Supplemental Material, Amoasii et al.

A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolism

Leonela 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

Supplemental 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 diet

Supplemental 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.

Supplemental Figure S3. Similar liver glycogen content in MED13-mKO mice as

CTL mice on NC and HFD. Data are represented as mean ± SEM.

Supplemental 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.

Supplemental 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

Supplemental 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.

Supplemental 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.

Supplemental 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).

Supplemental 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.

Supplemental 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.

Supplemental 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.

Supplemental 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<0.005

Supplemental 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 (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 (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 approval

All 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 (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 infection

Retrovirus production and C2C12 myotubes infection were performed as

previously described (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 analysis

RNA 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

(http://www.R-project.org/) with default parameters, unless stated otherwise.

Heatmaps were produced using the heatmap.2 function of the gplots package

(http://CRAN.R-project.org/package=gplots). 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 (http://www.ingenuity.com) was used to identify relationships among

genes and place them in categories.

Chromatin immunoprecipitation assays

ChIP assays were performed as previously described by Tuteja et al (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 assays

Luciferase assays were performed as previously described (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.

Immunoprecipitation

HEK293 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.

Histology

WAT, 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 chemistry

Blood 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 analysis

Lipids 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 analysis

After 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 (Sunny and Bequette

2010), organic and amino acid (Sobolevsky et al. 2003). 13C-mass isotopomer

enrichments were determined as previously described.

Glucose uptake and insulin tolerance

Glucose 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 measurements

A 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 clamps

Clamps were performed in conscious unrestrained animals as previously

described (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 exercise

Mice 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 analysis

Proteins 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).

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