Supplemental Material, Amoasii et al. A MED13-dependent skeletal muscle gene program controls systemic glucose homeostasis and hepatic metabolism Leonela Amoasii 1,3,4 , William Holland 2 , Efrain Sanchez- Ortiz 1,3,4 , Kedryn K. Baskin 1,3,4 , Mackenzie Pearson 2 , Shawn C. Burgess 5 , Benjamin R. Nelson 1,3,4 , Rhonda Bassel-Duby 1,3,4 and Eric N. Olson 1,3,4* 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)
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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,
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).
REFERENCES
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.