-glucose (CLM-1396 D-Glc-U-13C6 99% from Cambridge Isotope) was administered via IP injection. Tissues were collected at 60 min following glucose administration and were rapidly frozen in liquid nitrogen. Glucose ADDIN PAPERS2_CITATIONS <citation><uuid>39901DF2-A6BA-4873-B1BE-8B915A0D4A6A</uuid><priority>6</priority><publications><publication><volume>88</volume><publication_date>99201002251200000000222000</publication_date><number>3</number><doi>10.2527/jas.2009-2479</doi><startpage>912</startpage><title>Gluconeogenesis differs in developing chick embryos derived from small compared with typical size broiler breeder eggs</title><uuid>134D1203-1B2A-4139-8F87-AB36DFF7D9D5</uuid><subtype>400</subtype><endpage>921</endpage><type>400</type><url> of Animal Science</title><type>-100</type><subtype>-100</subtype><uuid>2A9F2C6C-D5FE-49DB-BA45-49CA2AFBA888</uuid></publication></bundle><authors><author><firstName>N</firstName><middleNames>E</middleNames><lastName>Sunny</lastName></author><author><firstName>B</firstName><middleNames>J</middleNames><lastName>Bequette</lastName></author></authors></publication></publications><cites></cites></citation>(Sunny and Bequette 2010),?organic and amino acid ADDIN PAPERS2_CITATIONS <citation><uuid>ECC825B1-5D7E-495A-97EA-726DD948983A</uuid><priority>7</priority><publications><publication><volume>26</volume><publication_date>99200311011200000000222000</publication_date><number>17</number><doi>10.1002/jssc.200301492</doi><startpage>1474</startpage><title>Comparison of silylation and esterification/acylation procedures in GC-MS analysis of amino acids</title><uuid>2C2BD41D-C746-4069-825A-5C05AE073F58</uuid><subtype>400</subtype><endpage>1478</endpage><type>400</type><url> of Separation Science</title><type>-100</type><subtype>-100</subtype><uuid>623EE239-EAA7-4EB0-8D3C-352672743B27</uuid></publication></bundle><authors><author><firstName>Tim</firstName><middleNames>G</middleNames><lastName>Sobolevsky</lastName></author><author><firstName>Alexander</firstName><middleNames>I</middleNames><lastName>Revelsky</lastName></author><author><firstName>Barbara</firstName><lastName>Miller</lastName></author><author><firstName>Vincent</firstName><lastName>Oriedo</lastName></author><author><firstName>Elena</firstName><middleNames>S</middleNames><lastName>Chernetsova</lastName></author><author><firstName>Igor</firstName><middleNames>A</middleNames><lastName>Revelsky</lastName></author></authors></publication></publications><cites></cites></citation>(Sobolevsky et al. 2003). 13C-mass isotopomer enrichments were determined as previously described.Glucose uptake and insulin toleranceGlucose tolerance test and insulin tolerance test were performed as previously described. For glucose tolerance test, mice were fasted for 6 h and injected intraperitoneally with a glucose solution (0.15 g/ml, 158968 from Sigma-Aldrich, St. Louis, MO) at 1.5 g/kg body weight. Blood glucose concentrations were measured before and 15, 30, 60 and 90 min after glucose injection. For insulin tolerance test, mice prefasted for 6 hours were injected intraperitoneally with insulin (Human insulin I9278 from Sigma-Aldrich, St. Louis, MO) at 1.0 U/kg body weight. Blood glucose concentrations were measured before and 15, 30, 60 and 90 min after insulin injection.Glycogen measurementsA glycogen colorimetric/fluorometric assay kit (Abcam 65620) was used as per the manufacturer’s protocol to measure the quadriceps glycogen content in CTL and MED13-mKO mice on HFD and NC diet.Hyperinsulemic euglycemic clampsClamps were performed in conscious unrestrained animals as previously described ADDIN PAPERS2_CITATIONS <citation><uuid>8D160FDD-7D9F-428D-81EA-FA1CC8CBD9CF</uuid><priority>8</priority><publications><publication><uuid>554EBF7B-6341-4C9B-847E-61EC42F47671</uuid><volume>55</volume><startpage>390</startpage><publication_date>99200602001200000000220000</publication_date><url> in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse.</title><location><html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"/><title>Sorry...</title><style> body { font-family: verdana, arial, sans-serif; background-color: #fff; color: #000; }</style></head><body><div><table><tr><td><b><font face=times color=#0039b6 size=10>G</font><font face=times color=#c41200 size=10>o</font><font face=times color=#f3c518 size=10>o</font><font face=times color=#0039b6 size=10>g</font><font face=times color=#30a72f size=10>l</font><font face=times color=#c41200 size=10>e</font></b></td><td style="text-align: left; vertical-align: bottom; padding-bottom: 15px; width: 50%"><div style="border-bottom: 1px solid #dfdfdf;">Sorry...</div></td></tr></table></div><div style="margin-left: 4em;"><h1>We're sorry...</h1><p>... but your computer or network may be sending automated queries. To protect our users, we can't process your request right now.</p></div><div style="margin-left: 4em;">See <a href="">Google Help</a> for more information.<br/><br/></div><div style="text-align: center; border-top: 1px solid #dfdfdf;">&copy; 2013 Google - <a href="">Google Home</a></div></body></html></location><institution>Vanderbilt-NIDDK (National Institutes of Diabetes and Digestive and Kidney Diseases) Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. julio.ayala@vanderbilt.edu</institution><number>2</number><subtype>400</subtype><endpage>397</endpage><bundle><publication><title>Diabetes</title><type>-100</type><subtype>-100</subtype><uuid>CBFC774A-A910-474A-8E02-A24BF5379449</uuid></publication></bundle><authors><author><firstName>Julio</firstName><middleNames>E</middleNames><lastName>Ayala</lastName></author><author><firstName>Deanna</firstName><middleNames>P</middleNames><lastName>Bracy</lastName></author><author><firstName>Owen</firstName><middleNames>P</middleNames><lastName>McGuinness</lastName></author><author><firstName>David</firstName><middleNames>H</middleNames><lastName>Wasserman</lastName></author></authors></publication><publication><uuid>F7BAF161-F973-4D13-9719-D80F85D9FB17</uuid><volume>560</volume><doi>10.1007/978-1-59745-448-3_15</doi><startpage>221</startpage><publication_date>99200900001200000000200000</publication_date><url> clamp to assess insulin sensitivity in vivo.</title><location><html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"/><title>Sorry...</title><style> body { font-family: verdana, arial, sans-serif; background-color: #fff; color: #000; }</style></head><body><div><table><tr><td><b><font face=times color=#0039b6 size=10>G</font><font face=times color=#c41200 size=10>o</font><font face=times color=#f3c518 size=10>o</font><font face=times color=#0039b6 size=10>g</font><font face=times color=#30a72f size=10>l</font><font face=times color=#c41200 size=10>e</font></b></td><td style="text-align: left; vertical-align: bottom; padding-bottom: 15px; width: 50%"><div style="border-bottom: 1px solid #dfdfdf;">Sorry...</div></td></tr></table></div><div style="margin-left: 4em;"><h1>We're sorry...</h1><p>... but your computer or network may be sending automated queries. To protect our users, we can't process your request right now.</p></div><div style="margin-left: 4em;">See <a href="">Google Help</a> for more information.<br/><br/></div><div style="text-align: center; border-top: 1px solid #dfdfdf;"><a href="">Google Home</a></div></body></html></location><institution>UMass Mouse Phenotyping Center, University of Massachusetts Medical School, 381 Plantation Street, Suite 200 Worcester, MA, USA. jason.kim@psu.edu</institution><subtype>400</subtype><endpage>238</endpage><bundle><publication><title>Methods in molecular biology (Clifton, N.J.)</title><type>-100</type><subtype>-100</subtype><uuid>377400EC-855B-42C1-9369-A0B48B817C0E</uuid></publication></bundle><authors><author><firstName>Jason</firstName><middleNames>K</middleNames><lastName>Kim</lastName></author></authors></publication></publications><cites></cites></citation>(Ayala et al. 2006; Kim 2009). Hyperinsulinemia was initiated via a primed continuous infusion of insulin (4 mU/kg/min) and glycemia was maintained (~140 mg/dL) via variable infusion of 50% dextrose. Glucose kinetics were assessed via a primed (0.5 uCi/min for 2 min), continuous infusion of 3H-Glucose. 0.05 uCi glucose/min was infused for 90 min prior to initiating hyperinsulinemia, and 0.1 uCi glucose/min was infused during the hyperinsulinemic period. 2-deoxyglucose uptake was assessed for 25 min following a bolus (13 uCi) of 14C-2-deoxyglucose during the clamped state.Voluntary wheel running Ten-week old MED13-mKO and corresponding CTL littermates were randomly assigned to housing in individual cages with or without a running wheel for a total of 6 weeks. Completed wheel revolutions and time spent running were continuously monitored and recorded. Run distance over 24-hour periods was determined at the end.Treadmill exerciseMice were run on Exer-6 treadmill apparatus (Columbus Instruments) with mild electrical stimulus at 5% inclination. Two days before the experiment, mice were acclimatized to a single lane treadmill by performing a 10 m/min run for 30 min. To test maximal running speed, mice were acclimated for a period of 30 min at 10 m/min followed by acceleration for 1 m/min until exhaustion. For endurance test, mice were acclimated for 60 min at 10 m/min followed by incremental acceleration (1 m/min every 5 min) to a maximum speed of 20 m/min until exhaustion. Exhaustion was defined by failure to run for greater than 10 sec. Time to exhaustion was determined.Immunoblot analysisProteins were extracted from skeletal muscle of mice. Muscles were homogenized in RIPA Buffer, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF and protease inhibitors tablet (Roche Diagnostics). Protein concentration was determined using a BCA protein assay kit (Thermo Scientific) and lysates analyzed by SDS–polyacrylamide gel electrophoresis and western blot analysis on PVDF membrane. Statistical Analysis All values are given as mean standard error. Differences between two groups were assessed using unpaired two-tailed Student’s t-tests. P<0.05 was regarded as significant. Statistical analysis was performed in Excel (Microsoft).REFERENCES ADDIN PAPERS2_CITATIONS <papers2_bibliography/>Ayala JE, Bracy DP, McGuinness OP, Wasserman DH. 2006. Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes 55: 390–397.Grueter CE, van Rooij E, Johnson BA, DeLeon SM, Sutherland LB, Qi X, Gautron L, Elmquist JK, Bassel-Duby R, Olson EN. 2012. A cardiac microRNA governs systemic energy homeostasis by regulation of MED13. Cell 149: 671–683.Kim JK. 2009. Hyperinsulinemic-euglycemic clamp to assess insulin sensitivity in vivo. Methods Mol Biol 560: 221–238.Li S, Czubryt MP, McAnally J, Bassel-Duby R, Richardson JA, Wiebel FF, Nordheim A, Olson EN. 2005. Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice. Proc Natl Acad Sci USA 102: 1082–1087.McKinsey TA, Zhang CL, Olson EN. 2000. Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5. Proc Natl Acad Sci USA 97: 14400–14405.Millay DP, O’Rourke JR, Sutherland LB, Bezprozvannaya S, Shelton JM, Bassel-Duby R, Olson EN. 2013. Myomaker is a membrane activator of myoblast fusion and muscle formation. Nature 499: 301–305.Sobolevsky TG, Revelsky AI, Miller B, Oriedo V, Chernetsova ES, Revelsky IA. 2003. Comparison of silylation and esterification/acylation procedures in GC-MS analysis of amino acids. J Sep Science 26: 1474–1478.Sunny NE, Bequette BJ. 2010. Gluconeogenesis differs in developing chick embryos derived from small compared with typical size broiler breeder eggs. J Anim Sci 88: 912–921.Tuteja G, Jensen ST, White P, Kaestner KH. 2008. Cis-regulatory modules in the mammalian liver: composition depends on strength of Foxa2 consensus site. Nucleic Acids Res 36: 4149–4157. ................
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