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Nitric Oxide-Dependent Regulation of AMP-Activated Protein Kinase and Peroxisome-Proliferator-Activated Receptor-Gamma C...

Permanent Link: http://ufdc.ufl.edu/UFE0022597/00001

Material Information

Title: Nitric Oxide-Dependent Regulation of AMP-Activated Protein Kinase and Peroxisome-Proliferator-Activated Receptor-Gamma Co-Activator 1Alpha in Skeletal Muscle
Physical Description: 1 online resource (102 p.)
Language: english
Creator: Lira, Vitor
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: 1alpha, alphaampk, ampk, c2c12, citrate, cyclicgmp, daf, enos, f1atpsynthase, fm, glut4, knockout, l6, mice, muscle, nitric, nnos, oxide, pgc, sirna, skeletal, synthase
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Over nutrition and the associated mitochondrial dysfunction in skeletal muscle are central players in the development of insulin resistance. PGC-1alpha, AMPK and NO are involved in mitochondrial biogenesis, and tend to be downregulated in insulin resistant states. Our lab has recently provided evidence for a positive feedback loop between NO and AMPK that upregulates GLUT4 expression in skeletal muscle. Our central hypothesis was that NO and AMPK act synergistically to upregulate PGC-1alpha mRNA expression, GLUT4 expression and stimulate mitochondrial biogenesis. We demonstrated for the first time that NO regulates PGC-1alpha transcriptionally and that normal alphaAMPK function is required for the NO-dependent increase in mitochondrial respiration in skeletal muscle cells. More specifically, we provide evidence for an important role of alpha1AMPK expression in the NO-mediated regulation of PGC-1alpha in L6 myotubes. Further, our observations suggest that NO does not require intact CRE and MEF2 sites at the PGC-1alpha promoter to induce its activity. In mouse skeletal muscle, ablation of the nNOS gene results in lower expression of GLUT4 in the glycolytic EDL only, paralleled by a trend towards reduced markers of AMPK-dependent signaling. Finally, we demonstrated that endogenous NO production is required for AMPK-dependent upregulation of PGC-1alpha, F_1ATP Synthase and Citrate Synthase in L6 myotubes. Altogether these results corroborate our previously proposed model that NO and AMPK interact through a positive feedback loop that impacts metabolic adaptations in skeletal muscle.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Vitor Lira.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Criswell, David S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022597:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022597/00001

Material Information

Title: Nitric Oxide-Dependent Regulation of AMP-Activated Protein Kinase and Peroxisome-Proliferator-Activated Receptor-Gamma Co-Activator 1Alpha in Skeletal Muscle
Physical Description: 1 online resource (102 p.)
Language: english
Creator: Lira, Vitor
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: 1alpha, alphaampk, ampk, c2c12, citrate, cyclicgmp, daf, enos, f1atpsynthase, fm, glut4, knockout, l6, mice, muscle, nitric, nnos, oxide, pgc, sirna, skeletal, synthase
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Health and Human Performance thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Over nutrition and the associated mitochondrial dysfunction in skeletal muscle are central players in the development of insulin resistance. PGC-1alpha, AMPK and NO are involved in mitochondrial biogenesis, and tend to be downregulated in insulin resistant states. Our lab has recently provided evidence for a positive feedback loop between NO and AMPK that upregulates GLUT4 expression in skeletal muscle. Our central hypothesis was that NO and AMPK act synergistically to upregulate PGC-1alpha mRNA expression, GLUT4 expression and stimulate mitochondrial biogenesis. We demonstrated for the first time that NO regulates PGC-1alpha transcriptionally and that normal alphaAMPK function is required for the NO-dependent increase in mitochondrial respiration in skeletal muscle cells. More specifically, we provide evidence for an important role of alpha1AMPK expression in the NO-mediated regulation of PGC-1alpha in L6 myotubes. Further, our observations suggest that NO does not require intact CRE and MEF2 sites at the PGC-1alpha promoter to induce its activity. In mouse skeletal muscle, ablation of the nNOS gene results in lower expression of GLUT4 in the glycolytic EDL only, paralleled by a trend towards reduced markers of AMPK-dependent signaling. Finally, we demonstrated that endogenous NO production is required for AMPK-dependent upregulation of PGC-1alpha, F_1ATP Synthase and Citrate Synthase in L6 myotubes. Altogether these results corroborate our previously proposed model that NO and AMPK interact through a positive feedback loop that impacts metabolic adaptations in skeletal muscle.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Vitor Lira.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Criswell, David S.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022597:00001


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1 NITRIC OXIDE-DEPENDENT REGULATION OF AMP-ACTIVATED PROTEIN KINASE AND PEROXISOME-PROLIFERATOR-ACTIVATED RECEPTORCO-ACTIVATOR 1 IN SKELETAL MUSCLE By VITOR AGNEW LIRA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Vitor Agnew Lira

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3 To my Parents Lucia and Edson, my Sister Cinthia, my Wife Ca rola, my Daughter Manuella, and my Friends who always supported me at both sides of the Equator.

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4 ACKNOWLEDGMENTS First and forem ost, I thank my mentor Dr David Criswell for the friendship and the unconditional support throughout my entire doctora l studies. I also would like to thank past members of the Criswells lab. I am also grateful for the fr iendship and support from Tammy, Jonathan and Joel Criswell. I thank Dr. Scott Powers and Lou Powers fo r the friendship and s upport, which started even before I came to the University of Florida. I would like to name some very important friends that I came to know here in Gainesville and that were very important for me during th e last few years. Bruno Maciel, McNair Bolstick, Carmen Valero Aracama and Luca Bolstick-Valer o, Gisele, Jens and Gabriel Schoene, Marcio, Belinda, Andr and Rebeca Pereira, Tnia Broi sler, Jos Francisco, Tony and Joo Pedro Figueiredo, Lucio, Valria and Jlia Gordan, Abrao, Leandra, Luca and Enzo Dopazo, Flavio, Catarina and Victoria Soares, Cludio, Beatriz and Joo Pedro Varella, Guilhermo and Karina Matias, Keith, Vera and Monique Blanchard, Thiago Resende, Luiz Augusto (Guto) and Camila de Paula, Tristan Hromnick, Sikitti Punak, Marcos Ivanowski, Ryan Caserta, Alvaro Gurovich, Carolina and Benjamin Valencia, Quinlyn Solt ow, Zsolt Murlasits, A ndreas Kavazis thank you so much for the friendship and support. I am also very thankful for the friendship and support from old time friends Fernando and Sirlaine Filgueiras, Geraldo and Michele Maranho Neto, Jos Ri cardo Vianna, Walace Monteiro, Sydnei Silva and famil y, Andr Siqueira Rodrigues and family, Jos Antonio Duarte and family, Mario Pitaluga and famil y, Flvio Ferreira Pinto and family. Finally, I am thankful for the experiences shared with my wife, daughter and family throughout my life and career.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................7LIST OF FIGURES .........................................................................................................................8ABSTRACT ...................................................................................................................... .............10 CHAP TER 1 INTRODUCTION .................................................................................................................. 122 LITERATURE REVIEW .......................................................................................................15Skeletal Muscle Plasticity and Metabolic Flexibility .............................................................16Regulation of GLUT4 Expression in Skeletal Muscle .................................................... 17Mitochondrial Biogenesis in Skeletal Muscle .................................................................18PGC-1 -dependent Regulation of GLUT 4 Expression and Mitochondrial Biogenesis .................................................................................................................... 20AMPK-dependent Regulation of GLUT4 Expression and Mitochondrial Function ....... 22Nitric Oxide Production and Impact on Skeletal Muscle Phenotype .............................. 23Summary ....................................................................................................................... ...253 MATERIALS AND METHODS ...........................................................................................27Experimental Designs .......................................................................................................... ...27Experiments 1 and 2 (Aim 1) Hypothesis 1a and 1b .......................................................27Experiments 3, 4, 5, 6 and 7 (Aim 2) Hypotheses 2a, 2b, 2c .......................................... 27Experiment 8 (Aim 3) Hypothesis 3 ................................................................................ 28Experiments 9 and 10 (Aim 4) Hypothesis 4 .................................................................. 29Experiments 11, 12 and 13 (Aim 5) Hypotheses 5a, 5b .................................................. 29Statistical Analysis .......................................................................................................... 30Protocols ..................................................................................................................... ............30Experiments 1, 3, 4, 5 and 13 .......................................................................................... 30Experiments 2 and 8 ........................................................................................................ 31Experiment 6 .................................................................................................................. .32Experiment 7 .................................................................................................................. .32Experiments 9 and 10 ...................................................................................................... 33Experiments 11 and 12 .................................................................................................... 34General Methods .....................................................................................................................34Animal Measurements ..................................................................................................... 34Body weight and Tissue weight. .............................................................................. 34Western Blot .............................................................................................................34Nuclear and Cytosolic Fractionation ........................................................................ 35

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6 Cell Culture Measurements ............................................................................................. 36Western Blot .............................................................................................................36Quantitative Real-time PCR ..................................................................................... 37Dual Luciferase Assay .............................................................................................38Fluorescence-Based Real-Time Measurement of NO Production ........................... 38Citrate Synthase Assay .............................................................................................39Cell Respiration ........................................................................................................394 RESULTS ....................................................................................................................... ........49Nitric Oxide, AMPK, PGC-1 and GLUT4 Expression in Skeletal Muscle ......................... 49Nitric Oxide Increases AMPK Phosphorylation and Upregulates PGC-1 and GLUT4 Gene Expression in Myotubes........................................................................ 49Both Nitric Oxide and cGMP Upregulate PGC-1 mRNA and Mitochondrial Enzyme Activity in Myotubes. .................................................................................... 50Nitric Oxide-Dependent Upregulation of PGC-1 Gene Expression and Mitochondrial Function Requires AMPK Activation. ................................................. 50Pharmacological Evidence ....................................................................................... 50Genetic Evidence ......................................................................................................51Presence of Either the CRE or the MEF2 site is Sufficient for the Nitric OxideDependent Upregulation of PGC-1 Promoter Activity ............................................. 53Observations in eNOS(-/-) and nNOS(-/-) Mice .................................................................. 53AMPK Activation Increases NO Production in Both L6 and C2C12 Myotubes ............ 55AMPK-Dependent Induction of PGC-1 and Mitochondrial Genes Requires Endogenous NO Production in L6 Myotubes. .............................................................555 DISCUSSION .................................................................................................................... .....76LIST OF REFERENCES ...............................................................................................................85BIOGRAPHICAL SKETCH .......................................................................................................100

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7 LIST OF TABLES Table page 4-1 Body weight and anatomical characteristics of eNOS(-/-) and nNOS(-/-) mice, as well as of their respective wildtypes (eWT and nWT)... ........................................................... 57

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8 LIST OF FIGURES Figure page 3-1 Experimental designs to address whe ther nitric oxide (NO) transcriptionally regulates PGC-1 expression.. ..........................................................................................41 3-2 Experimental designs to address whether nitric oxide (NO) and cGMP upregulate PGC-1 mRNA through AMPK activation.. ..................................................................... 42 3-3 Experimental designs to address whe ther nitric oxide (NO) upregulates mitochondrial function through AMPK activation.. .......................................................... 43 3-4 Experimental design to address which isoform of the catalytic subunit of AMPK is required for the NO-induced regulation of PGC-1 m RNA, ACC phosphorylation and HDAC5 phosphorylation.. .......................................................................................... 44 3-5 Experimental design to test whether both MEF2and CRE-binding sites in the PGC1 prom oter are required for the NO-induced upregulation of PGC-1 .. .........................45 3-6 Experimental designs to te st whether basal levels of ac tivation of AMPK, as well as phosphorylation, expression and localization of some of its downstream targets are altered in muscles from eNOS and nNOS knockout mice in comparison to their respective wildtypes. ......................................................................................................... .46 3-7 Experimental designs to test whethe r AMPK activation causes an increase in endogenous NO production.. ............................................................................................. 47 3-8 Experimental design to test whethe r endogenous NO production is required for AMPK-dependent upregulation of PGC-1 mRNA and the m itochondrial genes F1ATP Synthase and Citrate Synthase.. .............................................................................48 4-1 AMPK phosphorylation in response to NO tr eatm ents of 1 hour in L6 and C2C12 myotubes.. .................................................................................................................... ......58 4-2 PGC-1 mRNA expression in L6 m yotubes treated with NO donors for 3 hours, 8.5 hours and 16 hours.. .......................................................................................................... .59 4-3 PGC-1 mRNA expression in C2C12 m yotubes treat ed with NO donors for 3 hours, 8.5 hours and 16 hours.. ..........................................................................................59 4-4 GLUT4 mRNA expression in C2C12 myotubes treated with NO donors for 3 hours, 8.5 hours and 16 hours.. ..........................................................................................60 4-5 NO induces PGC-1 promoter activity in C2C12 myotubes. ...........................................60 4-6 PGC-1 mRNA expression in L6 m yotubes treated for 3 hours.. ..................................... 61

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9 4-7 Chronic NO and cGMP treatments increase maxim al Citrate Synthase activity in L6 myotubes.. .................................................................................................................... ......61 4-8 AMPK inhibition prevents chronic NO-de pendent upregulation of basal and m aximal mitochondrial respiration in L6 myotubes.. ....................................................................... 62 4-9 Effect of 1 AMPK siRNA and 2AMPK siRNA on AMPK and PGC-1 gene expression.. .................................................................................................................. ......63 4-10 Knockdown of 1AMPK prevents NO-depende nt increase in PGC-1 mRNA. ............. 65 4-11 NO induction of PGC-1 prom oter activity does not re quire intact CRE and MEF2 sites in C2C12 myotubes.. ................................................................................................. 66 4-12 eNOS and nNOS expression in EDL and Soleus muscles from eNOS(-/-) and nNOS(/-) mice and their respec tive wildtypes.. .............................................................................67 4-13 Representative blots of pr oteins assessed in EDL and So leus muscles of eNOS (-/-) and nNOS(-/-) mice and their respec tive wildtypes.. ...........................................................68 4-14 Protein expression of prot eins related to AMPK signali ng in the EDL m uscle from eNOS(-/-) and nNOS(-/-) mice and their resp ective wildtypes. ............................................. 69 4-15 Protein expression of protei ns related to AMPK signaling in the Soleus muscle f rom eNOS(-/-) and nNOS(-/-) mice and their respective wildtypes.. ............................................ 70 4-16 Representative blots of pr oteins assessed in nuclear a nd cy tosolic fractions of the Plantaris muscle of eNOS(-/-) and nNOS(-/-) mice and their resp ective wildtypes.. ............ 71 4-17 Protein expression of prot eins related to AMPK signali ng in nuclear and cytosolic fractions of the Plantaris muscle from eNOS(-/-) and nNOS(-/-) mice and their respective wildtypes.. .........................................................................................................72 4-18 AMPK activation stimulates NO production in L6 m yotubes.. ......................................... 73 4-19 AMPK activation stimulates NO production in C2C12 myotubes.. .................................. 74 4-20 NOS inhibition blunts AMPK-dep endent upregulation of PGC-1 F1ATP Synthase and Citrate Synthase mR NAs in L6 myotubes.. ................................................................ 75

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10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy NITRIC OXIDE-DEPENDENT REGULATION OF AMP-ACTIVATED PROTEIN KINASE AND PEROXISOME-PROLIFERATOR-ACTIVATED RECEPTORCO-ACTIVATOR 1 IN SKELETAL MUSCLE By Vitor Agnew Lira December 2008 Chair: David S. Criswell Major: Health and Human Performance Over nutrition and the associated mitochondrial dysfunction in skeletal muscle are central players in the development of insulin resistance. PGC-1 AMPK and NO are involved in mitochondrial biogenesis, and tend to be downregul ated in insulin resistant states. Our lab has recently provided evidence for a positive feedback loop between NO and AMPK that upregulates GLUT4 expression in skeletal muscle. Our cen tral hypothesis was that NO and AMPK act synergistically to upregulate PGC-1 mRNA expression, GLUT4 expression and stimulate mitochondrial biogenesis. We demonstrated for the first time that NO regulates PGC-1 transcriptionally and that normal AMPK function is required for the NO-dependent increase in mitochondrial respiration in skelet al muscle cells. More specifica lly, we provide evidence for an important role of 1AMPK expression in the NO-mediated regulation of PGC-1 in L6 myotubes. Further, our observations suggest that NO does not require intact CRE and MEF2 sites at the PGC-1 promoter to induce its activity. In mouse skeletal muscle, ablation of the nNOS gene results in lower expression of GL UT4 in the glycolytic EDL only, paralleled by a trend towards reduced markers of AMPK-depende nt signaling. Finally, we demonstrated that endogenous NO production is required for AM PK-dependent upre gulation of PGC-1 F1ATP

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11 Synthase and Citrate Synthase in L6 myotube s. Altogether these results corroborate our previously proposed model that NO and AMPK in teract through a positiv e feedback loop that impacts metabolic adaptations in skeletal muscle.

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12 CHAPTER 1 INTRODUCTION A striking w orldwide increase in the number of people with the metabolic syndrome, also known as insulin resistance syndrome, has ta ken place due to over nutrition and physical inactivity. This condition is associated with mitochondrial dysfunction an d dysregulation of the metabolic gene transcriptional co-factor, peroxisome-proliferator-activated receptorcoactivator 1 (PGC-1 ) in skeletal muscle (81, 99, 116, 131). Nitric Oxide has emerged as a critical regulator for mitochondrial biogenesis in different tissues. Further, endothelial Nitric Oxide Synt hase (eNOS) and the neuronal isoform of NOS (nNOS) are both reduced in muscle of rodent s and humans presenting peripheral insulin resistance (13, 128, 149, 165). AMP-activated protein kinase (AMP K) also induces mitochondrial biogenesis in skeletal muscle, and its activity seems to be reduced in obesity and diabetes (11, 136, 139). Although the AM PK-dependent regulation of PGC-1 the glucose transporter GLUT4 and mitochondrial genes is well accepted and remains a major focus of research, the mechanisms by which NO regulates m itochondrial biogenesis remain to be defined. It is currently accepted that the NO-dependent signal involves increased production of cyclicGMP (cGMP) and upregulation of th e transcription cofactor PGC-1 However, the mechanism by which NO affects PGC-1 expression is still elusive. Recently we have shown that both NO and cy clic GMP (cGMP), a downstream effector of the NO-dependent signal, upregulate GLUT4 expre ssion in skeletal muscle cells through AMPK activation. In addition, we also observed that endogenous NO is required for the upregulation of AMPK activity by the AMP mimetic, AICAR (89) These findings suggest that NO-induced mitochondrial biogenesis and PGC-1 regulation could be AMPK-dependent as well. Our central hypothesis is that NO modulates AMPK activity, upregulating PGC-1 activity and

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13 expression. Such signaling positively impacts GLUT4 expression and mitochondrial function. Our experiments were designed to ad dress the following specific aims. Specific Aim 1: To determine whether NO transc riptionally regulates PGC-1 Hypothesis 1a: NO will upregulate PGC-1 mRNA in both L6 and C2C12 myotubes. Hypothesis 1b: NO treatments will increase PGC-1 -luciferase promoter activity in C2C12. Specific Aim 2: To test whether NO upregulates PGC-1 mRNA, and mitochondrial function through AMPK activation. Rationale: We have shown that pharmacological inhibition of AMPK prevents NO-induced upregulation of GLUT4. Muscle Enha ncer Factor 2 (MEF2) serves as a transcription factor for both GLUT4 and PGC-1 Therefore, AMPK-mediated phophorylation of Histone Deacetylase 5 (HDAC5), causing its dissociati on from MEF2 and exclusion from the nucleus, could be essential for NO-induced upregulation of PGC-1 and mitochondrial genes. In addition, it has been shown that 2 is the most abundant cata lytic isoform of AMPK in adult skeletal muscle (2, 31, 78, 114), but some of the AMPK-dependent adaptations have been attributed to 1AMPK as well (114). We will address these issues with both pharmacological inhibition and genetic knockdown of the catalytic subunits of AMPK in L6 myotubes. Hypothesis 2a: Specific pharmacological inhibition of AMPK will prevent the NOdependent increase in PGC-1 mRNA. Hypothesis 2b: Chronic NO treatment will increase Citrate Synthase Activity and improve mitochondrial function. Phar macological inhibition of AMPK will prevent this adaptation. Hypothesis 2c: Genetic knockdown of the 2 subunit of AMPK will be sufficient to blunt the effect of NO on AMPK activity, assessed by means of Acetyl CoA Carboxylase (ACC) phosphorylation, and will prevent the NOmediated increase in HDAC5 phosphorylation and PGC-1 mRNA. Specific Aim 3: To ascertain whether NO-induc ed upregulation of PGC-1 promoter activity requires the MEF2-binding site and/or the CRE-binding site in the promoter.

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14 Rationale: Both the MEF2and the cAMP response element (CRE)-consensus sequences play a fundamental role in the activation of the PGC-1 promoter in response to exercise and other stimuli (4, 50, 51). We will use plasmids containing the mouse PGC-1 promoter with sitedirected mutagenesis of these sequences (i.e. MEF2 and CRE) to test their requirement for the NO-induced increase in PGC-1 mRNA in C2C12. Hypothesis 3: Only the MEF2-PGC-1 promoter will be unresponsive to NO and AICAR treatments. This will provide genetic evidence for the MEF2 role in the NO/AMPK regulation of PGC-1 and consequent mitochondrial biogenesis. Specific Aim 4: To test whether genetic ablation of either the nNOS, or the eNOS gene will affect GLUT4 and PGC-1 expression as well as basal levels of AMPK activation in skeletal muscle in vivo. Hypothesis 4: Basal levels of AMPK activatio n, phosphorylation of some of its downstream targets (e.g. ACC and HDAC5), as well as PGC-1 nuclear localization and GLUT4 expression will be reduced in muscles of both eNOS and nNOS knockout animals in comparison to their respective wildtypes. Specific Aim 5: To determine whether endogenous NO production is required for the AMP analog 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR)dependent upregulation of PGC-1 and mitochondrial genes. Rationale: AICAR, which serves as an AMP analog, has been the drug of choice to activate AMPK in adult skeletal muscle and in muscle cell lines due to its hi gh specificity (75, 78, 94, 132). We will test whether NOS inhibition affect s AMPK-dependent signaling in L6 myotubes. Hypothesis 5a: AMPK activation will induce increas ed NO production in both L6 and C2C12. Hypothesis 5b: NOS inhibition will blunt AICAR-induced up regulation of PGC-1 F1ATP Synthase, and Citrate Syntha se mRNAs in L6 myotubes.

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15 CHAPTER 2 LITERATURE REVIEW The num ber of people with the metabolic s yndrome, also known as insulin resistance syndrome, has been increasing dramatically over the last two decades (33, 116, 166, 169). Approximately 85% of people diagnosed with t ype 2 diabetes in the United States are overweight, and 55% are obese (97). In fact, type 2 diabetes and obe sity are just two examples of diseases comprising the metabolic syndrome, and therefore associ ated with insulin resistance (116). The economic burden of these metabolic diso rders is enormous. For example, the current estimate of 150 million people with diabetes is set to rise to 220 million by 2010 (166), and the direct medical costs in the United States alone are expected to increase from $92 billion in 2002 to $138 billion by 2020 (62). Skeletal muscle is extremel y plastic and accounts for 70-90% of glucose disposal under insulin-stimulated conditions (27, 28, 80). Insulin resistant states in humans are generally not associated with reductions in GLUT4 expressi on, which is the main glucose transporter in skeletal muscle (5, 39, 111). Instead, the compromised capacity to transp ort glucose seems to derive from a loss of sensitivity of the receptor and the associated downstream kinases to insulin (93, 102, 131). However, it has been shown that sk eletal muscle-specific overexpression of GLUT4 improves glucose disposal in animal models of insulin resistance (86, 119, 148). Therefore, the study of the mechanisms regulating GLUT4 expression could provide useful insights into therapeutic targets for the treatment of insulin resistant states. Over nutrition and the associated mitochondrial dysfunction in skeletal muscle are central players in the development of insulin resi stance (102, 109, 112, 116). M itochondrial dysfunction is also a common feature of both type 2 diab etes and obesity. Although it is still debatable whether mitochondrial dysfunction is the main cause for the development of insulin resistance,

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16 recent studies suggest that improving mitochondr ial function has a direct and positive impact on insulin sensitivity in peripheral tissues, including skeletal muscle (84, 102). Consequently, it is critical to understand the ce llular mechanisms involved in the regulation of mitochondrial volume and function. The focus of this disserta tion is to investigate a signaling pathway potentially linked to the regulati on of both GLUT4 and mitochondrial genes in skeletal muscle. In this chapter we will review skeletal musc le metabolic plasticity and how it affects the whole body metabolic status under insulin stimulated conditions. In this context we will discuss mechanisms involved in mitochondrial biogenesis and GLUT4 regulation, with special emphasis to the roles of peroxisome pr oliferator-activated receptor coactivator 1 (PGC-1 ), AMPactivated protein kinase (AMP K), and nitric oxide (NO). Skeletal Muscle Plasticity and Metabolic Flexibility Skeletal m uscle accounts for approximately 45% of body mass, and is the main site for glucose disposal under insulin s timulated conditions (28, 32). Duri ng exercise glucose transport into muscle occurs through mech anisms that are independent of the action of insulin (154, 164). However, one bout of exercise is sufficient to tr ansiently improve insulin sensitivity in muscle (16, 120), which seems to persist for as long as glycogen levels remain low (16). Exercise training on the other hand induces more pronounced phenotypic changes, which are mostly due to upregulation of several protei ns involved in both glycolytic and oxidative metabolisms. Such improved phenotype involves, but is not exclusive to, a shift in expression from fast-to-slow twitch-specific proteins (e.g. increased levels of type 1 myosin heavy chain and myoglobin), increased GLUT4 expression, and improved mito chondrial function and fuel oxidation capacity (45, 63-65, 84, 125, 144). Interestingl y, physical inactivity causes a fast reversal of these adaptations, thereby reducing insulin sensitivity and limiting metabolic flexibility in the tissue

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17 (108, 117). Therefore, regular phys ical activity has been used not only in the prevention of metabolic disorders, such as obesity and type 2 diabetes, but also as an adjunct therapy for insulin resistant states in general (45, 51, 56, 57, 59, 129, 145). Taken together these evidences represent an important motiva tion for scientists to understa nd the mechanisms involved in skeletal muscle adaptation to metabolic challe nging events, such as caloric restriction and exercise or increased contractile activity. Regulation of GLUT4 Expre ssion in Skeleta l Muscle Glucose transport in eukaryotic cells is mediated by a family of transporters (GLUT), and 13 isoforms have been identified and clone d (77, 79, 92). GLUT4 is the major glucose transporter in skeletal muscle, but is also expr essed in other insulin-responsive tissues, such as white and brown adipose, and heart (15, 79, 143, 150, 155, 172). The translocation hypothesis suggests that intracellular vesicles containing GLUT4, in response to insulin and other stimuli translocate and fuse with the plasma membrane, thereby allowing the contact of the transporter with the glucose molecule in the interstitial fluid (14, 24). Glucose, and other 6-carbon carbohydrates, is transported in an ATP-inde pendent manner, via a facilitative diffusion mechanism (71). The expression of GLUT4 is highly regulated in muscle, being stimulated during muscle regeneration and in response to increased cont ractile activity (101, 103, 171). Exercise induces a very rapid upregulation of GLUT4 expression in both rodent and hum an muscle. In rats most of the adaptive response occurs within 18h after a singl e bout of exercise, with reports showing that the entire response to chronic ex ercise (~twofold increase) can be achieved with two days of exercise (66-68, 121). In humans GLUT4 expr ession may increase by ~100% with exercise regimens ranging from 7 days to 14 weeks (49, 69, 70).

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18 Various transcription factors and cofactors are involved in GLUT4 transcription regulation, including myocyte enhancer factor 2 (MEF2) (90, 94, 100), GLUT4 enhancer factor (GEF) (107), PGC1(75, 95), MyoD (123), thyroid hormone receptor (TRalpha1) (123) and Kruppellike factor 15 (47). However, MEF2 seems to be the most important factor in GLUT4 regulation since it physically intera cts with many of the other factors at the GLUT4 promoter (94, 95, 123). Several lines of evidence suppor t the notion that MEF2 is involved in th e exercise/activitydependent regulation of GLUT4. Fi rst, MEF2 is regulated by calcium-related pathways and is involved in fiber-type determina tion in skeletal muscle (160). Second, MEF2 activity is higher in the oxidative soleus in comparison to the glycolytic EDL muscle, which correlates with the pattern of contractile activity of these muscles and the level of GLUT4 expression across fiber types (101, 171). Third, mutation of the MEF2 bindi ng site at the GLUT4 promoter drastically reduces the activity of the promoter in skeletal muscle (101). Fourth, ME F2 activity is repressed by its physical association with class 2 histone deacetylases (HDAC4-9). Recently HDAC5 has been identified as a critical re gulator of GLUT4 in response to AMPK activation (94). AMPK is activated in response to energy depriving events, such as incr eased contractil e activity. We hypothesize that MEF2 is involved in the NO-dependent regulation of GLUT4, and that NO regulates MEF2 binding ac tivity by activating AMPK a nd inducing the associated phosphorylation of HDAC5. Mitochondrial Biogenesis in Skeletal Muscle Mitochondria are unique organelles in the sense that they poss ess part of their own genetic m aterial in a circular dou ble-stranded DNA molecule (m tDNA) (124). Mitochondrial DNA encodes 13 protein subunits of th e respiratory chain complexes invol ved in the electron transport system. In addition, mtDNA encodes 22 tRNAs a nd 2 ribosomal RNAs. However, the vast

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19 majority of proteins involved in oxidative metabolism located in mitochondria are nuclear encoded and therefore synthesized outside of the organelle and subsequently transported to the mitochondria (21, 22, 153). As pointed out by Sc arpulla in a recent re view, mitochondria are genetically semiautonomous (124), and mitoc hondrial nuclear dependence can be further supported by the fact that mitochondrial transcri ption, translation and DNA replication are all controlled by factors encoded in the nuclear geno me and regulated at that level (9, 21, 22, 124). Mitochondrial dysfunction in muscle has been related to neurodegenerative disease (126), diabetes (91, 127), obesity (1) and aging (30). Conversely, the upregul ation of mitochondrial mass and function, also referred as mitochondrial biogenesis, is induced by a series of environmental conditions involving metabolic cha llenges, of which exercise, cold exposure and caloric restriction are the most used as research models (3, 4, 104, 106, 159). Mitochondrial biogenesis is regu lated by several tran scription factors a nd cofactors, such as mitochondrial transcription factor A (Tfam) (37, 38), mitochondrial transcription factor B (TFB1M and TFB2M) (35, 44), nuclear respirat ory factors (NRF-1 and NRF-2) (34, 124, 151), specificity protein 1 (Sp1) (23), early growth response gene-1 (Egr-1) (74, 96), estrogen-related receptor (ERR ) (72,73), peroxisome proliferator activated receptors (PPAR / ) (8, 43, 46, 73), PGC-1 (161) and others (124). Mitochondrial function is greatly influenced by muscle contractile activity. First, at an acute stage contraction induces a drama tic increase in substrate flux through -oxidation and Tricarboxylic Cycle (TCA) (84, 144). This increase in substrate flux is most likely the result of an initial change in intracellular AMP-to-ATP ratio leading to AMPK activation. AMPK phosphorylates and inhibits Acet yl Coa Carboxylase (ACC) causing the levels of its product Malonyl CoA to drop sharply. As a result Carnitin e Palmitoyl Transferase (CPT1) is released

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20 from the allosteric inhibition mediated by Malony l CoA, and fatty-acid transport to mitochondria is stimulated (55, 144). Secondly, chronic contrac tion, or exercise traini ng, causes increases in mitochondrial volume and improved oxidation capacity (63). More importantly, improvements in mitochondrial number and function in skeletal mu scle display a very strong correlation with improvements in whole body insulin sensitivity in type 2 diabetics ( 146) and obese patients (147). Mitochondrial bioegenesis in muscle can be induced by calcium-dependent pathways, AMPK activation, and NO. Although the extent of participation of each of these upstream players in the adaptive events related to exercise, caloric rest riction and cold exposure requires more investigation, the upregulation of PGC-1 mRNA and protein levels, with subsequent upregulation of other transcription factors su ch as Tfam, NRF-1 and NRF-2, seem to be a common feature. Therefore unders tanding the regulation of PGC-1 by these pathways, as well as determining the degree of redundancy among those pathways may bring about important insights for therapeutic a lternatives to improve mitochondrial function. In the following sections we will examine the roles of PGC-1 AMPK and NO in the regulation of mitochondrial biogenesis and GLUT4 expression in skeletal muscle. PGC-1 -dependent Regulation of GLUT4 Exp ression an d Mitochondrial Biogenesis The transcription coactivator PGC-1 stimulates transcri ption of nuclearand mitochondrial-encoded metabolic genes, as well as mitochondrial DNA replication. Such regulation is due to the ability of PGC-1 to increase the expressi on and activity of Nuclear Respiratory Factors 1 and 2 (NRF-1 and NRF-2, respectively), and Mitochondrial Transcription Factor A (Tfam or mTFA) (160) Ectopic expression of PGC-1 in transgenic mice induces a conversion from the fast-glycolytic type 2b/2x fibe rs to the mitochondria rich type 1/2a fibers

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21 (88). GLUT4 expression in muscle cells is also regulated by PGC-1 which can bind to and coactivate Muscle Enhancer Factor 2 (MEF 2) in the GLUT4 promoter (95). PGC-1 is upregulated by contraction and is involved in most of the metabolic adaptations that occur in muscle thereafter (3, 4, 125, 162). On the other hand, insulin resistance, diabetes and moderateto-severe obesity are related to lower PGC-1 levels in muscle and white adipose tissue (84, 111, 134, 136). In the last 8 to 9 years much attention has been dedicated to the study the PGC-1 promoter and the elements that stim ulate its activity. In terestingly, PGC-1 interacts with MEF2 in its own promoter and stimulates transcrip tion, thereby controlling its own expression (50). However, the PGC-1 promoter also has a cAMP responsive element (CRE), which together with the MEF2 binding-site is responsible for the PGC-1 induction mediated by calcineurin (CaN) and calcium/calmodulin dependent protei n kinase IV (CAMK IV) (50). More recent studies using site-directed mutagenesis in the PGC-1 promoter in exercising mice in vivo have shown that actually both binding sites must be int act for the exercise induction of the gene to take place (3, 4). Although the upregulation of PGC-1 expression seems to be important for the adaptive changes related to mitochondrial biogenesis and GL UT4 to occur, it does not seem to account for the initial upregulation of some mitochondrial protein mRNAs of very short half-life induced by increased contractile activity (159). In fact, recent studies have shown that PGC-1 is also posttranslationally regulated (75, 159). This seems to be the case of sirtuindependent regulation of PGC-1 in response to caloric restriction a nd AMPK-dependent regulation of PGC-1 in response to AICAR treatment (75). These evid ences raise the exciti ng notion that PGC-1 expression accounts for more delaye d responses, as well as long-term maintenance of the level of

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22 expression of mitochondrial proteins and GLUT 4, but that activation of existing PGC-1 activation should also be cons idered in an experimental setting. Since most of PGC-1 is found in the cytosol, but can translocate to the nucle us in response to certa in stimuli (159), nuclear localization can be used as an indirect measure of overall state of activation of the protein. AMPK-dependent Regulation of GLUT4 Expr ession and Mitochondrial Function The 5-AMP-activated protein kinase (AMP K) is also downregul ated in diseases characterized by an unbalanced metabolic state, su ch as diabetes and obesity (11, 136). Due to the adenylate kinase reaction in muscle, and also to its very low basal levels, AMP concentration is much more dramatically affected than that of ADP or ATP in response to increased metabolic rate. Therefore, AMP serves as a metabolic stre ss signal, to which AMPK is very sensitive to (40, 52-54). AMPK is a heterotrimeric enzyme ( 1, 2, 1, 2, 1, 2, 3), which is activated by both direct AMP allosteric regul ation and phosphorylation of its catalytic subunit, the latter being mediated by an upstream AMPK kinase (157 ). The major AMPK kinase in skeletal muscle is LKB-1 (82). AMPK is involved in acute and delayed responses that improve the cell capacity to resynthesize ATP. Acutely, AMPK stimulates glucose transport and fatty acid oxidation in skeletal muscle (6, 132). AMPK is also known to phosphorylate both the endothelial and neuronal isoforms of nitric oxide synthase (eNOS and nNOS, respect ively) (18-20, 26). The associated delayed responses, or adaptations, to AMPK activation include the upregulation of proteins involved in substrate availability (e.g. GLUT4) and oxidati on capacity (e.g. PGC-1 and mitochondrial genes) (158, 167). These chan ges represent importa nt adaptive responses triggered by metabolic challenges su ch as exercise, energy deprivation, and hypoxia (140, 158, 167, 170).

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23 Several studies have shown that the pattern of expression of the catalytic subunits of AMPK (i.e. 1 and 2), as well as basal levels of AMPK activity differ between slow-twitch and fast-twitch predominant muscles (2, 31, 114). Of not e, AMPK expression is also responsive to increased metabolic activity, with both 1 and 2 expression increasing in response to exercise (114). Due to its beneficial effects in muscle phenotype and metabolism, AMPK has been considered as a pharmacological target for interv entions directed to improve insulin sensitivity (41, 55). The AMP analog 5-aminoimidazole-4carboxamide-1-D-ribofuranoside (AICAR) is one of several drugs that can induce phosphoryl ation and activation of AMPK and has been widely used in research models investigating AMPK-dependent si gnaling in differe nt tissues (75, 78, 94,132). Nitric Oxide Production and Impact on Skeletal Muscle Phenotype NO is a reac tive nitrogen molecule that is formed enzymatically by nitric oxide synthases (NOS), via the conversion of L-arginine to L-c itrulline. Skeletal muscle expresses nNOS, eNOS, and the inducible iNOS isoforms (118, 137). In terestingly, eNOS and nNOS expressions are downregulated in insulin resist ant states and iNOS is upre gulated (42, 113, 128, 149, 165). Both eNOS and nNOS synthesize NO at low levels, wh ereas iNOS expression results in much higher NO production (76, 113, 137). Another interesting obser vation is the differential expression of the constitutive NOS isoforms (i.e. eNOS and n NOS) across fiber types. eNOS is preferentially expressed in slow-twitch fibers, whereas nNOS is more abunda nt in fast-twitch fibers (61, 137), suggesting that: a) they may play different roles in NO-mediated signaling, and b) their expression is related to the determination an d/or maintenance of muscle phenotype and the related differences in muscle physiology across fiber types. Our lab has provided some evidence

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24 for a role of NO in fiber type determination. NOS activity is required for functional overloadinduced increase in myosin heavy chain type 1 (MHC 1) in the plantaris muscle (133) and NO donors induce MHC 1 expression and enhance ca lcium-induced nuclear accumulation of the transcription factor Nuclear Factor of Activated T-Cells (NFAT) in skeletal muscle myotubes (29). NO synthesis increases during skeletal muscle contraction (7, 112, 130), and many of its signaling effects are mediated by activation of soluble guanylat e cyclase (sGC), leading to increased production of cGMP (85, 118). However, cGMP levels seem to increase in response to contraction mostly in fast-twitc h fibers (85). Such increase correlates with nNOS expression and is observed in eNOS(-/-), but not in nNOS(-/-) mice (85). Some evidence suggests that NO is involved in some early responses in gene e xpression induced by exercise in humans (137). Altogether these observations suggest that if NO were to play a role in exercise-related adaptations in skeletal muscle and the signals were conserve d across species, nNOS expression should be required. Interestingly, both NO and cG MP are involved in mitochondrial biogenesis in different cell types (104, 105), but mitochondr ial biogenesis is compromised only in eNOS(-/-) mice in response to cold exposure and caloric restriction (104-106). More recently, it was observed that neither nNOS nor eNOS is required for the exercise-induced increase in markers of mitochondrial biogenesis, including PGC-1 expression (152). We have recently shown that NO regulates GLUT4 expression via AMPK activation. Of note is the observation that sp ecific pharmacological activation of AMPK with AICAR, and the subsequent induction of GLUT4, is blunt ed by inhibition of endogenous NO production. Considering these findings together with re ports showing that AM PK phosphorylates and activates NOS (18-20, 26), we proposed the existence of a positive feedback mechanism between

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25 NO production and AMPK activity in skeletal muscle. According to this positive feedback loop, basal levels of NO are required for normal AM PK activation and signal, and once AMPK is activated NO production is incr eased, which increases AMPK activity even further (89). However, several questions remain to be addressed. For example, it is unknown whether this mechanism is present in adult skeletal muscle and if so, whether the involvement of nNOS and eNOS would be related to their level of expression across different fiber types. Skeletal muscle GLUT4 upregulation shares several similarities with mitochondrial biogenesis, such as AMPK participation and transcription stimulat ion by MEF2, and PGC-1 (95, 105, 167, 170). AMPK is known to phosphoryl ate Histone Deacetylase 5 (HDAC5), and thereby mediate its dissociation from MEF2 and nuc lear export. This pathway seems to be very important for the regulation of GLUT4 expression mediated by AMPK (94). Also, it has been shown that the HDAC5/MEF2-pathway is required for the PGC-1 regulation in cardiomyocytes (25). At present, it remains to be tested whether NO-mediated upregulation of PGC-1 GLUT4 and mitochondrial biogenesis are related and whethe r they require AMPK activation. Further, it is still unknown whether NO tran scriptionally regulates PGC-1 expression and if so, which ciselements at the PGC-1 promoter are required for the NOdependent induction of the gene. Finally, it is important to note that in the la st decade a clear progress has been made in understanding the NO-dependent signal in skeletal muscle. However, the role of the different NOS isoforms in muscle metabolism and adaptati on requires further investigation. We attempted to address some of these issues in the e xperiments that integrate this dissertation. Summary Skeletal m uscle plays a crucial role in w hole body glucose homeostasis due to its large mass and rapid response to insulin. For this reason a defect in the insulin signal within skeletal

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26 muscle, as observed in obese individuals, is a ma jor determinant of periph eral insulin resistance and the development of type 2 diabetes. Impr ovement of mitochondrial function and increased expression of the glucose transporter GLUT4 have been shown to improve muscle-dependent glucose uptake in response to insulin. NO has been associated with GLUT4 expression and mitochondrial biogenesis. These events share some common signaling molecules, such as AMPK activation and upregulation of PGC-1 expression and activity. Therefore we hypothesize that NO regulates GLUT4 and PGC-1 expression via activation of AMPK. Here we address this central question with experiments involving several models including culture of muscle cell lines, use of siRNAs to knockdown AMPK expression, analysis of PGC-1 promoter activity, and analysis of the expression of key prot eins related to AMPK activity in adult skeletal muscle of mice carrying null mutations of either nNOS or eNOS genes.

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27 CHAPTER 3 MATERIALS AND METHODS Experimental Designs Experiments 1 and 2 (Aim 1) Hypothesis 1a and 1b L6 and C2C12 m yoblasts were seeded, grown a nd differentiated in 6-well plates and 24well plates, respectively. L6 and C2C12 myotube cultures were randomly assigned to one of the following groups and treated for 3, 8, or 16 hours (Experiment 1) (Figure 3-1): Experiment 1: 1) Control, 2) SNAP (25 M), 3) DETA-NO (50 M). C2C12 were transiently transfected with a 2kb PGC-1 promoter-luciferase plasmid together with pRL-CMV plasmid to control fo r transfection efficiency, and then randomly assigned to one of the following groups and tr eated for 9 hours (C2C12, Experiment 2). Another set of cells was transfected with the control plasmid (pGL3) together with pRL-CMV. These cells served as a control for po ssible non-specific treatment effects on the luciferase gene (Figure 3-1): Experiment 2: 1) Control, 2) DETA-NO (25 M), 3) DETA-NO (50 M). Experiments 3, 4, 5, 6 and 7 (Aim 2) Hypotheses 2a, 2b, 2c L6 were seeded, grown and differentiated in 6 -well plates. Cells were randomly assigned to one of the following groups and treated for either 3h for subsequent mRNA quantification (Experiment 3, Figure 3-2), or for 48h (Experime nt 4, Figure 3-2). Anot her set of cells was treated for 12 hours/day during 5 consecutive days (Experiment 5) (Figure 3-3): Experiment 3: 1) Control, 2) Compound C (40 M), 3) DETA-NO (50 M), 4) 8-Br-cGMP (2mM), 5) Compound C (40 M) + DETA-NO (50 M); Experiment 4: 1) Control, 2) DETA-NO (50 M), 3) ODQ (1 M), 4) ODQ (1 M) + DETA-NO (50 M);

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28 Experiment 5: 1) Control, 2) SNAP (10 M), 3) 8-Br-cGMP (2mM); Experiment 6: 1) Control, 2) DETA-NO (50 M), 3) Compound C (20 M), 4) DETA-NO (50 M) + Compound C (20 M). For the siRNA experiment (Experiment 6), L6 myotubes were grown and differentiated in 6-well plates. After 2 days of differentiation ce lls were transfected wi th siRNAs for 48 hours, and then treated. Preliminary experiments were c onducted to test for tran sfection efficiency, and to optimize the concentration of each siRNA used (i.e. 1AMPK and 2AMPK), as well as to establish the duration of transf ection. Once those optimization e xperiments were concluded the actual experiments started with treatments conducte d for either 1h (protein measurements) or 3h (mRNA measurements) (Figure 3-4): Experiment 7: 1) Control, 2) unrelated siRNA+DETA-NO (50 M), 3) 1 siRNA+ DETANO (50 M), 4) 2 siRNA+ DETA-NO (50 M), 5) 1+ 2 siRNAs + DETA-NO (50 M). Experiment 8 (Aim 3) Hypothesis 3 C2C12 were seeded, grown and diffe rentiated in 24-well plates. Cells were transiently transfected with either an intact 2kb PGC-1 promoter-luciferase plasmid, or with mutated PGC1 promoters (either MEF2-, or CRE-luciferase plasmid) and then assigned to one of the following groups and treated for 9 hours. Another set of cells was transf ected with the control plasmid (pGL3) together with pRL-CMV. Thes e cells served as a control for possible nonspecific treatment effects on the PGC-1 promoter (Figure 3-5): Experiment 8: 1) Control, 2) AICAR (0.65mM), 3) DETA-NO (50 M).

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29 Experiments 9 and 10 (Aim 4) Hypothesis 4 Basal levels of expression a nd phosphorylation status of certain proteins were com pared in Soleus and EDL muscles from knockout animals (either eNOS-/-, or nNOS-/-) and their respective wildtypes (WTe and WTn). In a ddition, nuclear and cytosolic frac tions of the Plantaris muscles from the same animals were isolated and compared to provide some insight on differential localization of certain proteins between w ild-types and knockout animals (Figure 3-6): Experiment 9: 1) WTe, 2) eNOS-/Experiment 10: 1) WTn, 2) nNOS-/-. Experiments 11, 12 and 13 (Aim 5) Hypotheses 5a, 5b L6 and C2C12 were seeded, grown and differen tia ted in 24-well plates. Cells were then assigned to one of the following groups and tr eated for 30 minutes before DAF-FM addition to the media, and then monitored for 50 minutes (ref er to protocol for details) (Experiments 10 and 11, Figure 3-7). Of note, L-NAME and L-NMMA we re added to the wells 30-40 minutes before the addition of AICAR, and subs equent 30 minute-incubation: Experiment 11: 1) Control, 2) AICAR (1mM ), 3) AICAR (3mM), 4) Metformin (2mM) and 5) MAHMA-NO (1 M) [performed with L6]. Experiment12: 1) Control, 2) AICAR (1mM), 3) L-NAME (1mM), 4)L-NMMA (1mM), 5) AICAR+L-NAME, 6) AICAR+L-NMMA [performed with C2C12]. In Experiment 13, L6 were seeded, grown and differentiated in 6-well plates, and randomly assigned to one of the following groups and tr eated for 16h (mRNA measurements, Figure 3-8): Experiment 13: 1) Control, 2) AICAR (1 mM), 3) L-NAME (1mM), AICAR (1mM) +LNAME (1mM).

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30 Statistical Analysis Group sam ple size was determined with power analysis from our preliminary data. Comparisons between groups were performed us ing Student t test and one-way ANOVA when applicable. Fisher LSD test was used post hoc when appropriate. The Levene test for variance homogeneity was performed a priori and in cases where group vari ables presented significantly different variances, data was l og transformed before further analyses were conducted. Group values, however, are reported on units specified in text and figures. Sta tistical significance was established at P<0.05. Protocols Experiments 1, 3, 4, 5 and 13 L6 and C2C12 m yoblasts were seeded in ~4000cells/cm2 in 10% FBS DMEM containing 5mM glucose. Differentiation was induced by switching 80% confluent cells to medium containing 2% HoS. Treatments were performed when myotubes were evident (4-5 days of differentiation in C2C12, and 6-7 days in L6 ). The doses of AICAR, SNAP, DETA-NO, LNAME, L-NMMA, ODQ and Compound C are based upon previous investigations using muscle cells (36, 89, 105, 113). In groups treated with L-NAME, L-NMMA, ODQ, or Compound C, cells were pre-incubated with the drug for 30 min before the final treatment. In Experiments 1, 3, 4 and 13, myotubes were treated in 2% HoS from 3hours to 16hours, washed in ice-cold PBS and harvested in TRizol for RNA isolation. For Experiment 5, L6 cells were grown and di fferentiated as described above. Cells were treated for 48 hours after 6 days of differe ntiation. During treatm ent new medium with treatments was added every 24 hours. Immediat ely after the final 24 hours of treatment, myotubes were washed once in ice-cold PBS containing 1 M Na3VO4, and then harvested in Non-Denaturing Lysis Buffer ( NDLB) containing protease and phosphatase inhibitors as

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31 previously described (89). As described above cells were harvested in NDLB and Citrate Synthase Activity was assessed on the same day (refer to General Methods for details). Experiments 2 and 8 In experim ent 2, C2C12 were grown in 24-we ll plates and differentiated in similar conditions as described above for Experiment s 1, 3 and 12. When myoblasts became ~90-100% confluent they were tran sfected with 0.5g/well of either the 2kb PGC-1 -luciferase reporter plasmid (Addgene, Cambridge, MA; plasmid 8887) ( 50) or the pGL3 Control plasmid (Promega, Madison, WI; cat E1741). In addition, cells were always cotransfected with 0.01g/well of the pRL-CMV (Promega, Madison, WI). Transfections were when C2C12 myoblasts were ~90% confluent. Lipofectamine reagent was used as the lipid-mediated reagent for transfection (1 L/well, Invitrogen, Carlsbad, CA; cat. 11668027), and DNA-Lipofectamine complexes were formed according to the manufactur ers instructions. Before transfection, cells were washed in serum-free Opti-MEM (Invitrogen, Carlsbad, CA; cat. 31985), and then exposed to DNALipofectamine complexes also in serum-free Opti-MEM for 6 hours. During this period, plates were gently swirled every 2 hours. After that, 12%HoS Opti-MEM was added in a volume sufficient to bring the final concen tration of serum in wells to 6%HoS. Cells were then kept in 6%HoS Opti-MEM for 18 hours. At this po int cells were switched to 2%HoS DMEM. Treatments started 48 hours after the beginning of transfection and lasted for 9hours. Cells were treated in DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin. Immediately after treatment, cells were harveste d in Passive Lysis Buffer after being washed once in PBS at room temperature and Dual Luciferase Assay was subsequently performed according to the manufacturers instructi ons (Promega, Madison, WI; cat E1910).

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32 For Experiment 8, C2C12 will be grown, differentiated and trasnfected as described above, with the exception that two extra reporter plasmids with site-directed mutation in the PGC-1 promoter were transfected to independent sets of cells (i.e. MEF2, CRE, Addgene, Cambridge, MA; plasmids 8888 and 8889). Generation of these plasmids, as well as the relevance of these binding-sites in the PGC-1 promoter, has been previously documented both in vitro and in vivo (4, 50). Cells then treated and harves ted as described for Experiment 2. Experiment 6 L6 m yoblasts were seeded in 6-well plat es, grown and differentiated as previously mentioned. Treatments started after 2 days of differentiation and were performed in 2%HoS. Treatments lasted for 12 hours and were perf ormed in 5 consecutive days. Compound C (20 M) was added to cells always 30-40 min before the addition of DETA-NO (50 M). After each treatment media was withdrawn and replaced wi th fresh 2%HoS medium. Cells were harvested 10 hours after the last treatment. At that point cells had been in differentiation medium for 7 days and myotubes were fully confluent. Experiment 7 L6 cells were grown and differentiated as described above. Cells we re transfected with siRNAs for the 1 and 2 subunits of AMPK at 2 days of differentiation (when m yotubes reached 60 to 70% confluence). Transfection was performed exactly as described for Experiments 2 and 7. The siRNA sequences used were: AMPK 1 (GCA UAU GCU GCA GGU AGA UdTdT, nucleotides 738 to 756, acc. no. NM019142), AMPK 2 (CGU CAU UGA UGA UGA GGC UdTdT, nucleotides 865, acc. no. NM023991), and unrelated (control siRNA AUU CUA UCA CUA GCG UGA CUU) All siRNA oligonucleotides were purchased from Dharmacon (Lafayette, CO) (83, 113, 156), and transfection efficiency was tested with

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33 fluorophore-conjugated siRNAs (siGLO Green, cat. D-001630-01-05). Knockdown efficiencies at mRNA and protein levels were tested at 48 hours post-transfection. All treatments aiming at inducing NO/AMPK signaling were pe rformed after 48h of transfection. In the experiment assessing the phosphorylat ion status of AMPK and other proteins, cells were kept in serum-free DMEM (0.5% BSA) for 4 hours before and also during the 1 hour treatments to control for factors present in th e serum that could interfere with basal phosphorylation levels of these proteins. In the experiment assessing mRNA, cells were treated for 3hours in 2%HoS DMEM. Experiments 9 and 10 Young adult fe male mice (4 weeks old) homozygous for eNOS knockout (eNOS -/-, strain B6.129P2NOS3tm1Unc/J ) and nNOS knockout (nNOS-/-, strain B6.12954NOS1tm1P/h/J ) allele genes, as well as their respective wildtypes (eNOS+/+, strain C57BL/6J and nNOS+/+, strain B6.129SF2/J) were obtained from Jackson Laboratory. Animals were housed for 2 days in the University of Florida Animal Care Services Ce nter according to the guidelines set forth by the Institutional Animal Care a nd Use Committee. During this initial period animals were maintained on a 12:12 hour light-dar k cycle and provided food and water ad libitum. On the third day animals were brought to the lab, and were sa crificed in the followi ng morning. During this period animals were kept at the same conditions described above, with the exception that animals had access only to water within 2 hours before sacrifice. This procedure had the objective to minimize the variability and therefore the possi ble confounding effects of different circulating insulin and energy substrates levels on th e basal status of phos phorylation and nuclear localization of proteins. On the day of tissue collection, animals were anesthetized with 3-5% Isoflurane, and sacrificed by cervi cal dislocation. Immediately af terwards, muscles, pancreatic

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34 fat pad, heart were removed (with careful dissecti on and isolation of the le ft ventricle) weighed, frozen in liquid N2, and kept at 80 C until biochemical analyses were conducted. Experiments 11 and 12 C2C12 and L6 were seeded in 24-w ell plates grown and differentiated as previously mentioned. After 4 days of differentiation, cell s were washed once with phenol-red free and serum-free DMEM, and were treated on this sa me medium. L-NAME and L-NMMA were added to cells 30 minutes before the addition of AICA R. Cells were then incubated for further 30 minutes at 5%CO2 and 37C. Metformin, another well known ac tivator of AMPK (58, 168), and MAHMA-NO were also used in the same ma nner, but in different wells. Immediately afterwards, 10 M DAF-FM was added to each well while cells were protected from light. Monitoring of fluorescence starte d either approximately 5 minutes after the addition of DAF-FM and continued for 50 minutes (C2C12), or 25 minutes after the addition of DAF-FM and continued for 20 minutes (L6). Fluorescence wa s measured in intervals of 5 minutes. General Methods Animal Measurements Body weight and Tissue weight. NOS knockout anim als as well as their respec tive wildtypes were weighted after being anesthetized with Isoflurane. Immediately after sacrifice by su rgical dislocation, hind-limb muscles were then removed together with the pa ncreatic fat pad, and the heart with subsequent isolation of the left ventricle. All tissues were weighted and immediately frozen in liquid N2, and then kept in 80C for further analysis. Western Blot Muscle samples were homogenized in 20 m M Tris (pH7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM -glycerol phosphate, 1

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35 mM sodium orthovanadate, 1 g/ml leupeptin, 1 mM PMSF, and 10 g/ml aprotinin containing 1% vol/vol phosphatase inhibitor (p-5726) from Sigma. Protein concentrations were assessed using the DC Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA). Aliquots of muscle homogenates, (50-70 g) were then run in SDS-PAGE ge ls (either 8% or 4-20% Thermo Scientific Pierce Preci se Protein Gels) for -actin, eNOS, nNOS, iNOS, GLUT4, phosphor(Ser79)and total ACC, phosphor(Thr172)and total AMPK, phosphor(Ser498)and total HDAC5. Primary antibodies were used as follows: rabbit anti-eNOS (1:500, BD Biosciences), rabbit anti-nNOS (1:500, BD Biosciences), rabbit anti-iNOS (1:500, BD Biosciences), rabbit antiAMPK and anti-phosphoAMPK (Thr172) (1:1,500, Cell Signaling), rabbit anti-ACC (1:1500, Cell Signaling), rabbit anti-phosphoACC (Ser 79) (1:500; Upstate), goat anti-HDAC5 (1:1,000; Santa Cruz), rabbit an ti-phosphoHDAC5 (Ser498) (1:1500; Affinity Bioreagents), goat anti-GLUT4 (1:1200; Santa Cruz). Total levels of expr ession of proteins was normalized to either -actin (mouse anti-actin 1:5000, Abcam), or Ponc eau Stain to control for loading. Reactions were develope d by using enhanced chemiluminescence detection reagents (ECL Plus; Amersham Biosciences, Buckinghamshi re, UK), and protein leve ls were determined by densitometry (Kodak 1D Image An alysis Software version 3.6). Nuclear and Cytosolic Fractionation Fractionation of nuclear and cytosolic proteins was perform ed only in the Plantaris muscle of knockouts and wildtypes. Fractionation was performed using a specific kit (NE-PER, Thermo Scientific, Pierce Biotechnology) accord ing to the manufaturers intructions. Muscles were homogenized in Cyotoplasmic Extraction R eagent I (CER I,buffer containing 1% vol/vol protease inhibitors, and 1% vol/vol phosphatase inhibitors). The nuc lear isolation buffer contained protease and phosphatase inhibitors at the same con centrations reported above. The

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36 DC protein assay kit was used to quantify the protein concentrations of the samples. Appropriate fractionation of nuclear and cytosolic proteins was confirmed by western blot and subsequent probing of histone2B (rabbit anti-histone H2B 1:2000, Upstate) and CuZnSOD (rabbit antiCuZnSOD, Santa Cruz), respectively. Nuclear samples were run at 34-35 g/gel, whereas cytosolic fractions were run at 80 g/gel. PGC-1 AMPK, and phosphoAMPK were probed in both fractions in order to determine whether ablation of either nNOS, or eNOS, would interfere with the distribution of th ese proteins in skeletal muscle. Cell Culture Measurements Western Blot For total protein extracts, both C2C12 and L6 cells were washed twice in ice-cold PBS and harvested in nondenaturing lysis buffe r (NDL buffer) containing 1% vol/vol Triton X-100, 0.3 M NaCl, 0.05 M Tris base, 5 mM EDTA, 3.1 M NaN3, 95 mM NaF, 22 M Na3VO4, 0.1% vol/vol protease inhibitors (p-8340), and 1% vol/vol phosphatase inhibito rs (p-5726) from Sigma (St. Louis, MO). Protein co ncentrations were assessed using the DC Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA). Aliquots of cell lysates, (15 g) were then run in SDS-PAGE gels (either 8% or 4-20% Th ermo Scientific Pierce Precis e Protein Gels) and probed for -actin, phosphoand total ACC, phosphoand total AMPK, and phosphoand total HDAC5 using the same antibodies as described for the animal/muscle experiments. -actin blots were used to control for loading. Reactions we re developed by using enhanced chemiluminescence detection reagents (ECL Plus; Amersham Biosciences, Buckinghamshire, UK), and protein levels were determined by densitometry (Kodak 1D Im age Analysis Software version 3.6).

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37 Quantitative Real-time PCR Quantification of mRNAs fr om both L6 and C2C12 myotubes were performed with specific primer and probes using TaqMan technology designed by Applied Biosystems for Quantitative real-time PCR as described above. RT was performed using either the Verso cDNA Kit (Thermo Scientific UK) or SuperScript III First-Strand Synthesis System for RT-PCR according to the manufacturers instructions (L ife Technologies, Carlsbad, CA). Reactions were carried out using 1.5 g of total RNA.according to the manufactur ers instructions. Primers and probes used were: 1AMPK (RefSeq NM_019142.1, assay no. Rn00569558_m1), 2AMPK (RefSeq NM_023991.1, assay no. Rn00576935_m1), PGC-1 (RefSeq NM_031347.1, assay no. Rn00580241_m1 [rat] and RefSeq NM_008904.1, assay no. Mm00447183_m1 [mouse]), F1ATP Synthase (RefSeq NM_134364.1, assay no. Rn01756310_g1), Citrate Synthase (RefSeq NM_130755.1, assay no. Rn00756225_m1), GLUT4 (RefSeq NM_012751.1, assay no. Rn00562597_m1 [rat] and RefSeq NM_012751.1, assay no. Mm00436615_m1 [mouse]), Cito chrome C Oxidase (RefSeq NM_009941.2, assay no. Mm00438289_g1) and HPRT (RefSeq NM_013556.2, assay no. Mm01545399_m1). Primer and probe were obtained from Applie d Biosystems and their sequences are proprietary and therefore are not reported. Quantitative real -time PCR for the target genes were performed in the ABI Prism 7700 Sequence Detection System (Applied Bi osystems, Foster City, CA), using the 2CT method, where CT is threshold cycle. Hypo xanthine guanine phosp horibosyl transferase (HPRT) was used as the control gene for both cel ls lines. The primer and probe sequences for the rat HPRT used are: forward, 5-GTT GGATACAGGCCAGACTTTGT-3; reverse, 5AGTCAAGGGCATATCCAACAACAA-3; probe, 5-ACTTGTCTGGAATTTCA-3.

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38 Dual Luciferase Assay After the 9hr-treatm ents, C2C12 myotube cult ures were washed once in PBS at room temperature and then lysed by a ddition of 120 l passive lysis buffer (PLB, Promega DualLuciferase Reporter Assay System) as previously described (29). Plates were rocked at room temperature for 15 min. The lysate was then transferred to microcentrifuge tubes and centrifuged for 30 seconds at room temperature (2000x g) to sediment cellular debris. S upernatants were transferred to new tubes for subsequent use in the assay. Firefly lu ciferase (originating from transcriptional activity of the PGC-1 PGC-1 CRE, PGC-1 MEF2 or pGL3 vectors) and renilla luciferase activities (originating from the constitutively active uptake-control plasmid; pRL-CMV) were measured sequentially in the same 10-l volume of cell lysate. A manual luminometer (model FB12, Berthold) set to measure average light intensity in relative light units (RLU) over a 10-s measurement period was used for this assay. The level of transcription activity of the intact PGC-1 promoter, the mutated ones (PGC-1 CRE and PGC-1 MEF2), as well as of pGL3 were considered as the raw firefly luciferase activity (RLU) divided by the renilla luciferase activity (RLU). Values were always repo rted as relative to the average obtained for the control group. Fluorescence-Based Real-Time Me asurement of NO Production Intracellular NO was monitored with DAF-FM (Invitrogen, Carlsbad, CA), a pHinsensitive fluorescent dye that emits increased fluorescence after reaction with an active intermediate of NO formed during the spontaneous oxidation of NO to NO2 (110, 115). C2C12 myotubes were incubated at 37C for 30 min in phenol red-free, serum-free DMEM containing 10 M of DAF-FM diacetate. After loading was completed, cells were rinsed three times with phenol red-free, serum-free DMEM and then placed in a SpectraMax M5 multi-detection reader

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39 (Molecular Devices, Sunnyvale, CA ) for fluorometric analysis of live cells. NO fluorescence was measured every 5 minutes using excitation and emission wavelengths of 488 and 520 nm, respectively. Results consist in the difference between the fluorescence seen at a given minute minus the background (fluorescence obt ained on the first reading). Citrate Synthase Assay Citrate Synthase activity was assayed using a modified protocol from Srere (134). The assay buffer (230 L final volume) contained monobasic and dibasic potassium phosphate buffers (36.5 and 63.5 mM, respectively), EDTA (10mM), DTNB (0.2mM), acetyl-CoA (0.1mM) and Triton X-100 (0.05% v/v). The r eaction was initiated by the addition of 2 L of cell lysate and of 10 L of oxaloacetate. Absorbance at 412 nm (25 C) was measured during 5 minutes. Values were then normalized to protein content assessed with th e DC Protein Assay Kit as previously mentioned. Cell Respiration L6 cells were grown, differentiated, as describe d for Experiment 5 in the Protocols section. Harvesting and cell resp iration measurements we re performed as previously described (84). Basically, approximately 10 hours after the last treatment, myotubes were washed once in PBS, trypsinized, and centrifuged at 800x g for 3 minutes. Cell pellets were then washed twice and resuspended in 400 L of supplemented PBS containing 10mM glucose, 10mM HEPES, 0.2% BSA and pH 7.45 (10, 84). Cells were maintained at 37 C thereafter. First 300 L of supplemented PBS was added to the oxymeter ch amber (Hansatech Instruments, Norfolk, UK) and equilibrated for 1 min. Immediately afterwards, 350 L of the cell suspension solution was added to the chamber. Basal respiration rate was recorded for approximately 4 min. At this point, Oligomycin, an inhibitor of F1/F0 ATP Sy nthase was added to a final concentration of

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40 3 g/mL and non-mitochondrial respiration was m onitored for approx. 2 mins. Subsequently, uncoupled respiration, or maximal respiration, wa s then monitored for 1min. after the addition of 6 M FCCP. The highest respiration rates (observed in 20 sec-intervals) were considered as representative of basal respirati on (initial phase of the protocol) and uncoupled respiration (after the addition of FCCP). The most constant rate s (observed in 10 secintervals) during the Oligomycin incubation period were considered as representative of non-mitochondrial respiration and these values were subtracted from basal and uncoupled respiration rates before further analyses. Results were then normalized to protein concentration measured in the aliquot of the cell suspension solution remaining after th e respiration assay. Basically cells were lysed by 3 consecutive freezing and thaw cycles in liquid N2. No debris was observed even after a 5 min centrifugation at 350x g. Alliquots of these cell lysates were used to measured protein concentrations using the DC assay protein kit as described above.

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41 Figure 3-1. Experimental designs to address whether nitric oxide (NO) transcriptionally regulates PGC-1 expression. These experiments were used to test Hypotheses 1a and 1b from Aim 1. A) Experiment 1, both L6 and C2C12 were used, and cells were treated with either 25 M SNAP or 50 M DETA-NO, B) Experiment 2, a separate set of cells was transfected with the cont rol vector pGL3 (not shown in the figure) and exposed to the same treatments.

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42 Figure 3-2. Experimental designs to address whether nitric ox ide (NO) and cGMP upregulate PGC-1 mRNA through AMPK activation. These experiments were used to test Hypothesis 2a from Aim 2. A) Experiment 3, L6 myotubes were treated with DETANO (50 M) and the Guanylate Cyclase inhibitor (ODQ, 1 M), B) Experiment 4, L6 were treated with DETA-NO (50 M), the cGMP analog (8-Br-cGMP, referred as cGMP, 2mM) and a specific AMPK inhibitor (Compound C, referred as Cop C, 40 M).

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43 Figure 3-3. Experimental designs to address whether nitric oxid e (NO) upregulates mitochondrial function through AMPK activation. These expe riments were used to test Hypothesis 2b from Aim 2. A) Experiment 5, L6 were treated with the NO-donor SNAP and the cGMP analog (8-Br-cGMP, referred as cGMP ), B) Experiment 6, L6 were treated with the NO-donor DETA-NO (50 M) alone or in combination with the AMPK inhibitor Compound C (referred as Cop C, 20 M).

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44 Figure 3-4. Experimental design to address which isoform of the catalytic subunit of AMPK is required for the NO-induced regulation of PGC-1 mRNA, ACC phosphorylation and HDAC5 phosphorylation. This Experiment 7 was used to test Hypothesis 2c from Aim 2. Treatments with 50 M DETA-NO were started 48h after the beginning of siRNA transfection. Treatments lasted for 1h (protein extraction) or for 3h (mRNA isolation).

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45 Figure 3-5. Experimental design to test whether both MEF2a nd CRE-binding sites in the PGC1 promoter are required for the NO-induced upregulation of PGC-1 Experiment 8 was used to test Hypothesis 3 from Aim 3. Treatments consisted of 1mM AICAR and 50 M DETA-NO. A separate set of cells was transfected with the control vector pGL3 (not shown in the figure) a nd exposed to the same treatments.

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46 Figure 3-6. Experimental designs to test whether basal levels of activation of AMPK, as well as phosphorylation, expression and localization of some of its downstream targets are altered in muscles from eNOS and nNOS knockout mice in comparison to their respective wildtypes. These experiments we re used to test Hypothesis 4 from Aim 4. A) Experiment 9, muscles from eNOS(-/-) mice and their wildtype, B) Experiment 10, muscles from nNOS(-/-) mice and their wildtype.

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47 Figure 3-7. Experimental designs to test whether AMPK activation causes an increase in endogenous NO production. These experiments were used to test Hypothesis 5a from Aim 5. A) Experiment 11, L6 were treated with either AICAR, Metformin, or MAHMA-NO at the concentrations shown, B) Experiment 12, C2C12 were treated with AICAR (1mM), L-NAME (1mM), and L-NMMA (1mM) either alone or in combination, C) Protocol for experiments involved addition of inhibitors (L-NAME or L-NMMA) 30 minutes before the addi tion of AICAR to the media. DAF-FM (10 M) was added only 30 minutes af ter AICAR or Metformin.

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48 Figure 3-8. Experimental desi gn to test whether endogenous NO production is required for AMPK-dependent upregulation of PGC-1 mRNA and the mitochondrial genes F1ATP Synthase and Citrate Synthase. Cells were treated with AICAR and L-NAME (both at 1mM) either alone or in comb ination. Experiment 13 was used to test Hypothesis 5b from Aim 5.

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49 CHAPTER 4 RESULTS Nitric Oxide, AMPK, PGC-1 and GLUT4 Expression in Skele tal Muscle Nitric Oxide Increases AMPK Phosphorylation and Upr egulates PGC-1 and GLUT4 Gene Expression in Myotubes. Treatments of 1 hour with low levels of the NO-donors, SNAP and DETA-NO, caused an increase in AMPK phosphorylation in both L6 and C2 C12 myotubes. L6 myotubes were more sensitive to the NO-mediated effect than C2C12, with both NO donors causing a ~2-fold increase in AMPK phosphorylation in L6 versus a ~ 1.3to 1.5-fold in C2C12 (Figure 4-1). This trend was also observed in relation to PGC-1 mRNA, with the L6 and C2C12 presenting 2to 3.5-fold and 1.7to 1.9-fold increases, resp ectively, in response to 3 hour treatments with the NO-donors. Although upregulation of PGC-1 mRNA was only statistically significant in L6, in both cell types PGC-1 mRNA levels tended to remain elevated with up to 16 hours of treatments (Figures 4-2 and 4-3). Since the resp onses to either SNAP or DETA-NO were very similar in terms of AMPK activation and induction of gene expression in both cell types, for simplicity we elected to pool those samples for analysis of GLUT4 mRNA. A significant 5-fold increase in GLUT4 mRNA was onl y observed with 16 hours of treatments (Figure 4-4). Both PGC-1 and GLUT4 mRNA levels were back to contro l levels after 48 hours of treatments in both cell types (data not shown). To confirm that the effect of NO on PGC-1 mRNA was due to upregulation of transcription, we transfected C2C12 with a 2kb-promoter of PGC-1 driving luciferase (45) and performed reporter gene assays as describe d in the Methods section. The NO donor DETA-NO at two different con centrations (i.e. 25 M and 50 M) was effective in upregulating PGC-1

PAGE 50

50 promoter activity by ~ 1.8-fold. We did not observe any effect of NO treatment on the control vector pGL3 (Figure 4-5). Both Nitric Oxide and cGMP Upregulate PGC-1 mRNA and Mitochondrial En zyme Activity in Myotubes. First, we wanted to confirm that the NO-dependent effects on PGC-1 and mitochondrial function are related to activa tion of soluble Guanylate Cycl ase (sGC) and upregulation of intracellular cGMP levels. Both DETA-NO and th e cGMP analog (8-Br-cGMP) induced a small, but significant, increase in PGC-1 mRNA. The DETA-NO effect was prevented by cotreatment with the sGC inhibito r ODQ (Figure 4-6). In addition, 48hour treatments with either low levels of the NO donor (SNAP) or cGMP induced an approximate 40% increase in maximal Citrate Synthase activity (Figure 4-7). Nitric Oxide-Dependent Upregulation of PGC-1 Gene Expression an d Mitochondrial Function Requires AMPK Activation. To test whether the NO-dependent effect on PGC-1 gene expression and mitochondrial function was AMPK-dependent, we manipulated AMPK activity both pharmacologically and genetically. Pharmacological Evidence First, we observed that co-treatm ent of L6 myotubes with DETA-NO and the pharmacological inhibitor of AMPK Compound C totally prev ented the effect on PGC-1 mRNA seen with the NO-donor alone (Figure 4-6). Second, DETA-NO treatment for 5 days (12h/day) induced ~40% increase in L6 basal respiration rates, and a 15 to 20% increase in maximal (uncoupled) respiration rates in these cells. However, co-treatment with Compound C for the same period of time completely ablated the positive effect of lo w levels of chronic NO supplementation on the mitochondrial func tion of these cells (Figure 4-8).

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51 Genetic Evidence To inhibit the activity of the catalytic subunit of AMPK (i.e. AMPK) we used siRNAs for both 1AMPK and 2AMPK ( 1 siRNA a nd 2 siRNA, respectively), which sequences had been previously reported (83, 113, 156). The first set of experiments refers to the optimization of transfection and knocking down of AMPK isoforms assessed by mRNA and protein expression. In the first experiment, we transf ected L6 myotubes with a fluorophore-conjugated siRNA and observed an approxima te 90% uptake efficiency 24 hours after transfection (Figure 4-9, A). Then, we optimized the concentration of each siRNA by assessing their effect on the mRNA levels of each isoform of AMPK 48 hours after transfecti ons. As shown in Figure 4-9 (B) we observed that 1 siRNA was effective in knocking down 1AMPK mRNA by 50% to 70% at concentrations ranging fr om 120nM to 240nM. Surprisingly, 2 siRNA also caused a ~10% to 30% reduction in 1AMPK mRNA at concentrations ranging from 120nM to 200nM. At 240nM, however, 2 siRNA actually increased 1AMPK mRNA by 30%. To what concerns 2AMPK mRNA, 1 siRNA had little effect on its level of expression, except at the highest dose, which significantly re duced the expression of 2AMPK mRNA by 60% (Figure 4-9, C). The 2 siRNA caused a 95% or higher reduction in 2AMPK mRNA in all doses tested. In the following experiment, we tested the effect of both siRNAs used togeth er, but at different concentrations (Figure 4-9, D). We observed that all combinations were effective in significantly knocking down 2AMPK mRNA, but only the combination between 120nM of 1 siRNA and 200nM of 2 siRNA was effective in knocking down 1AMPK mRNA levels. Therefore, these concentrations were used in the subsequent expe riments. In the following experiment, we tested at which time point after transfection AMPK protein level was redu ced the most. We observed a 60% to 70% reduction in total AMPK protein expression at 48 h, 56h, and 64h after

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52 transfection (Figure 4-9, E). Based on the results of mRNA and prot ein expression we decided to perform the experiments involving NO treatments starting only 48 hours af ter transfection. Since our main goal was to test whether th e NO-dependent upregulation of PGC-1 required functional AMPK subunits, we tested the isolated effect of the AMPK siRNAs on PGC-1 gene expression. As shown in Figure 4-9 (F), the siRNAs either together or alone did not cause significant effects on basal PGC-1 mRNA. In the following experiments we were then able to test whether both AMPK isoforms were required for the NO-de pendent induction of PGC-1 As shown in Figure 4-10 (A and B) a significant reduction in total AMPK expression was only observed in cells treated with either 1siRNA, or both 1 and 2 siRNAs. This observation suggested that in L6 1AMPK is the mostly abundant isoform at the protein level. In fact, although expres sion of both genes was easily detected by real-time PCR, we obser ved a ~50% higher leve l of expression of 1AMPK mRNA in comparison to 2AMPK mRNA (Figure 4-10, A[inset]). However, regardless of the degree of total AMPK expression, DETA-NO treatment caus ed a 2to 2.5-fold increase in AMPK phosphorylation (Figure 4-10, C) Interestingly, only when total AMPK expression was unaffected by siRNA (i .e. unrelated siRNA and 2siRNA) was DETA-NO able to induce a significant increase in AMPK activity, indirectly assesse d by ACC phosphorylation (Figure 410, D). NO treatments did not affect HDAC5 phosphorylation (Figur e 4-10, E). Finally, consistent with the effects of NO on AMPK ac tivation and activity, we observed a 2-fold increase in PGC-1 gene expression only in cells which total AMPK levels were not reduced (i.e. Unrel siRNA and 2 siRNA groups) (Figure 4-10, F).

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53 Presence of Either the CRE or th e MEF2 site is Sufficient for the Nitric Ox ide-Dependent Upregulation of PGC-1 Promoter Activity It has been previously reporte d that AICAR, an AMP analog, at concentrations as low as 0.5mM induces AMPK activation and subsequent upregulation of the PGC-1 promoter activity in C2C12 (75). Therefore, we sought to test whether upregulation of PGC-1 promoter activity mediated by specific AMPK activation, via AICAR, and by NO would be affected similarly by different mutations at the promoter. As show n in Figure 4-11, both AICAR (0.65mM) and DETA-NO (50M) increased gene re porter activity of the intact promoter by 65% and 80%, respectively. Both treatm ents were again effective in increasing the activity of the CRE promoter, but the magnitude of the effect was slightly hi gher (i.e. 2.1and 2-5-fold respectively). However, AICAR did not induce an increase in the activity of the PGC-1 promoter lacking the MEF2 binding site, whereas DETA-NO caused an approximate 75% upregulation in the activit y of this promoter. Observations in eNOS(-/-) and nNOS(-/-) Mice Since our cell-based experiments indicated that NO transcriptionally regulates PGC-1 expression and that normal AMPK expression and activity is required for this pathway to be functional, we tested whether AMPK-d ependent signaling, as well as PGC-1 and GLUT4 expression would be affected at basal conditions in mice harboring ablation of either the eNOS or the nNOS gene. Our first observation was that eNOS(-/-) mice did not present any clear difference in whole body mass as well as in the mass of several skeletal muscles, heart, and pancreatic fat pad, when compared to their wildtype (eWT, Table 4-1). Conversely, nNOS(-/-) mice presented a 27.5% larger pancreatic fat pad and lower mass for the Soleus, Plantaris and EDL muscles by 34%, 13.8%, and 15.7%, respectively, compared to their wildtype (nWT). Next, we performed specific

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54 measurements of protein levels in both knoc kouts and compared their expression with the respective wildtypes. The ablation of eNOS in eNOS(-/-) mice and nNOS in nNOS(-/-) mice was confirmed by western blots in both the EDL and Soleus muscles (Figure 4-12). No overcompensation in the expression of the other constitutive NOS isoform wa s observed in either knockout. When examining each skeletal muscle more closely we observed very few differences between the knockouts and their w ildtypes (Figures 4-13, 4-14, 4-15, 4-16, and 4-17). In the EDL muscle, GLUT4 expression was reduced by approximately 30% in the nNOS knockouts only, although PGC-1 expression was normal in both knockouts (Figure 4-14, A and B). Levels of AMPK were slightly lower in eNOS(-/-) mice, but no difference in the expression of its surrogate ACC was observe d (Figure 4-14, C and E, respectively). Phospho-to-total AMPK was 32% lower in nNOS(-/-) mice, almost reaching statistical significance (P=0.14, Figure 4-14, D). This observation was parall eled by a trend of 35% reduction in ACC phosphorylation (P=0.30, Figure 4-14, F). In the Soleus muscle no difference in GLUT4 or PGC-1 was observed in both knockouts (Figure 4-15, A and B). The expression of AMPK was slightly reduced in nNOS(-/-) mice, but phospho-to-total AMPK presented a 2.2-fold increase (Figure 4-15, C and D). ACC expression was similar between knockouts and wildtypes (Figure 4-15, E). Phos pho-to-total ACC, however, pr esented a slight trend for reduction only in the eNOS(-/-) mice (Figure 4-15, F). It has been previously shown that both PGC-1 and AMPK translocate to the nucleus when activated (89, 114, 141, 159). Therefore, we tried to measure the distribution of these proteins across nuclear and cytosolic compartments in the plantaris muscle in order to test whether basal levels of activation of these proteins were altered in the knockout animals (Figures

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55 4-16 and 4-17). GLUT4 expression in the knockout s, assessed in the cytosolic fraction only, was very similar to wildtypes (Figure 4-17, A and E). In addition, no differences in AMPK and phospho-to-total AMPK were seen between knockouts and wildtypes both in the nuclear and cytosolic fractions (Figure 4-17, C, D, G and H). Total and cytosolic PGC-1 levels were elevated in nNOS(-/-) mice only (Figure 4-17, B and F). AMPK Activation Increases NO Producti on in Both L6 and C2C12 Myotubes In the first experim ent, which was performed in L6, the specificity of the DAF-FM probe for nitric oxide was confirmed by the fact that the highest levels of fluorescence were observed in cells treated with the NO-donor MAHMA-NO (Figure 4-18). In addition, AICAR at 1mM and 3mM, as well as Metformin (2mM), another AMPK activator, induced significant increases in DAF-FM fluorescence at 35 and 40 minutes of incubation with the probe. In the second experiment, we sought to test whether the sa me phenomenon would be present in C2C12, and also whether co-treatment of cells with th e NOS inhibitors L-NAME and L-NMMA, both at 1mM, would prevent the AICAR-i nduced increase in DAF-FM fluorescence. However, in order to avoid missing any response to DAF-FM in th e first minutes following its addition to the medium, we monitored DAF-FM fluorescence continuously during 45 minutes. As seen in Figure 4-19, AICAR treatment induced signi ficantly higher DAF-FM fluorescence than AICAR+L-NMMA starting at 30 minutes of in cubation. Further, DAF-FM fluorescence in response to AICAR treatment was significantly higher than all ot her groups from the minute 35 until the last minute monitored (i.e. 45th minute). AMPK-Dependent Induction of PGC-1 and Mitochondrial Genes Requires Endogenous NO Production in L6 Myotubes. We observed that L6 myotubes treated for 16h with AICAR presented a ~10-fold increase in PGC-1 and F1ATP Synthase mRNA levels. At the same conditions, Citrate Synthase

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56 mRNAs increased 4-fold. Co-treatment with the NOS inhibitor L-NAME blunted the AICAR effect on both PGC-1 and ATP Synthase gene expression, whereas it completely prevented the AICAR effect on Citrate Synt hase mRNA (Figure 4-20).

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57 Table 4-1. Body weight and anatom ical characteristics of eNOS(-/-) and nNOS(-/-) mice, as well as of their respective wildtypes (eWT and nWT). The average weight of left a nd right hindlimb muscles was considered as representative of each animal. Values are presented as meanSE calculated within groups. Values for percent differences (% differ.) represent percent differences between values observed in knockout animals versus wildtypes for any given variable.* Significantly different from nWT. Variable eWT eNOS ( / ) % differ. P nWT nNOS ( / ) % differ. P Body Mass (g) 16.382.3615.937.25 2.7 0.340 17.297.4816.983.38 1.8 0.620 Pancreatic Fat Pad (mg) 84.283.4885.367.14 + 1.3 0.880 76.180.1597.150.35 + 27.5 0.028* Heart (mg) 72.017.1169.967.41 2.9 0.440 70.100.9268.350.33 2.5 0.740 Left Ventricle (mg) 29.033.0729.117.09 + 0.3 0.980 25.767.4529.283.15 + 13.7 0.220 Soleus (mg) 4.675.17 4.500.12 3.7 0.420 4.100.21 2.708.11 33.9 0.001* Plantaris (mg) 9.167.38 9.017.34 1.6 0.770 10.083.51 8.692.37 13.8 0.051 EDL (mg) 6.258.20 5.908.19 5.6 0.220 5.842.26 4.925.21 15.7 0.001*

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58 Figure 4-1. AMPK phosphorylation in response to NO tr eatments of 1 hour in L6 and C2C12 myotubes. A) AMPK phosphorylation in L6 myotubes, B) AMPK phosphorylation in C2C12 myotubes. In both figures AMPK phosphorylation is normalized to total AMPK expression. P<0.05 in comparison to untreated (Control) cells.

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59 Figure 4-2. PGC-1 mRNA expression in L6 myotubes trea ted with NO donors for 3 hours, 8.5 hours and 16 hours. Values represent fo ld changes (meanSE) versus untreated (Control) cells (n=4-7/group). P<0.05 in comparison to untreated (Control) cells. Figure 4-3. PGC-1 mRNA expression in C2C12 myotubes treated with NO donors for 3 hours, 8.5 hours and 16 hours. Values represent fold changes (meanSE) versus untreated (Control) cells (n=4-8/group). There was a trend for upregulation of PGC-1 mRNA in the NO treated groups in comparison to untreated (Control) cells at 3 hours (P<0.08).

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60 Figure 4-4. GLUT4 mRNA expre ssion in C2C12 myotubes treated with NO donors for 3 hours, 8.5 hours and 16 hours. NO donors values represent fold changes (meanSE) of samples treated with either SNAP (25 M) or DETA-NO (50 M) versus untreated (Control) cells (n=7-13/group). P<0.05 compar ed to Control at the same time point. Figure 4-5. NO induces PGC-1 promoter activity in C2C12 myotubes. Cells were treated for 9 hours with different concen trations of the NO donor DETA-NO. Values represent fold changes (meanSE) versus untreated (Control) cells (n =4-12/group). *P<0.05 compared to Control.

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61 Figure 4-6. PGC-1 mRNA expression in L6 myotubes treate d for 3 hours. Values represent fold changes (meanSE) versus untreated (C ontrol) cells (n=4-5/group). P<0.05 in comparison to all groups except 8-Br-cGMP and ODQ+DETA-NO, #P<0.05 in comparison to all groups except DETA-NO. Figure 4-7. Chronic NO and cGMP treatments increas e maximal Citrate Synthase activity in L6 myotubes. Cells were treated for 48 hour s. Values represent enzyme activity normalized to protein concentration (m eanSE, n=6/group). P<0.05 compared to Control.

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62 Figure 4-8. AMPK inhibition prev ents chronic NO-dependent upreg ulation of basal and maximal mitochondrial respiration in L6 myotubes. Values represent respiration rates normalized to protein concentration (m eanSE, n=5/group). Respiration rates observed in the presence of Oligomycin were subtracted from both basal and uncoupled respiration before protein nor malization. *P<0.05 in comparison to all groups except Compound C, #P<0.05 in comparison to DETA-NO + Compound C (P=0.054 in comparison to Control).

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63 Figure 4-9. Effect of 1AMPK siRNA and 2AMPK siRNA on AMPK and PGC-1 gene expression. A) Pictures of L6 myotubes treated with Lipofectamine (control vehicle) or transfected with siGLO Green siRNA for 24 hours (Merged pictures are shown, Blue depicts nuclei stained with DAPI, Green depicts siRNA, magnification =200x), B) Effect of either 1AMPK siRNA or 2AMPK siRNA on 1AMPK mRNA expression (n=4/group, *P<0.05 in comparison to Control [open bar], #P<0.05 in comparison to other concentrations of 2AMPK siRNA), C) Effect of either 1AMPK siRNA or 2AMPK siRNA on 2AMPK mRNA expression (n=34/group, *P<0.05 in comparison to Control [open bar], # P<0.05 in comparison to other concentrations of 1AMPK siRNA), D) Effect of different concentrations of 1AMPK siRNA and 2AMPK siRNA used in combination on 1AMPK mRNA and 2AMPK mRNA expression (n=4/group, *P< 0.05 in comparison to Control and Lipofectamine), E) Effect of 1AMPK siRNA and 2AMPK siRNA used in combination on AMPK protein expression at differen t time points after transfection (n=2/group,*P<0.05 in comparison to Control), F) Effect of 1AMPK siRNA and 2AMPK siRNA used either in combination or separately on PGC-1 mRNA expression (n=3-4/group). Except for A, in all other experiments samples were harvested 48 hours af ter transfection.

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64 Figure 4-9. Continued

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65 Figure 4-10. Knockdown of 1AMPK prevents NO-depende nt increase in PGC-1 mRNA. A) Representative blots of pr oteins assessed and their phosphorylation status (Inset depicts the comparison between 1AMPK mRNA and 2AMPK mRNA in untreated cells [n=8]), B) AMPK expression (*P<0.05 in comparison to Control, Unrel. siRNA and 2AMPK siRNA, P=0.09 between Control and 1AMPK siRNA + 2AMPK siRNA groups), C) Phospho-to-total AMPK ratio, D)Phospho-to-total ACC ratio (*P<0.05 in comparison to Control and 1AMPK, P=0.07 between either Unrel.siRNA or 2AMPK siRNA and 1AMPK siRNA + 2AMPK siRNA groups), E) Phospho-HDAC5 expression, F) PGC-1 mRNA (*P<0.05 in comparison to Control, 1AMPK siRNA and 1AMPK siRNA + 2AMPK siRNA groups). Except for F, in all other experiments cells were treated for 1 hour after 48 hours of transfection. PGC-1 mRNA was measured after a 3h treatment with DETA-NO after 48 hours of transfection.

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66 Figure 4-10. Continued Figure 4-11. NO induction of PGC-1 promoter activity does not re quire intact CRE and MEF2 sites in C2C12 myotubes. Cells were treated for 9 hours with either AICAR (0.65mM) or DETA-NO (50 M). Values represent fold changes (meanSE) versus untreated (control) cells (n=1215/group in pGL3 control & PGC-1 experiments; n=8-12 in CRE & MEF2 experiments). *P<0.05 compared to Control, #P<0.05 compared to Control and AICAR.

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67 Figure 4-12. eNOS and nNOS expression in EDL and Soleus muscles from eNOS(-/-) and nNOS(-/-) mice and their respective wildtypes. A) Representative blots of eNOS, nNOS and Ponceau stain for EDL and Soleus muscles from eNOS(-/-) and respective wildtype, B) Representative blots of e NOS, nNOS and Ponceau stain for EDL and Soleus muscles from nNOS(-/-) and respective wildtype.

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68 Figure 4-13. Representative blot s of proteins assessed in EDL and Soleus muscles of eNOS(-/-) and nNOS(-/-) mice and their respective wildtypes. A) Representative blots of proteins assessed in the EDL, B) Representative bl ots of proteins assessed in the Soleus.

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69 Figure 4-14. Protein expression of proteins related to AMPK si gnaling in the EDL muscle from eNOS(-/-) and nNOS(-/-) mice and their respective wildt ypes. A) GLUT4 expression, B) PGC-1 expression, C) AMPK expression, D) phospho-to-total AMPK ratio (P=0.14 between WT and nNOS(-/-)), E) ACC expression, F) phospho-to-total ACC ratio (P=0.3 between WT and nNOS(-/-)). All proteins were normalized to Ponceau stain. *P<0.05 in comparis on to respective wildtype.

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70 Figure 4-15. Protein expression of proteins related to AMPK signali ng in the Soleus muscle from eNOS(-/-) and nNOS(-/-) mice and their respective wildt ypes. A) GLUT4 expression, B) PGC-1 expression, C) AMPK expression, D) phospho-to-total AMPK ratio, E) ACC expression, F) phospho-to-total ACC ra tio. All proteins were normalized to Ponceau stain. *P<0.05 in comparison to respective wildtype.

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71 Figure 4-16. Representative blots of proteins assessed in nuclear and cytosolic fractions of the Plantaris muscle of eNOS(-/-) and nNOS(-/-) mice and their respective wildtypes. A) Representative blots of pr oteins assessed in eNOS(-/-) mice and their respective wildtypes, B) Representative blots of proteins assessed in nNOS(-/-) mice and their respective wildtypes, C) Ponceau stain a nd representative blots of CuZnSOD and Histone H2B demonstrating succes sful fractionation of samples.

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72 Figure 4-17. Protein expression of proteins related to AMPK si gnaling in nuclear and cytosolic fractions of the Plantaris muscle from eNOS(-/-) and nNOS(-/-) mice and their respective wildtypes. A, B, C a nd D) Expression of GLUT4, PGC-1 AMPK, and phospho-to-total AMPK ratio, respectively, in muscles from eNOS(-/-) mice and their respective wildtypes; E, F, G, and H) Expression of GLUT4, PGC-1 AMPK, and phospho-to-total AMPK ratio, respectively, in muscles from nNOS(-/-) mice and their respective wildtypes. A ll proteins were normalized to Ponceau stain. *P<0.05 in comparison to respective wildtype.

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73 Figure 4-17. Continued Figure 4-18. AMPK activation stimulates NO production in L6 myotubes. Values represent the fluorescence at each 5 minute-interval minus the background fluorescence assessed at 20 minutes ( fluorescence) and are presented as meanSE of (n=6/group). *P<0.05 MAHMA-NO in comparison to Control, #P<0.05 AICAR (1mM and 3mM) and Metformin (2mM) in co mparison to Control.

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74 Figure 4-19. AMPK activation stimulates NO production in C2C12 myotubes. Values represent the fluorescence at each 5 minute-interv al minus the background fluorescence assessed at minute zero ( fluorescence) and are pr esented as meanSE of (n=6/group). *P<0.05 AICAR in compar ison to AICAR+L-NMMA, #P<0.05 AICAR in comparison to all other groups.

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75 Figure 4-20. NOS inhibition blunts AMPK-dependent upregulation of PGC-1 F1ATP Synthase and Citrate Synthase mRNAs in L6 myotubes. Values represent fold change versus untreated (Control) cells (meanSE, n=5-7/ group). A) Effect of AICAR treatment alone or together with L-NAME on PGC-1 mRNA, B) Effect of AICAR treatment alone or together with L-NAME on F1ATP Synthase mRNA, C) Effect of AICAR treatment alone or together with L-NAM E on Citrate Synthase mRNA. *P<0.05 in comparison to all other groups.

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76 CHAPTER 5 DISCUSSION Several studies have shown that NO induces m itochondrial biogenesis in different tissues, including skeletal m uscle, and that PGC-1 upregulation is involved in this process (104-106). These reports provide strong evidence for th e influence of NO, and eNOS expression, on mitochondrial function. However, they provide li mited information on the mechanisms of PGC1 regulation in response to NO. Our lab has recently shown that NO regulates GLUT4 expression via AMPK stimulation in skeletal muscle (89). Since GLUT4 expression can also be regulated by PGC-1 (95), and AMPK-dependent signaling induces PGC-1 expression and activation (75, 170), our central hypothesis was that NO regulates mitochondrial biogenesis and GLUT4 expression via AMPK activation, and subsequent induction of PGC-1 We addressed our central hypothesis with 5 Specific Aims, encompassing several experiments that involved L6 and C2C12 myotubes, and adu lt skeletal muscle of eNOS(-/-) and nNOS(-/-) mice as well. The reasons for studying both L6 and C2C12 are two-fold. First, L6 cells have been frequently used in investigations involving skeletal muscle metabolic adaptations, especially in relation to NO and AMPK signaling (83, 89, 105, 156). Second, the mechanisms involved in PGC-1 transcription regulation have been mos tly studied with gene reporter assays using the mouse PGC-1 promoter transfected to C2C12 cells (50, 75, 95). Therefore, the use of both cell types was intended to improve our cap acity of discussion and comparison of results with previous studies investigating similar signaling pathways. In Specific Aim 1 we addressed whether NO would upregulate PGC-1 gene expression in both L6 and C2C12, and whether th e NO-dependent regulation of the gene would occur at the transcription level. The first set of expe riments demonstrated that NO induces PGC-1 in both L6 and C2C12 within 3 hours of treatments, and th at AMPK activation occu rs very rapidly (i.e.

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77 within 1 hour of treatmen ts). Although not statisti cally significant, PGC-1 mRNA tended to remain increased even with longer treatments of 8.5 and 16 hours. We are unaware of other studies looking at short-te rm regulation of PGC-1 mediated by NO. However, Nisoli et al. (105) reported a comparable increase in PGC-1 mRNA (~80%) in response to a 6-day treatment with 50M DETA-NO in L6 cells. In addition, we also demonstrated that NO donors were able to upregulate GLUT4 mRNA with 16 hours of treatm ents in C2C12, similarly to what was previously observed in L6 (89). These experiments served as proof of principle that similar mechanisms of regulation of PGC-1 and GLUT4 gene expression were in place in these two cell types. Of note, we used two different NO donors, namely SNAP (5h half-life at 37 C and pH 7.4) and DETA-NO (20h half-life at 37 C and pH 7.4) (12). DETA-N O releases 2 moles of NO per mole of compound, whereas SNAP releases 1 mole of NO per mole of compound. The combination of NO-release kinetic s and initial concen trations of these two donors (i.e.25M of SNAP and 50M DETA-NO) should result in ve ry similar NO release during the first hour (~3.3 and 3.4M at 1h, respectively) and second hour of treatments (6.1 and 6.8M at 2h, respectively). However, afte r 16 hours of treatments, 25 M SNAP will have released approximately half as much NO as 50 M DETA-NO (i.e. 22.3 and 42.6 M, respectively). Therefore, considering that the ov erall pattern of induction of PGC-1 upregulation of GLUT4 and activation of AMPK was very similar betw een NO donors, these data suggest that these cellular responses are re lated to events occurring early (w ithin 1 to 2 hours of treatments). Further, the fact that NO upregulated PGC-1 much more rapidly than GLUT4, suggests that increased expression of PGC-1 may be required for the NO-de pendent upregula tion of GLUT4 in myotubes. However, further studies are requi red to appropriately address this question. In addition, our gene reporter assays showed a ~1.8fold stimulation of the intact 2kb promoter of

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78 PGC-1 in response to NO treatments, which was c onsistent with the effects observed at the mRNA level, also in C2C12 myotubes. In Specific Aim 2 we tested whether the NO-dependent regulation of PGC-1 and mitochondrial function required f unctional AMPK. In agreement with previous reports, we observed that both NO and cGMP increased PGC-1 mRNA, whereas AMPK pharmacological inhibition prevented the NO-dependent upregulation of the gene. We have recently reported that Compound C has the same effect on the NO-de pendent upregulation of GLUT4 mRNA (89). Altogether these findings support th e notion that NO upr egulates PGC-1 and GLUT4 mRNAs via AMPK activation. Our results also provide clear evidence not only for the NOand cGMPdependent regulation of Citrate Synthase activity, but also for the dependence on functional AMPK for the NO-mediated improvement in mitochondrial function in skeletal muscle myotubes. The ~40% increase in the activity of a mitochondrial enzyme with a 48hour treatment with either a NO donor, or a cGMP analog, is co nsistent with: a) ~ 40% increase in basal respiration rates of L6 with a NO treatment fo r 5 days (12h/day), and b) the magnitude of increase in PGC-1 mRNA. Interestingly, Nisoli et al (105) observed a 140% increase in basal respiration rates of L6 treated wi th a cGMP analog continuously for 6 days. It is possible that a continuous manipulation of a more distal player in the signal (i .e. cGMP versus NO) may induce more pronounced adaptations. However, Nisoli et al. (105) did not report when in the differentiation process of L6 their treatments started, making it difficult to draw any conclusion. Koves et al. (84) reported a ~150% increase in mitochondrial respiration rates of L6 cells transfected with an adenoviru s containing the mouse PGC-1 Therefore, our results seem to be consistent with a lower, but physio logical, degree of stimulation of this adaptation pathway.

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79 To gain further insight on the AMPK role and more specifically on the role of AMPK catalytic subunits, in the NO-de pendent regulation of PGC-1 we performed experiments with siRNAs for both 1AMPK and 2AMPK. A surprising finding wa s that although both isoforms of AMPK were detectable at the mRNA level, and that both siRNAs were effective in reducing the expression of their target mRNAs, when used alone only 1siRNA caused a reduction in total AMPK protein expression. These findings suggest that 1AMPK is the main isoform expressed at protein levels in L6 myotubes. Previous studies using the same siRNA sequences reported similar reductions in total AMPK protein expression (83, 113, 156), but to our knowledge this is the first study to try to knockdown each AMPK separately. Therefore, comparisons between their results and ours are not possible at this point. Although protein measurements of each AMPK would be ideal to confirm these results, this is consistent with our observation of a higher level of 1AMPK mRNA in these cells Indeed, others have re ported the expression of both AMPK proteins in L6, but their level of expression was not compared (17). Since we were not able to reduce total AMPK protein expression with the 2si RNA alone, we were not able to test whether the 2AMPK isoform is involved in the NO-dependent signaling. However, our resu lts clearly show that 1AMPK is required for the NO-dependent regulation of AMPK activity and PGC-1 expression. This is in agre ement with the observation that sodium nitroprusside preferentially activates 1AMPK in isolated rat EDL muscles (60). Another interesting observation was that DETA -NO caused very similar levels of AMPK activation across siRNA treated gr oups, but a measurable increase in AMPK activity, indirectly assessed by phospho-to-total ACC quantific ation, was only induced when total AMPK expression remained at control levels. This obser vation suggests that: a) reductions of ~40-60% of AMPK expression are sufficient to physiological ly compromise the enzymes function in

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80 these cells, even with normal activation by upstream signals taking place, and b) total AMPK expression should also be considered when one is assessing AMPK-dependent signaling in skeletal muscle. Indeed, a recent report shows that 1AMPK and 2AMPK are expressed at higher levels in the more oxidative and metaboli cally active type IIa fibers, and that chronic electrical stimulation of the TA muscle increases their expression (114). Finally, in contrast with the increase in PGC-1 mRNA seen in groups displaying no rmal AMPK expression that were treated with NO, we failed to detect a parallel increase in HDAC5 phosphorylation. This finding suggests that the NO-mediat ed regulation of PGC-1 may not rely on HDAC5 phosphorylation and liberation of MEF2 trans-activity, but ra ther on AMPK direct phosphorylation of PGC-1 instead (75). In Specific Aim 3 we assessed whether the MEF2 or CRE sites at the PGC-1 promoter would be required for the NO-de pendent upregulation of PGC-1 Contrary to what we initially hypothesized, NO was capable of inducing reporter activity of the intact PGC-1 promoter, and also of both CREand MEF2-PGC-1 promoters, whereas AICAR was ineffective in increasing luciferase activity of the MEF2-PGC-1 promoter. This finding is consistent with the observation that NO treatment does not seem to be involved in significant upregulation of HDAC5 phosphorylation, and subsequent increase in MEF2 availability. To our knowledge this is the first study to examine the requireme nt of these binding sites of the PGC-1 promoter for NOand AMPK-dependent induction of PGC-1 Spiegelmans group has shown that calciumcalmodulin dependent protein kinase (CAMKIV) and calcineurin (CaN ) induction of PGC-1 require both sites to be intact, whereas PGC-1 may regulate its ow n expression through interaction with MEF2 at its own promoter (50). Akimoto et al. (3, 4) also reported that both sites are required for the exercise -mediated induction of PGC-1 in adult skeletal muscle in vivo In

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81 this context the fact that NO requires either one of these sites is unique. We can not offer a definitive explanation for this phenomenon at this moment. As previouosly mentioned, it was recently demonstrated that AMPK can directly phosphorylate PGC-1 increasing its activity, which further stimulates PGC-1 transcription (75). In addition, AMPK is known to phosphorylate and activate CREB in skeletal muscle (142). Therefore, it is possible that our results reflect different degrees of AMPK activation (i.e lower le vels of AMPK activation with 0.65mM AICAR versus 50M DETA-NO). In add ition, it is also possible that NO-dependent regulation of PGC-1 involves other mechanisms that are not dependent on AMPK signaling. Further studies are required to confirm these possibilities and also test the effect of NO supplementation, or NOS inhibition, on CREB-re lated adaptations in skeletal muscle. In Specific Aim 4 we ascertained whether geneti c ablation of either the eNOS or the nNOS gene would affect PGC-1 and GLUT4 expression, as well as basal levels of AMPK-dependent signaling in skeletal muscle. We did not observe an overcompensation of the other constitutive NOS isoform in the muscles of either knockout. Recently, Wadley et al. (152) reported an overcompensation of eNOS in nNOS(-/-) mice and of nNOS in eNOS(-/-) mice only in the EDL muscle. Although we can not provide a clear reason for the differences between their results and ours, it is possible that the ag e of the animals may have an influence. We studied very young mice (4-weeks old), whereas Wadley et al. used 16-week old mice. It is possible that compensations become more evid ent once these animals become a dults, as a result of a longer challenge to their normal systemic physiology impos ed by the lack of either the nNOS or the eNOS genes. The most striking findings, however, were the lower GLUT4 expression observed in the EDL muscle, the higher pancreatic fat pad depot, and the lower mass of the EDL, Plantaris and

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82 Soleus muscles in nNOS(-/-) mice. The reduction in muscle mass is corroborated by Wadley et al. (152). We are unaware of previous studies reporting an increase in visceral fat in such an early age in these mice. The lower expression of GLUT4 in a predominantly fast-twitch muscle is consistent with nNOS being more abundant in glycolytic skeletal muscle (85, 152). This observation supports our previous findings where inhibition of nNOS and iNOS prevented the AMPK-dependent upregulation of GLUT4 in the plantaris muscle in vivo (89). Although PGC1 protein levels were not altered in the EDL muscle of these animals, both phospho-to-total AMPK as well as phospho-to-total ACC showed a trend for reduction. Whether this trend is exacerbated as the animals age, deserves further investigation. However, nNOS knockouts display signs of peripheral insulin resistance (128). Therefore, it is possible that the increase in visceral fat accumulation, as well as lower e xpression of GLUT4 in gl ycolytic fibers, may contribute to this phenomenon. On the other hand, mitochondrial dys function is observed preferentially in eNOS knockout mice (46, 98, 104, 106, 149), consistent with the observation that these animals develop both central and peripheral insulin resistance (128). However, in agreement with Wadley et al (152), we fa iled to detect any reduction in basal PGC-1 expression in both knockouts. In addition, we also did not observe a significant difference in nuclear localization of PGC-1 in the plantaris muscle of these anim als. Considering that in slow-twitch muscle the array of signa ls contributing to PGC-1 and GLUT4 regulation is constantly more active than in fast-twitch muscle, one could expect that inhibition of one of the positive regulators (i.e. NO production) would have a larger impact in fa st glycolytic muscle. Therefore, although we observed that at a very early age n NOS gene ablation causes structural metabolic changes in glycolytic muscle only, represented by a significant reduction in GLUT4, we interpret these results as evidence for a role of NO in the maintenance of normal GLUT4 levels in skeletal

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83 muscle. This interpretation s hould be confirmed in models where normal physiology would be more severely challenged, such as in response to chronic high-fat diet. Future studies addressing this issue in nNOS knockout mice are still lacking and will defin itely contribute for a better understanding of the interaction between NOand AMPK-dependent si gnaling in skeletal muscle. In our final Specific Aim we addressed wh ether AMPK-dependent regulation of PGC-1 and mitochondrial genes would be affected by inhibition of NO production. Similarly to our observations with GLUT4 (89), NOS inhibition blunted AMPK induction of PGC-1 F1ATP Synthase, and Citrate Synthase mR NA in L6 myotubes. This observa tion is consistent with the findings that AMPK activation, via AICAR a nd Metformin, increase NO production in L6. In general our results support our centr al hypothesis that NO regulates PGC-1 and GLUT4 expression via AMPK activation. We demons trated for the first time that NO regulates PGC-1 transcriptionally and that normal AMPK function is required for the NO-dependent increase in mitochondrial respira tion in skeletal muscle cells. More specifically, we provide evidence for an important role of 1AMPK expression in the NO-me diated regulation of PGC1 in L6 myotubes. Regarding the regulation of PGC-1 by NO, our observations suggest that NO does not require intact CRE and MEF2 sites at the PGC-1 promoter to induce its activity. In addition, we also observed that ab lation of the nNOS gene results in lower expression of GLUT4 in the glycolytic EDL muscle only, paralleled by a trend towards reduc ed markers of AMPKdependent signaling. Finally, we demonstrated that endogenous NO production is required for AMPK-dependent upregulation of PGC-1 a marker of mitochondrial biogenesis, as well as of F1ATP Synthase and Citrate Synthase in L6 myot ubes. Altogether these results corroborate our proposed model that NO and AMPK interact through a positive feedback loop that impact

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84 metabolic adaptations in skeletal muscle (89). A ccording to this model, low and basal levels of NO production are required for normal AMPK activ ity, and stimulation of AMPK causes small increases in NO production from the constitutive NOS isoforms in skeletal muscle. Future studies to test whether this si gnaling mechanism is involved in sk eletal muscle adaptations to metabolic challenging conditions, su ch as cold exposure, exercise and caloric restriction are still required.

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100 BIOGRAPHICAL SKETCH The experience I have b een accumulating since I completed my bachelors degree in physical education continues to influence my goals for the future as a professional. Therefore, throughout this text I would like to briefly allude to a couple of facts in my career and discuss how they helped to shape what I no w see as my professional future. My interest and curiosity for the effects of exercise on wellbeing and prevention of certain diseases started when I was still pursuing my undergraduate degree. Back then, I was very interested in knowing how exercise could positively aff ect the immune system, and whether that could be beneficial for HIV-positive patients. I wa s fortunate to be able to perform a relatively long-term study with sixteen patients that were ra ndomly assigned to the c ontrol or the exercise group. I personally was responsible for monitoring and developing the training sessions of the exercising group (8 patients), which occurred thr ee times a week for six months, and consisted of lowto moderate-intensity endurance exercises. Before the training program started and every two moths thereafter we performe d physical fitness and wellbeing assessments, and also assessed white blood cell counts, as a rough measure of immune function. By the end of the 6-month program of exercise we observed no clear immune improvement and only a mild increase in general physical fitness (i.e. ae robic capacity, flexibility and strength all slightly improved). However, the most striking result was the dram atic decrease in anxiet y, depression and stress levels in the exercise group. This rich experience taught me that exercise definitely added life to those 6 months for each of the patients involved in the training program. This wonderful experience served as a great motivation for me to try to understand even further how changes in activity levels induc e adaptations in skeletal muscle. My next step as a professional was to pursue a masters degree with emphasis in physical fitness assessment. During my masters I worked on the development of a test called Sitting-

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101 Rising test, which basically grades the actions of sitt ing and rising from the ground, independently, using a scale from 0 to 4. The idea was to develop an evaluation tool that could assess functional fitness in a very simple way, an d therefore have the poten tial of broad use with different populations (e.g. obese individuals, elderly) and in diffe rent settings (e.g. on the field, rehab centers, clinics, etc.). Interestingly, the sc ores in those two very simple tasks correlated very well with leg strength and flexibility, and were adversely affected by body mass index (BMI) and adiposity (body fat %) in people of differe nt ages and fitness levels. Of note, for some individuals safely sitting and rising from the ground, without the help of hands or arms for support, were very challenging tasks. This has clearl y drawn my attention to the fact that if these simple tasks were so challenging for some indivi duals, exercise at a level that could bring about important improvements in muscle function would pr obably be as well. Actually this is the case for some elderly, and individuals affected by muscle and joint probl ems, neurological disorders, as well as those with extremely high body fat. As I started to understand more and more about physiology in general, I also bega n to realize that for one to have a palpable notion of the exercise effects on certain organs or tissues (e.g. skeletal muscle) it is important to be trained in more specific measurements in humans, but also understand and use animal models to address some questions in further detail. These observations led me to search for a be tter education and traini ng on the cellular and molecular mechanisms by which exercise can impr ove muscle function. My expectation was that once having this kind of training I would be able to work on the identification of proteins (e.g. enzymes) and/or molecules that could serve as pharmacological targets fo r the induction of some of the exercise-induced adaptations. This could be of great help in th e treatment of obesity, diabetes and also for the elderly. As a result I applied to the Ph.D program in Exercise

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102 Physiology here at the Department of Applie d Physiology and Kinesiology of the College of Health and Human Performance of the University of Florida. Since the beginning of my Ph.D program I have been learning about the mechanisms involved in exercise-induced ad aptations in skeletal muscle, and also about some molecular biology tools that can be used in th is type of study. This is mainly because of the interaction with Dr. Criswell, but it is also because of the healthy environment and the great amount of information that we are exposed to in the Center of Exercise Science. In Dr. Criswells lab we have been performing experiments with mice and ra ts, muscles in baths, as well as muscle cell culture to investigat e some of skeletal muscle adaptations to different stimuli. More specifically, my project focus on the role of nitric oxide, a free radical produced at higher levels in skeletal muscle during contraction, in th e regulation of mitochondrial f unction and expression of the major glucose transporter in muscle (i.e. GLUT4). This study is directed towards the development of possible treatment alternatives for chronic diseases related to over nutrition. Diabetes, obesity and coronary heart disease are all related to deficient glucose transport and mitochondrial function in skeletal muscle. In the near future I intend to pursue pos t-doctoral training focu sing on the mechanisms involved in the improvement of metabolic functi on in skeletal muscle mediated by exercise. Ultimately, I intend to pursue a career in academ ia, where I would like to be involved with both teaching and research. I enjoy doing experiments with animal and cell culture models to address some adaptations in skeletal muscle. However, I also would like to collaborate with other investigators that perform human st udies. I believe this type of inte raction is critical for a more prompt development of better strategies for treatm ent and prevention of chr onic diseases such as obesity and diabetes.