Baf60c drives glycolytic muscle formation and improves glucose homeostasis through Deptor-mediated Akt activation

Transcription

1 Baf6c drives glycolytic muscle formation and improves glucose homeostasis through Deptor-mediated Akt activation Zhuo-Xian Meng,2, Siming Li,2, Lin Wang,2, Hwi Jin Ko 3, Yongjin Lee 3, Dae Young Jung 3, Mitsuharu Okutsu 4-6, Zhen Yan 4-6, Jason K Kim 3 & Jiandie D Lin,2 Life Sciences Institute, and 2 Department of Cell and Developmental Biology,University of Michigan Medical Center, Ann Arbor, Michigan, USA. 3 Program in Molecular Medicine and Division of Endocrinology, Metabolism and Diabetes, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. 4 Department of Medicine, 5 Department of Pharmacology, and 6 Center for Skeletal Muscle Research at Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia, USA. Corresponding Author: Jiandie Lin, Ph.D Life Sciences Institute University of Michigan 2 Washtenaw Avenue Ann Arbor, MI jdlin@umich.edu Office: (734) Fax: (734)

2 Plantaris Soleus a b Percentage (%) Percentage (%) I IIa IId/x IIx IIb I IIa IId/x IIx IIb c d Percentage (%) ,,2 2,4 4,6 6, , Fiber cross-sectional area (μm 2 ) Supplementary Figure. Skeletal muscle fiber type and size analyses. (a,b) Representative images of plantaris (a) or soleus (b) muscle frozen sections from and MCK-Baf6c transgenic () mice immunostained for MHC I (red), IIa (green), and IIb (Blue). Type IId/x fibers remain unstained. Quantitation of each fiber type is shown below (n = 4 5 mice per group). Data represent mean ± s.e.m.; P <.5, vs.. (c) Crosssections of tibialis anterior (TA) muscle from and mice were immunostained with anti-laminin antibody (green) and DAPI (blue). Shown are representative images. (d) Immunostained images of and TA muscle sections were analyzed by CyteSeer software (Vala Sciences). Distribution of myofiber cross-sectional area is shown. Data represent mean ± s.e.m. (n = 4 mice per group).

3 a Hrt LDH5 (A4) LDH4 (A3B) LDH3 (A2B2) LDH2 (AB3) LDH (B4) Relative activity LDH LDH2 LDH3 LDH4 LDH5 b c Myoglobin pampk-72 VLDLR Total-AMPK GAPDH CD36 Ndufb8 Uqcrc2 Atp5a HA Tubulin Supplementary Figure 2. Skeletal muscle gene expression and LDH isoenzyme analysis. (a) Left, enzymatic staining of lactate dehydrogenase (LDH) isoenzymes following non-denaturing polyacrylamide gel electrophoresis of whole cell extracts from normal heart (Hrt, serve as a positive control) or plantaris muscles. Right, quantitation of LDH isoenzyme activity shown on the gel image. Note the shift from LDH-B to LDH-A isoenzymes in the transgenic muscles. Data represent mean ± s.e.m. (n = 5 6 mice per group); P <., vs.. (b) Immunoblots of total quadriceps protein lysates from and mice. GAPDH was used as a loading control. Note the phosphorylation levels of AMPK were similar between two groups. (c) Immunoblots of total quadriceps protein lysates from and mice. HA antibody was used to detect FH-Baf6c. Tubulin was used as a loading control.

4 Supplementary Figure 3. Regulation of skeletal muscle gene expression by Baf6c. qpcr analysis of genes in plantaris and soleus muscles from and mice (n = 6 8 mice per group). Genes significantly elevated in muscles are shown as red whereas those significantly decreased as blue cells. Gray cells indicate no significant changes.

5 a c Glycogen content (μmol glycosyl residue g ) Muscle triglycerides (mg g tissue) b Glycerol (mg ml ) β-hydroxybutyrate (mm) NEFA (mm) Triglycerides (mg ml ) d Supplementary Figure 4. Metabolic parameters and gene expression. (a) Baseline TA muscle glycogen content in chow diet fed mice. (b) Plasma metabolite concentrations in HFD-fed (open) and MCK-Baf6c (filled) mice. (c) Gast muscle triglyceride content in HFD-fed mice. (d) qpcr analysis of myokine and cytokine gene expression in gastrocnemius muscles from HFD-fed (open) and (filled) mice. a d, Data represent mean ± s.e.m. (n = 5 8 mice per group); P <.5 vs..

6 Liver BAT WAT Supplementary Figure 5. qpcr analyses of gene expression. Total RNA was isolated from liver, brown adipose tissue (BAT) and white adipose tissue (WAT) from HFD-fed (open) and MCK-Baf6c (filled) mice. Data represent mean ± s.e.m. (n = 5 8 mice per group); P <.5 vs..

7 a Gast Quad Plantaris Soleus #9 2 # #9 2 #5 3 #9 2 #5 3 #9 2 #5 3 b Deptor HA Tubulin Gast Quad Plantaris Soleus #9 #5 #9 #5 #9 #5 #9 #5 c Supplementary Figure 6. Baf6c regulates Deptor gene expression in transgenic muscles and C2C2 myotubes. (a) qpcr analysis of Deptor mrna expression in indicated muscles from and lines #9 and #5. Data represent mean ± s.e.m. (n = 3 mice per group); P <.5 vs.. Gast, gastrocnemius; Quad, quadriceps. (b) Immunoblots of pooled total protein lysates from indicated muscles. HA antibody was used to detect FH-Baf6c. Tubulin was used as a loading control. (c) qpcr analyses of Deptor expression in transduced C2C2 myotubes. Data represent mean ± s.d.; P <.. Vec, Vector; Scrb, Scramble.

8 a d Vec Baf6c b e pakt-38 c Vec BAF6c Baf6c pakt-473 Total-Akt Deptor HA Tubulin GFP Baf6c Supplementary Figure 7. Baf6c stimulates Deptor gene expression and Akt phosphorylation independently of muscle differentiation. (a) H&E staining of Gast muscle. (b) qpcr analysis of muscle gene expression in plantaris from and mice. Data represent mean ± s.e.m. (n = 7 8 mice per group); P <.5. (c) qpcr analysis of muscle gene expression in transduced myotubes. Data represent mean ± s.d.; P <.5. Note that the expression of muscle differentiation markers is largely unaffected in Baf6c overexpressing C2C2 myotubes and transgenic skeletal muscle. (d) Myotube morphology in transduced C2C2 cells after differentiation revealed by myostin (MF 2) immunofluorescent staining. (e) Immunoblots of total protein lysates from differentiated C2C2 myotubes transiently transduced with GFP or Baf6c adenoviruses. HA antibody was used to detect FH-Baf6c. Tubulin was used as a loading control.

9 a Scrb sibaf6c b Vehicle Insulin pakt-38 8 pakt-473 Total-Akt Baf6c Deptor Glycolytic flux (nmol h mg protein) Tubulin Insulin (nm) Scrb SiBAF6c sibaf6c c Vec FH-Deptor Scrb sideptor pakt-38 pakt-473 Total-Akt FH-Deptor Deptor Tubulin Supplementary Figure 8. Baf6c and Deptor regulate Akt phosphorylation in cultured myotubes. (a) Transduced and differentiated C2C2 myotubes were starved in DMEM containing.% BSA overnight and treated with different doses of insulin for 3 min. Total protein lysates were analyzed by immunoblotting with indicated antibodies. (b) Glycolytic flux measurements using D-[5-3 H]-glucose in transduced C2C2 myotubes treated with vehicle or insulin (6 nm) for h. (c) C2C2 myoblast were transduced with retroviral vectors expressing vector (Vec), FH-Deptor, Scramble shrna (Scrb), or Deptor shrna (sideptor), and differentiated into myotubes. Total protein lysates were isolated for immunoblotting with indicated antibodies. Note that Akt phosphorylation on both T38 and S473 sites was enhanced by Deptor overexpression but attenuated by Deptor RNAi knockdown.

10 Supplementary Table. ChIP qpcr primers Relative to TSS Forward primer Reverse primer Deptor promoter -,85 ATGGGTGAATGAAACAAATCTCAA CACTATGCCCAGCTGCTGGT -,5 TCCCTCTGTCACTTAATGTTTATGCT TTATAAGACCTGAACACCATGCTCTG 3 CGCATGGCTGAAGTCCTAGTT GGGCATAGGGCAATCGAGA 6 CACCACTCAGTGCGTGCAA ATCCGAAATCTCTTCTTTTTGGAG Smarcd3 promoter -3 GCGAATTCCCTAACCGAGAAA CCACGGGAGGTGACAAGG CAGAAAAGGGAAAGAAAGAGGAAGA GGTGGGCTCAGCGGC 2 GCGGACGAAGTTGCCG GACCAGAAACTCAAAAAGTTTGCTT TSS, Transcriptional start site

11 Supplementary Table 2. Gene expression qpcr primers Mouse gene primers Transcriptional factors Smarcd TGGACCCAAATGACCAGAAAA TCTTGTTGTCTAGAGTGGCGATCT Smarcd2 GAAGCTGGACCAGACCATCG CGCAGTTCCCGCATTATCTC Smarcd3 AGGCTTACATGGACCTCCTAG CATCAGAGTCTTCCGCATCAG Pgc-α AGCCGTGACCACTGACAACGAG GCTGCATGGTTCTGAGTGCTAAG Pgc-β GCTCCAGGAGACTGAATCCAGAG CTTGACTACTGTCTGTGAGGC Nr4a GCGTGACCACCTGACCGGTGAT AACAAGCTGAGGAGCACGGCTG Tbx AATCAGCTGGGCACCGAG AGCTTCACTTGGAACGTGGG Tbx5 ACCTCCTGCTCCTTCAGCACTCA CTTCCAGAGGTCAGCACATTGCA PPARγ CCGTAGAAGCCGTGCAAGAG GGAGGCCAGCATCGTGTAGA PPARα GCAGTGCCCTGAACATCGA CGCCGAAAGAAGCCCTTAC Srebpc GGAGCCATGGATTGCACATT GGCCCGGGAAGTCACTGT Glucose metabolism Glut4 TTCATTGTCGGCATGGGTTT ACGGCAAATAGAAGGAAGACGTA HK GAGGTCTACGACACCCCAGA GAAGTCTCCGAGGCATTCAG HK2 CCGCCGTGGTGGACAAGATA AGCAGTGATGAGAGCCGCTC Gpi AATCGCCTCCAAGACCTTCA CGAGAAACCACTCCTTTGCTGT Pfkm CGTGGGAGAGCGTGTCTATGA CTCGCTCTCGGAAGTCCTTG Fbp2 GGTTCCATGGTGGCTGATGT GGCCACAGGATTGCATTCAT AldoA AATGTTCTGGCCCGTTATGC GGAGAATTTCAGGCTCCACAAT Tpi CAAACCAAGGTCATCGCAGA GCCCACACAGGTTCATAGGC Gpd CCCATGAGCGTGCTGATG GTGATGCGAAAGTTGGGTGTCT Gpd2 CACGCACCATCCTATTCC GACATCCCCTCTTCTCACTTC Pgk GCTGTTCTCCTCTTCCTCATC CCTTTGGTTGTTTGTTATCTGG Pgam2 GAGAGTGCTTATTGCAGCCCA GGTCGGACATCCCTTCCAG Eno3 CGACACATCGCAGATCTTGC CCGTTGATCACATTAAAGGCAG Pkm2 ATCATTTGTACCATTGGGCCTG TTCATTCCAGACTTAATCATCTCCTTC Ldha TGCCTACGAGGTGATCAAGCT GCACCCGCCTAAGGTTCTTC Ldhb AGTCTCCCGTGCATCCTCAA AGGGTGTCCGCACTCTTCCT Pdk4 GAGTATAAAGACACCTGCACAG CCGTCTTTGAGTCACTGAATATG Phka CATCTCTGCGCCTCTACCGTA TGCTCATGTCGCCTTCACTG Pygm CTCCTCAACTGCCTGCACATC ACCTATTGGGCTCCCTTTTGAT Pppr3c TCCAATGAGCTGCACCAGAAT GGCATGACGGAACTTGTCAAA Gys GATGAATTCGACCCCGAGAA GATGCCACCCACCTTGTTG Phkg ACCGAGAAGGAAACCAGAAAGA ATGTTGAGTTTGTGCAGGGTACA PEPCK CATATGCTGATCCTGGGCATAAC CAAACTTCATCCAGGCAATGTC G6Pase ACACCGACTACTACAGCAACAG CCTCGAAAGATAGCAAGAGTAG Lipid metabolism CD36 TTAGATGTGGAACCCATAACTGGA TTGACCAATATGTTGACCTGCAG VLDLR AGAGCCTGCCTCCATAGCTG CGCCCCAGTCTGACCAGTAA Cptα GAGAAATACCCTGACTATGTG TGTGAGTCTGTCTCAGGGCTAG Cptβ ACAGACTTGCTACAGCACCTC CGTCGAGGATTCTCTGGAAC Acc2 CCCAGCCGAGTTTGTCACT GGCGATGAGCACCTTCTCTA Acadm GCTGGAGACATTGCCAATCA GGCGTCCCTCATCAGCTTCT Ech AAGATAAGGACGCCATGCTGAA TCCAGGTGGCCATGTAGTCA Acox GCCTGCTGTGTGGGTATGTCATT GTCATGGGCGGGTGCAT Acaa2 GATCTCAAGCTGGAAGATAC ACCTCTGCTGAGACTGCAAG Hmgcs2 GACATCAACTCCCTGTGCCTG GATGTCAGTGTTGCCTGAATC Acc TAATGGGCTGCTTCTGTGACTC CTCAATATCGCCATCAGTCTTG Scd GCTGGAGTACGTCTGGAGGAA TCCCGAAGAGGCAGGTGTAG FAS GGTTACACTGTGCTAGGTGTTG TCCAGGCGCATGAGGCTCAGC Dgat GCTCTGGCATCATACTCCATC CGGTAGGTCAGGTTGTCTGG GPAT TGCTGAAGTGGCTGCGGAGTTG TTAGTAACACCCAGCCAGTCAG S32 GCTCCACCTCCACAAAGGAC GATGCCAAACACCCCAGAG Fsp27 TCGACCTGTACAAGCTGAACCCT AGGTGCCAAGCAGCATGTGACC

12 Mitochondrial function Ndufb8 GGCACGTGTTCCCTTCCTAC CCGCTCCAGGTACAGATTATTGT Sdhb CGACACGGACCTCAGCAAA CGACACGGACCTCAGCAAA Uqcrc2 TGGCTCTGGTTGGACTTGGT TGGCTCTGGTTGGACTTGGT Mtco CTACTATTCGGAGCCTGAGC GCATGGGCAGTTACGATAAC Atp5a TTTGCCCAGTTTGGTTCTGAT CCCGTACACCCGCATAGATAA Ndufv2 CTCCTGAGAATAACCCAGAT TCATAGCGGAGATAGGTAGC Ndufa2 TGCGTGAGATTCGCGTTCAC CTGCACCTCCGAGCATTCGC Cox7a GTCTCCCAGGCTCTGGTCCG CTGTACAGGACGTTGTCCATTC Muscle related Myoglobin CATGGACAGGAAGTCCTCATCG CTGTGAGCACGGTGCAACCATG MHC-β GAGGAAGAGTGAGCGGCG GCCGCAGTAGGTTCTTCCTGT MHC-IIa TACAACCTCAAAGAGCGTTATGCA AAGGGTTGACGGTGACACAGA MHC-IIx ACAGATCGGGAGAACCAGTCTATT CGTTTCGTGTTCACAGTCTTCC MHC-IIb GAGATCGATGATCTCGCTAGTAACAT AGGGTGCGGCACATCTTC Fndc5 TCCTCTTCATGTGGGCAGGT GGGCTCGTTGTCCTTGATGATA Lrg CTTGAGGACAGACATAGAGGAGCA GTCTTGAGATCCTGGAGGCTTC IL6 AGTTGCCTTCTTGGGACTGA TCCACGATTTCCCAGAGAAC Acta CGACGGGCAGGTCATCA ACCGATAAAGGAAGGCTGGAA Casq2 ACATCAAAGACCCACCCTACGT CGATGTGGATCCCATTCAAGT MEF2c GAGCGTGCTGTGCGACTGT CGTGCGGCTCGTTGTACTC MyoD CCTTTGAGACAGCAGACGACTTC CGTGCTCCTCCGGTTTCA SERCA2 GGTCCTGGCAGATGACAACTTC CTGTCACCAGATTGACCCAGAG Tnnc GCAGGAGATGATTGACGAAGTAGA CGAACCATCATGACAAGAAACTCA Tnnt2 AGATGCTGAAGAAGGTCCAGTAGAG CACCAAGTTGGGCATGAAGA Inflammation TNFα AGCCCCCAGTCTGTATCCTT CTCCCTTTGCAGAACTCAGG ILβ TGGCAACTGTTCCTGAACTCAA AGCAGCCCTTCATCTTTTGG INFγ TCAAGTGGCATAGATGTGGAAGAA TGGCTCTGCAGGATTTTCATG inos GAGGCCCAGGAGGAGAGAGATCCG TCCATGCAGACAACCTTGGTGTTG IL2p4 CCAGAGACATGGAGTCATAG AGATGTGAGTGGCTCAGA GT Mgl ATGATGTCTGCCAGAGAACC ATCACAGATTTCAGCAACCTTA Mgl2 CAGAACTTGGAGCGGGAAGAG TTCTTGTCACCATTTCTCATCTCCT Arg ACACGGCAGTGGCTTTAACC TGGCGCATTCACAGTCACTT IL GCCAAGCCTTATCGGAAATG CACCCAGGGAATTCAAATGC CCL5 TGCCCACGTCAAGGAGTATTT TTCTCTGGGTTGGCACACACT CCL2 AGGTCCCTGTCATGCTTCTG TCTGGACCCATTCCTTCTTG Others Deptor GACGGCGATAAAACTCATGCA CCTTGTGCTCATCACACACGT Ddit4l GGCTATCACCCTGGGAGTCTG CCTCGTTGAGGTTGGGCTC Ddit4 GGACAGCAGCAACAGTGGC CTCAAAGTCGGGCAGGGA UCP TGCCCACGTCAAGGAGTATTT TTCTCTGGGTTGGCACACACT Prdm6 CGGAAGAGCGTGAGTACAAATG TCCGTGAACACCTTGACACAGT CideA GCAGCCTGCAGGAACTTATCAGC GATCATGAAATGCGTGTTGTCC Cebpα AGACATCAGCGCCTACATCGAC GGGTAGTCAAAGTCACCGCCGC Adiponectin GCCCAGTCATGCCGAAGATGAC AGTGCCATCTCTGCCATCACGG Fbxo32 CTGTGCTGGTGGGCAACAT CGGTGATCGTGAGGCCTTT Hspaa GCGACGCCAAGATGGACAAG ATCAGGATGGCCGCCTGCAC Human gene primers SMARCD3 ACTACCAGCCTCCCCAGTTCAA TTGGTCTTCACATACTGCCACAG DEPTOR TCATCCAGCATGTGTCCAACA CGCCGGAAGTTCATTCTGAA IL6 GTACATCCTCGACGGCATCTC TGCTGCTTTCACACATGTTACTCTT