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Nitric Oxide Synthase Activity Mediates Activation of AMP-Associated Protein Kinase in Isolated Mouse Skeletal Muscle

Permanent Link: http://ufdc.ufl.edu/UFE0022199/00001

Material Information

Title: Nitric Oxide Synthase Activity Mediates Activation of AMP-Associated Protein Kinase in Isolated Mouse Skeletal Muscle
Physical Description: 1 online resource (54 p.)
Language: english
Creator: Brown, Dana
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ampk, edl, lnmma, no, nos, soleus
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nitric oxide(NO) production and signaling has an important role in exercise-induced metabolic and biochemical adaptations. Current research indicates that low levels of NO produced enzymatically by the nitric oxide synthases (NOS), regulates 5-AMP-activated protein kinase(AMPK) in cell culture. The physiological significance of this regulatory mechanism in adult skeletal muscle is unknown. Goal: This research was designed to determine if NOS activity is necessary for induction of AMPK phosphorylation in skeletal muscles isolated from adult mice. Hypotheses: Incubation of isolated extensor digitorum longus (EDL) and soleus muscles with the non-isoform specific NOS inhibitor, L-NMMA, will prevent activating phosphorylation of AMPK, and phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC). Approach: EDL and soleus muscles were surgically isolated bilaterally from young adult mice and incubated in micro tissue baths. AMPK was activated by 10 min of contractile activity or a 20-min incubation with the AMP analog, AICAR. The EDL and soleus muscle from the contralateral limb was treated identically, except for the addition of L-NMMA to the incubation media throughout the experiment. Results: The main findings of this study are: 1) electrical stimulation or AICAR incubation increases the phospho-AMPK/total AMPK ratio; 2) NOS inhibition prevents this increase; 3) AMPK activation by electrical stimulation and AICAR treatment is greater in the fast twitch EDL muscle than the slow twitch soleus muscle; and 4) L-NMMA treatment tends to increase AMPK phosphorylation status, independent of electrical stimulation or AICAR effects. Significance: AMPK is an important regulator of skeletal muscle metabolism and the acute and chronic adaptations to exercise training. Activation of AMPK, pharmacologically or by exercise, increases oxidative capacity and improves insulin sensitivity leading to a metabolically active phenotype in skeletal muscle that effectively counters the metabolic syndrome. Understanding how AMPK is regulated in adult muscle fibers and the potential role of nitric oxide in mediating AMPK activation is important for development of more potent and efficient treatments for metabolic disorders.
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 Dana Brown.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Criswell, David S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022199:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022199/00001

Material Information

Title: Nitric Oxide Synthase Activity Mediates Activation of AMP-Associated Protein Kinase in Isolated Mouse Skeletal Muscle
Physical Description: 1 online resource (54 p.)
Language: english
Creator: Brown, Dana
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ampk, edl, lnmma, no, nos, soleus
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nitric oxide(NO) production and signaling has an important role in exercise-induced metabolic and biochemical adaptations. Current research indicates that low levels of NO produced enzymatically by the nitric oxide synthases (NOS), regulates 5-AMP-activated protein kinase(AMPK) in cell culture. The physiological significance of this regulatory mechanism in adult skeletal muscle is unknown. Goal: This research was designed to determine if NOS activity is necessary for induction of AMPK phosphorylation in skeletal muscles isolated from adult mice. Hypotheses: Incubation of isolated extensor digitorum longus (EDL) and soleus muscles with the non-isoform specific NOS inhibitor, L-NMMA, will prevent activating phosphorylation of AMPK, and phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC). Approach: EDL and soleus muscles were surgically isolated bilaterally from young adult mice and incubated in micro tissue baths. AMPK was activated by 10 min of contractile activity or a 20-min incubation with the AMP analog, AICAR. The EDL and soleus muscle from the contralateral limb was treated identically, except for the addition of L-NMMA to the incubation media throughout the experiment. Results: The main findings of this study are: 1) electrical stimulation or AICAR incubation increases the phospho-AMPK/total AMPK ratio; 2) NOS inhibition prevents this increase; 3) AMPK activation by electrical stimulation and AICAR treatment is greater in the fast twitch EDL muscle than the slow twitch soleus muscle; and 4) L-NMMA treatment tends to increase AMPK phosphorylation status, independent of electrical stimulation or AICAR effects. Significance: AMPK is an important regulator of skeletal muscle metabolism and the acute and chronic adaptations to exercise training. Activation of AMPK, pharmacologically or by exercise, increases oxidative capacity and improves insulin sensitivity leading to a metabolically active phenotype in skeletal muscle that effectively counters the metabolic syndrome. Understanding how AMPK is regulated in adult muscle fibers and the potential role of nitric oxide in mediating AMPK activation is important for development of more potent and efficient treatments for metabolic disorders.
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 Dana Brown.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Criswell, David S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022199:00001


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NITRIC OXIDE SYNTHASE ACTIVITY MEDIATES ACTIVATION OF AMP-
ASSOCIATED PROTEIN KINASE IN ISOLATED MOUSE SKELETAL MUSCLE




















By

DANA LEIGH BROWN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008



































2008 Dana Leigh Brown




























To my family for their unwavering support and all the teachers and professors who taught and
mentored me along the way.









ACKNOWLEDGMENTS

This work was completed with the help and support from many people. First and

foremost I would like to thank Vitor Lira for teaching me the majority of the lab techniques used

for this study and for working with me to collect data. Without him I undoubtedly would not

have completed this project.

I would like to thank Dr. David Criswell, my supervisory committee chair, for allowing

me to work in his lab even though lab space and funding was tight. He stimulated my interest in

exercise physiology as an undergraduate and continued to teach me until I finished my Master's

degree.

I would like to thank my committee members, Drs. Scott Powers and Steve Dodd from

the Department of Applied Physiology and Kinesiology, College of Health and Human

Performance, University of Florida, for allowing me to present my research.

I thank my fellow Molecular Physiology labmates (Vitor, Quinlyn Soltow, Jason

Drenning, Liz Zeanah, Carlos Carmona, Claire Canon and Lauren Valera) who helped me in the

lab and kept me motivated to write this paper. Also to Dr. Jenna Leigh Jones Betters who

allowed me to assist with a portion of her data collection. This gave me the opportunity to

familiarize myself with the techniques I used for my project.

I thank all the professors in the Applied Physiology and Kinesiology department for

teaching and mentoring me and for continuously surpassing their own achievements.

This work could not have been done without the love and motivation from my friends

and family. I would especially like to thank my parents, Mike and Linda Brown, for supporting

and encouraging me when they did not exactly understand what I was doing.










TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IST O F TA B LE S ......... .... ........................................................................... 7

LIST OF FIGURES .................................. .. ..... ..... ................. .8

A B S T R A C T ......... ....................... .................. .......................... ................ .. 9

CHAPTER

1 INTRODUCTION ............... ................. ........... ................................. 11

B a c k g ro u n d .................................................................................................................... 1 1
Problem Statement ................................................................ .................. 12
V a riab le s in S tu d y ............................................................................................................. 12
H ypotheses.................................................................13
Definition of Terms ................................................................... 13
Limitations/Delimitations/Assumptions ................................. ................................... 14
S ig n ifican ce of th e Stu dy ................................................................. ...............................15

2 L IT E R A T U R E R E V IE W ................................................................ ...............................16

Enzymatic Production of Nitric Oxide(NO) ................................................................. 16
Nitric Oxide Synthase(NOS) Isoforms............................................. 16
Substrate Availability ................................... ................ ...................... 17
C alciu m -C alm o du lin ................................................................................................. 17
P hy sical A activity ....................................................... 18
N O Signaling and Skeletal M uscle...................................................... 18
M mechanism s of A action ......................................... .............................. 18
A cu te E effects .............................................................................19
C h ron ic A d aptation s ............................... ......................... ............... ............... 2 0
Adenosine Monophosphate Activated Protein Kinase(AMPK) is a Key Metabolic
R egulator in Skeletal M u scle ........................................................................................ 2 1
A M PK Structure and A ctivation ............................................................................... 2 1
A M PK Signaling E effects ............................................................................. 22
Interaction Between NO and AMPK ......................................25
AMPK-Induced Phosphorylation of nNOS and eNOS ................................................25
NO Facilitates AMPK Activation in Cell Culture ................ ......................... ..25
Conclusions......................... .. ................................ .....................26

3 M E T H O D S .................................................................................................................2 9

E x p erim mental D design ..................................................................................................2 9
Anim als........................ .. ............ .....................29









A natom ical D issection ......... ............................................................................. ....... ...... 29
Experimental Protocol ..................................... ................... ..........30
Western Blotting ................................................................ ........... ........ 31
Statistical A analysis ................................................. 31

4 R E SU L T S .............. ... ................................................................34

E electrical Stim ulation ....................................................... 34
Phospho-/Total-AMPK Ratio ......................... ............................34
Total-AMPK, Phospho-AMPK, and Phospho-Acetyl Co-A Carboxylase(ACC) ..........34
5-aminoimidazole4-carboxamide-l-P-D-ribofuranoside(AICAR) .....................................35
Phospho-/T otal-A M P K R atio .............. .. ...... ............ ...........................................35
Total-AMPK, Phospho-AMPK, and Phospho-ACC.....................................................35

5 D IS C U S S IO N ........................................................................................................4 2

M ain F in din g s .................................................................................................................. 4 2
Electrical Stimulation and AICAR Treatment Induces AMPK Activation............................42
NOS Inhibition Decreases Electrical Stimulation- and AICAR-Induced AMPK
A ctivation .......... .........................................................43
ACC Phosphorylation.................... ..... ... .. ..... ..... ..............45
Lim stations and Future D directions .............. ................................................. ............... 45
C onclu sions.......... ..........................................................46

L IST O F R E FE R E N C E S ......... .. ............. ....................................... .....................................48

B IO G R A PH IC A L SK E T C H .............................................................................. .....................54









LIST OF TABLES


Table page

4-1 Quantification of phospho-AMPK, total AMPK and phospho-ACC/P actin levels for
the EDL and soleus for electrical stimulation experimental groups.................................36

4-2 Quantification of phospho-AMPK, total AMPK and phospho-ACC/P actin levels for
the EDL and soleus for the AICAR experimental groups. ..............................................37









LIST OF FIGURES


Figure page

2-1 Metabolic processes regulated by AMPK................... .. ........ ............... 27

2-2 Exercise-induced signaling cascades ....................................................................... 28

3-1 Experim ental design flow chart.32 ............................................... ............................ 32

3-2 Experim mental groups chart ........................................... .......................... ............... 33

4-1 EDL electrical stimulation phospho-AMPK/aAMPK ratio .............................................38

4-2 Soleus electrical stimulation phospho-AMPK/aAMPK ratio. .........................................39

4-3 EDL AICAR phospho/total AM PK ratio.. ............................................... ............... 40

4-4 Soleus AICAR phospho/total AM PK ratio. ............................................. ............... 41

5-1 Proposed model illustrating potential role of nitric oxide in the activation of AMPK. ......47









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

NITRIC OXIDE SYNTHASE ACTIVITY MEDIATES ACTIVATION OF AMP-
ASSOCIATED PROTEIN KINASE IN ISOLATED MOUSE SKELETAL MUSCLE

By

Dana Leigh Brown

August 2008

Chair: David Criswell
Major: Applied Physiology and Kinesiology

Nitric oxide (NO) production and signaling has an important role in exercise-induced

metabolic and biochemical adaptations. Current research indicates that low levels of NO produced

enzymatically by the nitric oxide synthases (NOS), regulates 5'-AMP-activated protein kinase

(AMPK) in cell culture. The physiological significance of this regulatory mechanism in adult

skeletal muscle is unknown. Goal: This research was designed to determine if NOS activity is

necessary for induction of AMPK phosphorylation in skeletal muscles isolated from adult mice.

Hypotheses: Incubation of isolated extensor digitorum longus (EDL) and soleus muscles with the

non-isoform specific NOS inhibitor, L-NMMA, will prevent activating phosphorylation of AMPK,

and phosphorylation of the AMPK substrate, acetyl CoA carboxylase (ACC). Approach: EDL and

soleus muscles were surgically isolated bilaterally from young adult mice and incubated in micro

tissue baths. AMPK was activated by 10 min of contractile activity or a 20-min incubation with the

AMP analog, AICAR. The EDL and soleus muscle from the contralateral limb was treated

identically, except for the addition of L-NMMA to the incubation media throughout the experiment.

Results: The main findings of this study are: 1) electrical stimulation or AICAR incubation

increases the phospho-AMPK/total AMPK ratio; 2) NOS inhibition prevents this increase; 3) AMPK

activation by electrical stimulation and AICAR treatment is greater in the fast twitch EDL muscle









than the slow twitch soleus muscle; and 4) L-NMMA treatment tends to increase AMPK

phosphorylation status, independent of electrical stimulation or AICAR effects. Significance:

AMPK is an important regulator of skeletal muscle metabolism and the acute and chronic

adaptations to exercise training. Activation of AMPK, pharmacologically or by exercise, increases

oxidative capacity and improves insulin sensitivity leading to a metabolically active phenotype in

skeletal muscle that effectively counters the metabolic syndrome. Understanding how AMPK is

regulated in adult muscle fibers and the potential role of nitric oxide in mediating AMPK activation

is important for development of more potent and efficient treatments for metabolic disorders.









CHAPTER 1
INTRODUCTION

The metabolic syndrome, which manifests itself in pathophysiological conditions such as

insulin resistance, hypertension and dyslipidemia, is increasing at astounding rates throughout

the United States and developing countries due in large part to physical inactivity. These

conditions are coupled with increased morbidity and mortality rates and place an excessive

burden on healthcare systems. Physical activity has long been hailed as a necessary component

for a healthy lifestyle. Skeletal muscle is an exceptionally plastic tissue with a remarkable

capacity to adapt to external stresses by making fine metabolic adjustments. Contracting skeletal

muscle acutely increases insulin sensitivity in both healthy individuals and in people with Type 2

diabetes, and regular physical exercise is a cornerstone in the treatment of the disease. Exercise

training alters the biochemical signaling cascades that regulate the metabolic properties of

skeletal muscle. At present, the exact molecular mechanisms for these adaptations are poorly

understood.

Background

During exercise skeletal muscle is placed under enormous stress in order to maintain

energy levels and contractile function. 5'-AMP-activated protein kinase (AMPK) is a cellular

regulator that responds to changes in the AMP-to-ATP ratio. AMPK is activated allosterically

by AMP binding to the enzyme, and by phosphorylation of the catalytic a subunit by upstream

AMPK kinases (AMPKK). AMPK has an important role in numerous biochemical pathways

and is responsible for regulating energy metabolism processes. Chronic AMPK activation is an

important signal for upregulation of glucose transporter molecules. Recent research has

suggested that AMPK activation may be influenced by nitric oxide (NO), produced









enzymatically by the nitric oxide synthases (NOS). At present, it is not known if NO is

necessary for AMPK activation in adult skeletal muscle.

Problem Statement

The benefits of exercise in metabolic disease prevention and therapy have been well-

documented in literature but the specific mechanistic signaling pathways that exert the protective

effects remain elusive. The aim of this study was to determine whether the presence of NO is

required for AMPK activation and whether AMPK activation differs between skeletal muscle

fiber types.

Variables in Study

Independent variables: We will simulate contractile load to isolated extensor digitorum

longus (EDL) (primarily fast twitch muscle) and soleus muscles (primarily slow twitch muscle)

by means of electrical stimulation in vitro, and by contraction-independent stimulation of AMPK

via 5-aminoimidazole-4-carboxamide-1-P-D-ribonucleoside (AICAR) administration in vitro.

NOS activity will be inhibited via N(G)-monomethyl-L-arginine (L-NMMA) administration to

determine the necessity of NO production for AMPK activation.

Dependent variables: We will measure phosphorylation of metabolic proteins AMPK

and acetyl CoA carboxylase (ACC).


Control variables: Sex and age were controlled as all animals used within the study were

young female adult mice (-4 months old) from the ICR strain. Animals were brought to the lab

at least 12 hours prior to surgery to allow acclimatization to the new environment. All surgeries

were conducted at the same time of day. Isolated muscles were set to their optimal length using

a micromanipulator and individual isometric twitches.









Extraneous variables: Individual differences in daily activities such as ambulation and

feeding were not controlled.

Hypotheses

We hypothesize that:

1. Ten minutes of electrical stimulation, in vitro, will induce phosphorylation of AMPK and
ACC in the isolated EDL and soleus muscles.

2. Twenty minutes of AICAR treatment, in vitro, will also induce phosphorylation of
AMPK and ACC in these muscles.

3. Addition of L-NMMA to the incubation media will significantly decrease NO
biosynthesis in muscles at rest and during electrical stimulation, decreasing AMPK
phosphorylation.

4. AMPK phosphorylation will be higher in fast twitch muscles than slow twitch muscles.

Definition of Terms

5'-AMP-activated protein kinase (AMPK). A heterotrimeric protein kinase that

regulates cellular energy homeostasis and is largely responsible for exercise-induced skeletal

muscle adaptations to endurance training.

5-aminoimidazole-4-carboxamide-l-P-D-ribonucleoside (AICAR). AMP mimetic drug

capable of activating AMPK.

Acetyl CoA carboxylase (ACC). Rate-limiting enzyme involved in fatty acid synthesis,

and a known target of AMPK activity.

AMP-activated protein kinase kinase (AMPKK). A collective group of enzymes

responsible for phosphorylating AMPK.

Electrical stimulation. A technique that uses platinum electrodes to generate a field

current in an electrolyte buffer-filled chamber for the purpose of directly activating an isolated

mouse skeletal muscle, suspended in the buffer solution.









Optimal length (Lo). The length at which maximum tetanic tension is generated by

skeletal muscle.

N(G)-monomethyl-L-arginine (L-NMMA). A nonspecific competitive inhibitor of

NOS activity.

Nitric oxide (NO). A short-lived endogenous mediator that acts as a signaling molecule

and plays a role in a variety of biological reactions in virtually all mammalian tissues under

certain physiological or pathophysiological conditions.

Nitric oxide synthase (NOS). A family of isozymes responsible for NO production.

Three forms have been identified in mammalian skeletal muscle: neuronal NOS (nNOS),

endothelial NOS (eNOS) and inducible NOS (iNOS).

Limitations/Delimitations/Assumptions

Limitations: Due to the invasive nature of this research, the use of humans as

experimental subjects was not possible. Instead, a mouse model was chosen due to genetic

similarity to provide results for the researcher that would mimic those seen in humans.

The in vitro experimental design allows isolation of intrinsic muscle responses. It is

recognized that extrinsic responses to contractile activity, which are not considered in our design,

likely contribute to skeletal muscle responses in vivo.

Delimitations: The study is restricted to female mice.

Assumptions:

1. L-NMMA, at the dose used, specifically inhibits NOS activity in skeletal muscle, and has
negligible non-specific effects.

2. AMPK activity correlates well with ACC activity.









Significance of the Study

Whilst genetic predisposition for the metabolic syndrome cannot be modified by

behavior, obesity, the largest risk factor, is largely preventable. Failure to curb increased obesity

lies in large part with our lack of understanding of metabolic government in healthy and disease

states. To maintain metabolic function, intracellular ATP concentrations must remain within

acceptable limits. AMPK preserves ATP levels by managing energy utilization for metabolic

pathways based on necessity. AMPK can also regulate exercise responsive genes leading to

long-term metabolic stability. Therefore, it is clear that a dysfunction in AMPK signaling can

induce rapid and contrary responses that directly affect metabolic function. The AMPK pathway

is of particular interest within the clinical community because of its potential in treating and

preventing the metabolic syndrome. Understanding the mechanism by which muscle contractions

induce AMPK activity will aid in development of new strategies to prevent or treat metabolic

disorders.









CHAPTER 2
LITERATURE REVIEW

Nitric oxide (NO) is a short-lived endogenous mediator that acts as a signaling molecule

and plays a role in a variety of biological reactions in virtually all mammals under certain

physiological or pathophysiological conditions. Since its declaration as a cardiovascular

homeostatic mediator, NO has emerged as a fundamental signaling device regulating

intracellular processes and cell-cell interactions. NO production is primarily dependent on a

group of heme-containing proteins known as nitric oxide synthases (NOS). NO regulates

processes in skeletal muscle that include force production, autoregulation of blood flow,

mitochondrial biogenesis, mitochondrial respiration and glucose transport (6,15, 25, 29, 45, 57).

AMPK is a key metabolic enzyme that has emerged as a metabolic master switch. AMPK

activity is involved in many of the training adaptations acquired with chronic exercise. Recent

studies provide evidence that AMPK activation is dependent upon NO (8, 34). At present, a

complete understanding and grasp of NO participation in homeostatic functions and

pathophysiological conditions has not yet been reached. This chapter will review the recent

literature concerning nitric oxide and AMPK effects in skeletal muscle and potential interaction

between these two important signaling molecules.

Enzymatic Production of Nitric Oxide(NO)

Nitric Oxide Synthase(NOS) Isoforms

Three isoforms of NOS produce NO from the amino acid, L-arginine (L-arg), and require

oxygen and NADPH as substrates with citrulline as a by-product. Nitric oxide is synthesized in

normal skeletal muscle by neuronal NOS (nNOS) and endothelial NOS (eNOS). NOS is

constitutively expressed in rodent skeletal muscle (56, 66). Localization of the nNOS isoform

varies greatly among cell types and is directly associated with the dystrophin complex in skeletal









muscle. It is located in close proximity of the sarcolemma, and primarily in fast-twitch fibers of

rodents. nNOS expression in humans appears to be similar, except for a more widespread

distribution across fiber types (63). eNOS is uniformly distributed in muscle fibers and localized

in the mitochondria and also in vessel walls (63). nNOS and eNOS are highly regulated by Ca2

and calmodulin. The third isoform, inducible NOS (iNOS), is not produced at significant levels

in skeletal muscle. Its expression is regulated by transcription in response to cytokines and

other inflammatory agents and is largely Ca2+ independent.

Substrate Availability

NOS activity varies between muscles. Generally, higher activity of NOS is measured in

fast twitch fibers (56). The availability of cofactors and co-substrates as well as subcellular

localization can influence the identity of the enzymatic product (57). It has been hypothesized

that cellular supply of L-arginine is a rate-limiting step for NOS activity. Ogonowski and

colleagues suggest that L-arginine transport by y+ transporters may be the limiting step (41).

McConell et al (37) performed a study that infused L-arginine into humans while cycling at 72%

VO2 Max. They reported improved glucose disposal with arginine infusion during an exercise

protocol. This group theorized that L-arginine supplementation upregulated NO production

during skeletal muscle contraction and increased GLUT4 translocation.

Calcium-Calmodulin

Calmodulin binding is an essential requirement for NO production by all NOS isoforms.

iNOS is tightly bound to calmodulin in an almost irreversible fashion (21, 56), therefore, the

activity of this isoform is essentially independent of calcium. nNOS and eNOS, however, bind

to calcium-calmodulin and, therefore, rely on a calcium signal for upregulation of enzymatic

activity. Upon release of calcium from the sarcoplasmic reticulum during contraction, calcium

concentration increases and facilitates the interaction of nNOS and eNOS with calmodulin.









Jagnandan et al. (21) illustrated the calcium-dependence of eNOS by limiting access to calcium

and measuring NO production. Calcium blockade essentially eliminated NO production by

eNOS but had no significant effect on NO production by iNOS.

Physical Activity

Increasing research findings show that NO is an important metabolic regulator during

exercise. Furthermore, physiological adaptations such as improved functional ability and

cardioprotection result from chronic physical activity. At rest skeletal muscle produces

relatively small amounts of NO. However, there are significant changes in NO production in

response to exercise and physical activity. Exercise upregulates NOS activity and nNOS gene

expression (3, 29).

NO Signaling and Skeletal Muscle

Mechanisms of Action

Because NO is a short-lived free radical with a half life of 2 to 5 seconds (13), regulation

of signaling occurs largely at the level of NO biosynthesis. It can act locally on the cell that it is

produced within or it can permeate cell membranes of adjacent cells. Research has shown that

NO is involved in such diverse activities as vasodilation, metabolic regulation, immune function,

cellular signaling, contractile regulation and glucose homeostasis, to name a few (3, 45, 55).

There are a number of mechanisms whereby NO effects are mediated.

sGC and cGMP. Activation of soluble guanylate cyclase (sGC), a heme-containing

heterodimer, is a primary mechanism mediated by NO. NO binds to the heme increasing the

catalytic activity of cGC several hundredfold increasing the levels of the second messenger

cyclic guanylyl monophosphate (cGMP) (54). The resulting increase in cGMP concentration

serves to signal a multitude of complex physiological operations. cGMP can directly activate its

downstream effectors including Protein Kinase G (PKG), Cyclic Nucleotide Gated Channels









(CNG) and Cyclic nucleotide phosphodiesterases, all of which govern the operation of many

proteins involved in physiological functioning (35).

Protein nitrosylation. Following production, NO translocates to nearby cells and interacts

with the interior of the cell at metal sites within proteins. Arguably the most influential

mechanism of action is nitrosylation, the reaction of NO with cysteine residues in proteins or by

interactions with heme or non-heme copper and iron. A continuum between nitrosative and

oxidative stress must be maintained to reduce hazardous levels of stress which has been

connected to muscle fatigue and cell injury (57).

Acute Effects

Glucose transport. Exercise acutely increases glucose transport in both healthy

individuals and those with Type 2 diabetes. NO signaling markedly increases glucose transport

in isolated skeletal muscle fibers (5). Translocation of intracellular GLUT4, the primary glucose

transporter isoform found in skeletal muscle, to the plasma membrane is the underlying

mechanism by which exercise causes increased glucose transport (23). Recent studies have

shown a link between NO and AMPK responsible for exercise-induced glucose clearance.

Contractile function. At present, complete functional significance of the NOS isoforms

and skeletal muscle remains elusive. However, NO does play a pivotal role in maintenance of

skeletal muscle contractile ability. This regulation appears to be mediated through cGMP-

dependent and -independent mechanisms. Skeletal muscle fibers exposed to NO have shown

reduced actin-myosin cross-bridge cycling in vitro (18, 26). Lau et al (32) provided evidence of

the linkage between NO and cGMP. When electrically stimulated, the cGMP content of wild

type mouse extensor digitorum longus muscle cGMP content increased approximately 250%.

It has recently been discovered that colocalization of cGMP and nNOS at the sarcolemma

inhibits excitation-contraction coupling in skeletal muscle by impaired Ca2+ activation of thin









filaments (26). NO also interferes with contraction by inhibiting creatine kinase, and the

sarcoplasmic reticulum Ca2+-ATPase in fast-twitch and slow-twitch muscle fibers.

Mitochondrial respiration. NOS activity has been associated with inhibition of

mitochondrial respiration. Identified respiration targets of NO include cytochrome-c oxidase,

creatine kinase and Ca2+-ATPase in skeletal muscle (57). NO binds to cytochrome-c oxidase,

inhibiting comples IV of the electron transport chain and mitochondrial oxygen consumption.

This disruption in cellular respiration can result in alterations of calcium flux. Disruptions in

mitochondrial function can drastically alter energy levels. Redox balance is necessary for

homeostasis and optimal functioning of numerous physiological interactions.

Satellite cell activation Muscular injury due to exercise, mechanical stretch or blunt force

mobilizes satellite cells from quiescence to serve as antecedents for new muscle formation. At

present NO and hepatocyte growth factor (HGF) are the only known activators of satellite cells.

Satellite cell activation is defined as mobilization and entrance into the G1 interphase of the cell

cycle (1). Studies have shown that release of HGF from the extracellular matrix is NO-

dependent and inhibition of NOS activity reduces HGF release and satellite cell activation (1).

Chronic Adaptations

Mitochondrial biogenesis. In arterioles, NO vasodilates smooth muscle and increases

blood flow and 02 delivery to the tissues. NO contributes to the regulation of mitochondrial

respiration by inhibiting cytochrome-c oxidase at complex III of the electron transport chain.

This leads to decreased levels of cellular ATP and increase in levels of ADP, AMP, GDP and Pi

(13).

Adult skeletal muscle experiences a transformation from fast to slow fiber type with

exercise training. eNOS and neuronal nNOS isoforms may be differentially involved in the

regulation of mitochondrial biogenesis in skeletal muscle (66). Evidence to support this was









presented by Wadley et al. NO donor experiments in rodents lead to increased expression of

peroxisome proliferator-activated receptor coactivator 1 (PGC-1), nuclear respiratory factor 1

(NRF-1), and mitochondrial transcription factor A (mtTFA), all of which are markers of

mitochondrial biogenesis (66).

GLUT 4 expression. Regular aerobic exercise is associated with biochemical changes to

numerous metabolic genes. It is well established that chronic exercise is a cornerstone in the

treatment and prevention of Type 2 diabetes due in large part to increased insulin sensitivity.

Insulin stimulation leads to recruitment of the GLUT 4 transporter to the surface of the cell to

transport glucose intracellularly (24). AMPK has recently been linked to upregulation of

GLUT4 transporter gene expression.

Adenosine Monophosphate Activated Protein Kinase(AMPK) is a Key Metabolic Regulator
in Skeletal Muscle

AMPK Structure and Activation

The AMPK molecule is a heterotrimeric protein kinase composed of a catalytic a subunit

and regulatory P and X subunits (61). The a and 0 subunits each have two genes and the X

subunit has three, yielding at least 12 possible heterotrimeric sequences (17). The presence of

AMPK in primitive organisms suggests that this molecule has served an important genetic role

throughout evolution (12).

AMPK is an exercise-responsive gene that acts to preserve cellular energy levels (22, 40,

61). AMPK is activated during times of cellular energy stress including hypoxia, glucose

depletion and exercise. It is well established that exercise is characterized by heightened energy

turnover. AMPK is activated during times of elevated metabolism and ATP consumption. Once

activated, AMPK turns on catabolic processes that generate ATP while concurrently turning off

anabolic processes such as cellular growth and proliferation. Two independent articles were









published in 1973 describing protein fragments that had the capacity to inactivate acetyl-CoA

carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, 2 enzymes

necessary for lipid synthesis. It was later discovered that the protein fragments were composed

of protein kinases and that ACC kinase and HMG-CoA reductase kinase were activated by 5'-

AMP. In 1987 Carling et al realized that the same kinase was responsible for activation of both

functions (7). This molecule was named AMPK.

AMPK is activated by AMP in one of two ways: phosphorylation by upstream kinases or

allosteric activation. Phosphorylation of AMPK by upstream kinases occurs within the catalytic

a subunit at Thr172 (53). LKB1 and Ca2+/calmodulin-dependent protein kinase kinase 1

(CaMKKP) have been identified as well-suited upstream kinases capable of activating AMPK

(53, 61). Activation of AMPK during ex vivo electrical stimulation is dependent upon

modification of the a-Thr172 subunit by AMP phosphorylation making it a desirable substrate for

LKB 1 (52). Direct activation of AMPK occurs allosterically by AMP binding to the X subunit.

This renders AMPK a more suitable substrate for upstream kinases and a less suitable substrate

for phosphatases which can deactivate AMPK (61).

The level of metabolic stress placed on the body affects the level at which AMPK is

activated (17). Interestingly, AMPK is activated during strength training in endurance athletes

and during endurance training in strength athletes. It is reasoned, therefore, that AMPK is

upregulated during training at intensity levels greater than the individual is adapted to.

AMPK Signaling Effects

AMPK was first discovered in the liver and evidence suggested that the enzyme was

likely present in other tissues of the body. Protein expression analysis found mRNA levels of

AMPK expression is greatest within skeletal muscle (64). Over the past years extensive research

has been conducted with inconclusive results to decipher which particular subunits are expressed









in animal and human muscle. Within skeletal muscle, AMPK is known to exhibit regulatory

effects on glucose transport, fatty acid oxidation and mitochondrial biogenesis as exhibited in

Figure 2-1.

Glucose transport. Many of the downstream effects of AMPK homestatic-preservation

were first discovered using the AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-P-D-

ribofuranoside (AICAR) (17). It was initially found by Merrill et al in 1997 that muscle

incubated with AICAR exhibited increased glucose transport and AMPK activity (38). Ex vivo

experiments with AICAR incubation in transgenic animals where AMPK signal is greatly

decreased or nonexistent completely diminished glucose transport.

The mechanism for exercise-induced skeletal muscle glucose transporter (GLUT4)

upregulation appears to be influenced by NO. The interaction between AMPK and NOS is not

certain but is very intriguing. Acutely, AMPK influences glucose transport. Long term

adaptations show AMPK to be a mediator of exercise-induced glucose transport by increasing

GLUT4 concentrations at the cell surface (38). Balon and colleagues demonstrated that

incubation of skeletal muscle with a NOS inhibitor decreased glucose transport (4). Recently,

Lira et al reported that NO and cGMP were active partners in inducing GLUT4 expression in

skeletal muscle. They also proposed the novel idea that NOS activity is required upstream and

downstream of AMPK to induce GLUT4 expression (20).

Fatty acid oxidation. Accumulation of fatty acids within the endothelium is a major

contributor to atherosclerosis and, therefore, the metabolic syndrome. Fatty acid clearance is

necessary for normal endothelial function and accumulation can cause excess production of

damaging radicals. It has been shown that fatty acid oxidation accounts for approximately 40%

of ATP production by endothelial cells (10). Both resting and active skeletal muscle metabolize









fatty acids to produce energy. Several studies have indicated that AMPK activation can augment

fatty acid oxidation. AMPK is responsible for inhibitory phosphorylation of acetyl-CoA

carboxylase (ACC). ACC is responsible for fatty acid synthesis and when inactivated reduces

accumulation of triglycerides by stimulating mitochondrial uptake of fatty acids. AMPK activity

is markedly reduced in obese animals and activation of AMPK reduces intracellular

accumulation of triglyceride levels (40).

Mitochondrial biogenesis. Chronic endurance exercise is associated with increased

mitochondrial oxidative capacity. One of the mechanisms responsible for mitochondrial enzyme

gene transcription involves upregulation of AMPK during times of skeletal muscle contraction.

AMPK promotes mitochondrial biogenesis by increasing PGC-la and NRF expression. In an

experiment by Winder et al. (69) 28 days of chronic AICAR treatment in rats resulted in

increased AMPK activation as well as levels of citrate synthase, succinate dehydrogenase, malate

dehydrogenase and cytochrome c, all mitochondrial enzymes. AMPK clearly effects

mitochondrial regulation during times of energy deprivation by promoting expression of

mitochondrial enzymes and biogenesis markers.

Inhibition of muscle growth. Mammals have two distinct signaling cascades responsible

for exercise-induced adaptations: one which is responsible for increases in cardiovascular

endurance and another which signals protein synthesis and muscular growth. These two

pathways are exhibited in Figure 2.2. Increases in cardiovascular endurance are primarily

signaled through AMPK. Muscle hypertrophic response to exercise is signaled through the

mammalian target of rapamycin (mTOR) pathway due to its ability to stimulate protein

translation and synthesis. Protein synthesis begins with insulin stimulating the PI 3-kinase/Akt

pathway leading to increased mTOR activity (11). Inhibition of mTOR immediately before









muscle overload prevents protein synthesis, (31) suggesting that this pathway is necessary for

normal muscle hypertrophy. AMPK is a negative regulator of the mTOR pathway. The a2 and

X3 subunits of AMPK have been identified as the complexes responsible for inhibiting mTOR

activity (11). These two subunits have the ability to phosphorylate and thus deactivate the

mTOR signaling cascade.

Interaction Between NO and AMPK

AMPK-Induced Phosphorylation of nNOS and eNOS

The interaction between NOS and AMPK is not fully understood. It is known that

AMPK has the ability to phosphorylate and activate eNOS and nNOS isoforms (8, 9). In

addition, the AMP mimetic drug 5-aminoimidazole-4carboxamide-1-P-D-ribofuranoside

(AICAR) upregulates NOS activity in H-2Kb muscle cells (14). Altogether, the activation of

AMPK in response to exercise or hypoxia is a critical mechanism required for normal metabolic

regulation as well as nNOS and eNOS activity.

NO Facilitates AMPK Activation in Cell Culture

Current research indicates that NO produced enzymatically by the nitric oxide synthases

regulates AMPK in cell culture. Lira et al (34) found that NOS activity was necessary for

AMPK-induced upregulation of the GLUT4 transporter gene expression. L6 myotubes were

treated with AICAR or AICAR and the non-specific NOS inhibitor L-NAME. Cotreatment with

AICAR and L-NAME ablated 70% of the AICAR effect on GLUT4 mRNA, suggesting that NO

is necessary downstream of AMPK for normal function. They also suggest that NO is necessary

upstream of AMPK. Low concentrations of SNAP, an NO donor, at 1 and 10 pM increased

GLUT4 mRNA expression and AMPK phosphorylation; in contrast, high concentrations of

SNAP at 100 and 300 [iM did not affect AMPK or GLUT4 mRNA expression. Furthermore,

they propose the idea of a positive feedback system between NOS and AMPK enzyme activity.









Conclusions

The metabolic syndrome, characterized by the clustering of cardiovascular risk factors

that leads to Type 2 diabetes, is increasing at astounding rates throughout the United States and

developing countries due in large part to physical inactivity. NO production and signaling has an

important role in exercise-induced metabolic and biochemical adaptations. AMPK is an

important regulator of skeletal muscle metabolism and the acute and chronic adaptations to

exercise training. Activation of AMPK, pharmacologically or by exercise, increases oxidative

capacity and improves insulin sensitivity leading to a metabolically active phenotype in skeletal

muscle that effectively counters the metabolic syndrome. Understanding how AMPK is

regulated in adult muscle fibers and the potential role of nitric oxide in mediating AMPK

activation is important for development of more potent and efficient treatments for metabolic

disorders.












Mitochondrial
Biogenesis


Fatty Acid
Oxidation


Glucose
Transport


AMPK


Fatty Acid
Synthesis


Protein
Synthesis


Figure 2-1. Metabolic processes regulated by AMPK. Arrows indicate activation of pathways,
whereas lines with a bar at the end indicate inhibition of pathways.










Cardiovascular/
Endurance
Training

4I


Mitochondrial
Biogenesis



Increased
cardiovascular
capacity


Protein Synthesis





Hypertrophy


Figure 2-2. Exercise-induced signaling cascades. Arrows indicate the signaling events for
adaptations to occur whereas a line with a bar at the end indicates processes
inhibited.









CHAPTER 3
METHODS

Experimental Design

Extensor digitorum longus (EDL) and soleus muscles were dissected from young mice for

in vitro bath manipulation. One EDL and one soleus muscle were subjected to 10 minutes of

electrical stimulation or exposed to 5-aminoimidazole-4-carboxamide- 1-P-D ribonucleoside

(AICAR) for 20 minutes. Contralateral EDL and soleus muscles of the mice were treated with

N(G)-monomethyl-L-arginine (L-NMMA) to inhibit NOS activity. The experimental design

protocol is depicted in Figure 3-1.

Animals

The University of Florida Institutional Animal Care and Use Committee approved the

protocol of this study. The subjects were young (-2 months old) female ICR mice purchased

from Harlan Sprague Dawley, Inc. (Indianapolis, IN). All animals were housed in the J. Hillis

Miller Animal Science Center and fed the same diet (chow and water ad libitum) throughout the

experiment. They were kept on a 12 hr light:dark photoperiod. Animals were brought to the lab

approximately 12 hr prior to surgery to allow acclimatization to their new environment. Animals

were divided into 8 treatment groups as illustrated in Figure 3-2.

Anatomical Dissection

Surgical removal of the extensor digitorum longus (EDL) and soleus muscles was

necessary for ex vivo manipulation. The mice were anaesthetized with 2-5% isoflurane with

oxygen as the carrier gas. Once anaesthetized, both hindlimbs were skinned and the Achilles

tendon of the right leg cut. Because of its oxidative nature, the soleus was removed first to avoid

hypoxia and tissue death. An incision was made along the fascia of the gastrocnemius muscle

beginning at the distal end up to the posterior aspect of the fibula. The gastrocnemius/soleus









muscle complex was reflected to expose the deep soleus muscle and the proximal tendon. The

proximal tendon was cut and the soleus carefully maneuvered away from the gastrocnemius.

The distal tendon was then cut and the muscle immediately placed in the oxygenated buffer. The

same surgical procedure was repeated on the left leg.

The distal ends of the tibialis anterior (TA) and EDL tendons were located and cut from the

phalanges. An incision was made along the fascia of the TA up to the proximal end of the tendons at

the fibula. The TA/EDL complex was removed as a whole and the EDL carefully maneuvered away

from the TA and immediately transferred to the oxygenated buffer.

Experimental Protocol

Soleus and EDL muscles were surgically removed from fed anaesthetized mice and

immediately transferred to a bath containing Krebs-Henseleit (KH) buffer supplemented with 25

mM sodium bicarbonate, 5 mM Hepes, 2.54 mM calcium chloride and 100 uM L-arginine (pH

7.15) continuously bubbled with 95% 02, 5% CO2. The tendons of each muscle were then

clamped in micro tissue clamps and suspended separately between platinum field electrodes in

water-jacketed micro tissue baths containing oxygenated buffer solution and maintained at 290C.

After a 30 min equilibration period, each muscle was set to its optimal length (Lo) by repeated

isometric twitches while gradually increasing muscle length. After determining Lo the buffer

was washed from the baths and reloaded and equilibrated for an additional 10 min. One EDL

and one soleus muscle of each animal was subjected to electrical stimulation or 5-

aminoimidazole-4-carboxamide-l-P-D-ribonucleoside (AICAR) treatment. Following the

equilibration period, contraction was induced by electrical stimulation delivered at 10 Hz, 13 V

for 10 min. For AICAR treatment, 25 mM AICAR solution was added to the baths with buffer

for an incubation of 20 min. The contralateral EDL and soleus muscles served as non-contracted

or non-AICAR treated controls. Muscles from half of the mice were treated with ImM N(G)-









monomethyl-L-arginine (L-NMMA) to inhibit NOS activity during electrical stimulation or

AICAR treatment. Immediately following the experimental treatment, muscles were blotted and

frozen for subsequent protein isolation and quantification.

Western Blotting

Protein levels of total AMPK, phosphorylated AMPK, and phosphorylated acetyl CoA

carboxylase (ACC) were determined by standard immunoblotting technique. All muscles were

homogenized using glass-on-glass technique in 2X homogenizing buffer: 20 mM Tris (pH7.5),

150 mM NaC1, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1

mM P-glycerol phosphate, 1 mM sodium orthovanadate, 1 tlg/ml leupeptin, 1 mM PMSF, and 10

tlg/ml aprotinin containing 1% vol/vol phosphatase inhibitor (p-5726) from Sigma. Protein

concentrations were measured using the DC Protein Assay Kit (Bio-Rad Laboratories,

Richmond, CA). Aliquots of muscle homogenates (16-78tlg) were run in SDS-PAGE gels for

phospho-ACC, phospho- and total uAMPK blots. The primary antibodies used are as follows:

rabbit anti-uAMPK and anti-phospho-uAMPK (1:1000 dilution; Cell Signaling), rabbit anti-

phosphoACC (1:500 dilution; Upstate). Ponceau stain and P-actin blots were used to control for

loading. Reactions were developed by using the enhanced chemiluminescence detection reagents

(ECL Plus; Amersham Biosciences, Buckinghamshire, UK), and protein levels were determined

by densitometry (Kodak ID Image Analysis Software version 3.6).

Statistical Analysis

Treatment main effects were analyzed by 2-way ANOVA (E-stim or AICAR vs. L-

NMMA) with repeated measure on E-stim or AICAR. Individual group differences were

assessed by paired t-tests with Bonferroni's correction for multiple tests applied.












Electrical Stimulation Protocol


30 minute 10 minute 10 minute
equilibration incubation contraction


AICAR Incubation Protocol


30 minute 10 minute 20 minute
equilibration incubation incubation


SHang muscles Find Lo; Wash baths, Freeze
Hang muscles Find Lo; Electrical Freeze
in bath wash baths, add buffer, muscle
in bath wash baths stimulation muscle add buffer L-NMMA
add buffer L-NMMA
add buffer
add/obur Land/or L- and/or
an NMMA AICAR
NMMA


Figure 3-1. Experimental design flowchart. Electrical stimulation muscles were subjected to 10 minutes of stimulation; half of the
muscles were treated with N(G)-monomethyl-L-arginine (L-NMMA) to inhibit NOS activity. 5-aminoimidazole-4-
carboxamide-1-P-D ribonucleoside (AICAR) muscles were subjected to 20 minutes of AICAR treatment; half of the
muscles were co-treated with L-NMMA to inhibit NOS activity.
















Experiment #1
Electrical Stimulation
L-NMMA

+

EDL EDL
(n=5) (n=5)
Soleus Soleus
E-Stim (n=5) (n=5)
EDL /EDL
+ (n=5) (n=5)
Soleus Soleus
(n=5)/ (n=5)


Experiment #2
AICAR Incubation
L-NMMA

+

EDL EDL
(n=7) (n=7)
Soleus Soleus
AICAR (n=6) (n=6)
EDL EDL
+ (n=7) (n=7)
Soleus Soleus
(n=6) (n=6)


Figure 3-2. Experimental groups chart. Animals were divided based on electrical stimulation,
AICAR treatment or L-NMMA treatment.









CHAPTER 4
RESULTS

Electrical Stimulation

Phospho-/Total-AMPK Ratio

EDL. To examine the effects of electrical stimulation and NOS inhibition on AMPK

activation, a 2-way repeated measures ANOVA was conducted. The main effect of electrical

stimulation on the ratio of phospho-to-total AMPK was near significant (p = .082) in the EDL.

Further, we observed a trend of L-NMMA increasing the phospho-AMPK/total AMPK ratio at

rest in the EDL though there was not a significant main effect of NOS inhibition. Comparison of

individual groups revealed that electrical stimulation caused a significant, 4 fold, increase in

the phospho-AMPK/total AMPK ratio, while NOS inhibition by L-NMMA abrogated the

electrical stimulation effect in the muscle (Figure 4-1).

Soleus. Analysis of the soleus revealed a significant main effect of electrical stimulation

on phospho-AMPK/total AMPK ratio; however, a minimal increase in AMPK activation with

electrical stimulation alone was observed (Figure 4-2). There was not a statistical main effect of

L-NMMA on AMPK activation (p = .065). Interestingly, individual group comparison revealed

that L-NMMA treatment during electrical stimulation increased AMPK activation above L-

NMMA treatment alone.

Total-AMPK, Phospho-AMPK, and Phospho-Acetyl Co-A Carboxylase(ACC)

Quantification of phospho-AMPK, total AMPK and phospho-ACC data (means + SEM)

normalized to the control mean for the EDL and soleus is provided in Table 4-1.

Electrical stimulation and NOS inhibition of the EDL did not produce significant main

effects on these proteins, although phospho-AMPK was elevated in the electrical stimulation, L-

NMMA, and electrical stimulation plus L-NMMA groups, compared to control levels.









In the soleus, electrical stimulation and NOS inhibition did not have a significant effect

on total AMPK levels. In contrast, there was a significant effect of electrical stimulation and

NOS inhibition on phospho-AMPK. Although no changes in phospho-ACC reached statistical

significance in the electrical stimulation study, the electrical stimulation main effect on phospho-

ACC in the soleus was nearing significance (p = .051).

5-aminoimidazole4-carboxamide- 1--D-ribofuranoside(AICAR)

Phospho-/Total-AMPK Ratio


EDL. To examine the effects of AICAR treatment and NOS inhibition on AMPK

activation a 2-way repeated measures ANOVA was conducted. There was a significant main

effect of AICAR treatment on phospho-AMPK/total-AMPK ratio (p = .013) in the EDL. There

was not a significant main effect of L-NMMA on AMPK activation. AICAR treatment caused a

significant, 2 fold increase in the phospho-AMPK/total AMPK ratio. However, NOS inhibition

by L-NMMA incubation blunted the AICAR effect (Figure 4-3).

Soleus. Analysis of the soleus revealed no significant main effects of AICAR treatment

or NOS inhibition on AMPK phosphorylation status. However, it is important to note that the

AICAR main effect was nearing significance (p = .107). (Figure 4-4).

Total-AMPK, Phospho-AMPK, and Phospho-ACC

Quantification of phospho-AMPK, total AMPK and phospho-ACC data (means+SEM)

normalized to the control mean is provided in Table 4-2 for the EDL and soleus. AICAR or L-

NMMA treatment did not have a significant effect on these proteins in the EDL or soleus.









Table 4-1. Quantification of phospho-AMPK, total AMPK and phospho-ACC/P actin levels for
the EDL and soleus for electrical stimulation experimental groups. Data were
normalized to the mean of the control group. Values represent mean SEM.
EDL=extensor digitorum longus, E-stim=10min of in vitro electrical stimulation (see
text for details), L-NMMA=L-NG-monomethyl Arginine citrate. *Significantly
different from control.
Control E-stim L-NMMA E-stim + L-
NMMA
EDL
p-AMPK 1.000 5.877 2.566 3.169
.754 1.939* .754 1.939
t-AMPK 1.000 1.054 1.108 1.334
.201 .250 .201 .250
p-ACC 1.217 1.113 .841 + 1.534
.409 .44 .366 .393

Soleus
p-AMPK 1.000 + .899 1.328 2.162
.305 .192 .341 .215
t-AMPK 1.000 + .965 1.376 1.388
.295 .300 .330 .335
p-ACC 1.000 + .844 .659 2.322
.253 .370 .326 .478*









Table 4-2. Quantification of phospho-AMPK, total AMPK and phospho-ACC/P actin levels for
the EDL and soleus for the AICAR experimental groups. Data were normalized to the
mean of the control group. Values represent mean + SEM. AICAR = 5-
aminoimidazole-4-carboxamide-l-8 -D-ribofuranoside (1 mM).
Control AICAR L-NMMA AICAR + L-
NMMA
EDL
p-AMPK 1.009 + 1.289 + 1.668 + 1.673 +
.318 .269 .318 .269
t-AMPK 1.000 + .862 .935 .897
.105 .114 .105 .114
p-ACC 1.000 1.741 + 1.319+ 1.193 +
.273 .392 .273 .392

Soleus
p-AMPK 1.000 3.176 2.547 + 3.117
.827 1.193 .906 1.307
t-AMPK 1.000 1.804 1.588 1.482
.474 .517 .520 .567
p-ACC .965 2.826 2.076 3.661
.555 1.634 .555 1.634















phospho-AMPK


aW


SaM


I lControl
a E-Stim


Figure 4-1. EDL phospho-AMPK/aAMPK ratio for electrical stimulation experiment. (A)
Representative immunoblot from control, electrical stimulation, L-NMMA and L-
NMMA + electrical stimulation for phospho-AMPK and total(a) AMPK. (B)
Quantification of immunoblots for phospho-AMPK/total AMPK ratio. Values
represent mean SEM. L-NMMA = L-monomethyl Arginine citrate. E-stim =
electrical stimulation. *Significantly different from control.


UAMPK


E


>o
'a



Z
E
0
z














phospho-AMPK


- I


aAMPK


C*
E ""I Control
S2.0 E-stim

P 1.5

*E 1.0
E


3.0
Control L-NMMA

Figure 4-2. Soleus phospho-AMPK/aAMPK ratio for electrical stimulation experiment. (A)
Representative immunoblot from electrical stimulation, L-NMMA and L-NMMA +
electrical stimulation for phospho-AMPK and totalAMPK. (B) Quantification of
immunoblots for phospho-AMPK/total AMPK ratio. Values represent mean SEM.
*Significantly different from control.

















phospho-AMPK


aAMPK


E
S







E

Z
0.
U

z


m


a


- w a


T


Control


"I Control
MAICAR


L-NMMA


Figure 4-3. EDL phospho/total AMPK ratio for AICAR experiment. (A) Representative
immunoblot from control, AICAR, L-NMMA and L-NMMA + AICAR for phospho-
AMPK and total AMPK. (B) Quantification of immunoblots for phospho-
AMPK/total AMPK ratio. Values represent mean SEM. AICAR =5-
aminoimidazole-4-carboxamide- 1--D ribonucleoside (1 mM). *Significantly
different from control.















phospho-AMPK


- -


uAMPK S f 1n W 'I1SM


C

E


I&
>o


4=
E
0
z


o.o-


C lControl
T ~AICAR


Control


L-NMMA


Figure 4-4. Soleus phospho/total AMPK ratio for AICAR experiment. (A) Representative
immunoblot from control, AICAR, L-NMMA and L-NMMA + AICAR for phospho-
AMPK and total(a). (B) Quantification ofimmunoblots for phospho-AMPK/total
AMPK ratio. Values represent mean + SEM. AICAR =5-aminoimidazole-4-
carboxamide-1-P-D ribonucleoside (1 mM). *Significantly different from control.









CHAPTER 5
DISCUSSION

Main Findings

Our observations are based on relatively short-term exposure of isolated skeletal muscles

to AMPK-activating stimuli. The data support our hypotheses that electrical stimulation or

AICAR treatment is sufficient to increase AMPK activation, in vitro, in the fast-twitch EDL

muscle, and that NOS activity is necessary for this affect. Inhibition of NOS abrogated electrical

stimulation- and AICAR-induced activation of AMPK in the EDL; however interpretation is

complicated by a trend for L-NMMA treatment to induce phosphorylation of AMPK. The main

findings of this study are: 1) electrical stimulation or AICAR incubation increases the phospho-

AMPK/total AMPK ratio; 2) NOS inhibition prevents this increase; 3) AMPK activation by

electrical stimulation and AICAR treatment is greater in the fast twitch EDL muscle than the

slow twitch soleus muscle; and 4) L-NMMA treatment tends to increase AMPK phosphorylation

status, independent of electrical stimulation or AICAR effects.

Electrical Stimulation and AICAR Treatment Induces AMPK Activation

Activation of AMPK in response to exercise or hypoxia is a critical mechanism required

for normal metabolic regulation as well as nNOS and eNOS activity (8, 9, 14). Our data, in

agreement with several studies (14, 22, 38, 60) demonstrates that AMPK is an exercise-

responsive gene, activated by increases in intracellular AMP/ATP concentrations. The greater

the metabolic stress placed on skeletal muscle fibers, the greater the activation of AMPK (17).

Our stimulation protocol induced greater AMPK activation in the EDL compared to the soleus.

The level of metabolic stress placed on the oxidative fibers of the soleus was not great enough to

induce a significant increase in AMPK activation.









NOS Inhibition Decreases Electrical Stimulation- and AICAR-Induced AMPK Activation

NOS inhibition decreased the level of AMPK activation in the EDL following electrical

stimulation and AICAR treatment. We recently reported experiments in cultured L6 myotubes

demonstrating that AICAR-induced AMPK phosphorylation is nitric oxide-dependent (34).

Further, we found that nitric oxide donors are sufficient to induce AMPK phosphorylation (34).

Our working hypothesis to explain these data involves the convergence of AMP binding to

AMPK (induced experimentally be the AMP mimetic, AICAR) and NO-dependent activation of

AMPK kinases. Both of these events seem to be required to induce AMPK phosphorylation and

downstream signaling (Figure 5-1). The current experiments were designed to confirm that these

mechanisms are functional in adult skeletal muscle. We predicted that L-NMMA treatment

would prevent AMPK phosphorylation induced by either contractile activity or AICAR

treatment in adult skeletal muscle, in vitro. The current data indicate that NO plays an important

role in the regulation of AMPK phosphorylation in contracting or metabolically active muscle

fibers.

It appears that NOS inhibition caused a nonspecific effect on the phospho-AMPK/total

AMPK ratio in the soleus. Understanding this interaction is complicated as NO is intricately

involved in numerous physiological processes. The exact mechanisms whereby NO exerts its

effects on skeletal muscle contractility and metabolism remain uncertain. It is well established

that NOS inhibition increases force production by slowing cross bridge cycling, decreasing

ATPase activity and AMP levels and increasing oxidative phosphorylation. Logically, this would

suggest that basal AMPK activation would be reduced following treatment with a NOS inhibitor.

Nevertheless, our results suggest the opposite effect. Interestingly, NOS inhibition decreases

maximum shortening velocity in mixed fiber types but has no affect on the velocity of unloaded

shortening (determined by fast fibers) suggesting the possibility of a fiber-type-specific, NO-









dependent augmentation of ATPase activity (2, 36, 39) In contrast, NO thiol nitrosylation can

reduce ATPase activity and force production (28, 62).

Redox balance is necessary for homeostasis and optimal functioning of numerous

physiological interactions and may be an underlying condition affecting our results. The role of

NO as a signaling molecule has been well described in skeletal muscle (57). Most of the

physiological actions of NO are the result of its ability to stimulate guanylate cyclase, thereby

increasing the production of cGMP (57). Nevertheless, NO is a free radical capable of inducing

oxidative stress. Although it is not highly reactive itself, it can react with other compounds to

produce more toxic species (46, 70). Most notably, NO reacts with superoxide to produce

peroxynitrite, a highly reactive species that can oxidize lipids, proteins and nucleic acids (46).

Paradoxically, due to NO's low redox potential and its ability to combine with more reactive

species and convert them to less reactive products, it is capable of acting as an antioxidant as

well as a pro-oxidant (46, 70). The disparate effects of NO in tissue (i.e. positive signaling and

antioxidant effects vs. negative pro-oxidant effects) appear to be primarily determined by

concentration, and perhaps localization of the NO signal. The constitutive, calcium-dependent

NOS isoforms (nNOS and eNOS) produce nanomolar concentrations of NO, which are sufficient

for activation of guanylate cyclase, and produce antioxidant effects (46, 57, 70). Conversely,

iNOS which is expressed by neutrophils or macrophages, or by muscle cells in response to

oxidative stress, endotoxin, or inflammatory cytokine signaling, produces 100 to 1000 fold

higher concentrations of NO leading to nitrosylation and oxidant stress (50, 71). We believe that

NO concentration regulates the equilibrium between reduced and oxidized states within cells,

similar to that proposed by other research teams in relation to satellite cell activation and NO

concentration (1). At low and high concentrations of NO, the cell is placed in a state of oxidized









stress. An optimal concentration of NO exists creating an environment wherein skeletal muscle

metabolism can transpire without placing excess stress on the cell. This idea could explain why

lowering NO concentration by treatment with L-NMMA (a non-isoform-specific NOS inhibitor)

tended to increase basal metabolic stress and induce AMPK phosphorylation. Meanwhile,

activation of AMPK during contractile activity or elevated AMP concentration (mimicked by

AICAR) requires NO.

ACC Phosphorylation

Our electrical stimulation and AICAR treatment protocols were not sufficient to induce

significant main effects on phosphorylation of the AMPK target, ACC, in the soleus or EDL,

most likely due to the relatively short incubation times (10 min for E-stim and 20 min for

AICAR). The only significant group difference was observed between control and electrical

stimulation groups in the L-NMMA-treated soleus muscles. Although contractile performance

data was not recorded for this study, we consistently observed an increased force production

during electrical stimulation plus L-NMMA treatment in the soleus (-50% increase) compared to

electrical stimulation alone. Consistent with this greater force production, there were strong

trends for AMPK and ACC phosphorylation to be elevated in these samples above L-NMMA

alone (p = .051). Apparently, there is a fiber type-specific effect of NOS inhibition during

electrical stimulation since only the soleus demonstrated this synergistic effect of contractile

activity and NOS inhibition on AMPK activation. This could be due to a general mitochondrial

dysfunction in the mitochondria-rich slow-twitch fibers of the soleus leading to exaggerated

metabolic stress during contractile activity.

Limitations and Future Directions

The electrical stimulation protocol was not sufficient to cause significant changes in

AMPK activation in the soleus most likely because of its oxidative phenotype. Stimulation for a









longer period of time most likely would have activated AMPK to a higher degree. Future studies

need to increase the time of stimulation to see these changes.

Our limited ability to probe for oxidative enzymes and other proteins made it difficult to

interpret our results. Data on ATPase, total ACC and cytochrome-c oxidase may have given

additional insight as to what changes occurred as a result of our protocol.

A dose-response experiment needs to be performed to test our theory that NO

concentration regulates a balance between reduction and oxidation in healthy cells. In addition,

future studies should examine the effects of a high-intensity electrical stimulation protocol

imitating a strength or power exercise to determine how exercise intensity affects AMPK

activation.

Conclusions

NOS activity is involved in activation of AMPK during chronic electrical stimulation or

AICAR treatment. To date, the full extent of NOS involvement in AMPK activation and

exercise-induced metabolic adaptations is not known. Further studies concentrated on NOS

activity and AMPK are needed to investigate this relationship.












Contractile
Activity (ATP


AMP


+A


AICAR -


Electrical
Stimulation


+ AMPKK
Phosphatases



AMPK

4-


Mitochondrial biogenesis, GLUT 4 upregulation


Figure 5-1. Proposed model illustrating potential role of nitric oxide in the activation of AMPK.
NO may facilitate activation of AMPK by increasing AMPK kinases and/or
decreasing phosphatases. Inhibiting NOs metabolic signal regulation may have
affected our results.









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BIOGRAPHICAL SKETCH

Dana Brown was born in Fort Walton Beach, Florida, in 1984. She graduated in the top

3% of her high school in 2002. She received a Bachelor of Science degree in exercise and sport

sciences in 2006 from the University of Florida, where she was a member of the Golden Key

Honor Society and Social Entrepreneurship and an employee of UF's Department of

Recreational Sports. Dana will begin an additional Master of Science degree in business

entrepreneurship at the University of Florida beginning in the summer of 2008. After finishing

school Dana plans to continue working in the field of health and fitness.





PAGE 1

1 NITRIC OXIDE SYNTHASE ACTIVITY MEDIATES ACTIVATION OF AMPASSOCIATED PROTEIN KI NASE IN ISOLATED MOUSE SKELETAL MUSCLE By DANA LEIGH BROWN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Dana Leigh Brown

PAGE 3

3 To my family for their unwavering support and a ll the teachers and prof essors who taught and mentored me along the way.

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4 ACKNOWLEDGMENTS This work was completed with the help and support from many people. First and foremost I would like to thank V itor Lira for teaching me the majo rity of the lab techniques used for this study and for working with me to co llect data. Without him I undoubtedly would not have completed this project. I would like to thank Dr. David Criswell, my supervisory committee chair, for allowing me to work in his lab even though lab space and f unding was tight. He stimulated my interest in exercise physiology as an undergraduate and continued to teach me until I finished my Masters degree. I would like to thank my committee members, Drs. Scott Powers and Steve Dodd from the Department of Applied Physiology and Kinesiology, College of Health and Human Performance, University of Florida, fo r allowing me to present my research. I thank my fellow Molecular Physiology labmates (Vitor, Quinlyn Soltow, Jason Drenning, Liz Zeanah, Carlos Carmona, Claire Ca non and Lauren Valera) who helped me in the lab and kept me motivated to write this paper. Also to Dr. Jenna Leigh Jones Betters who allowed me to assist with a portion of her data collection. This gave me the opportunity to familiarize myself with the techniques I used for my project. I thank all the professors in the Applied Physiology and Kinesiology department for teaching and mentoring me and for conti nuously surpassing their own achievements. This work could not have been done without the love and motivation from my friends and family. I would especially like to thank my parents, Mike and Linda Brown, for supporting and encouraging me when they did not exactly understand what I was doing.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT.....................................................................................................................................9 CHAP TER 1 INTRODUCTION..................................................................................................................11 Background.............................................................................................................................11 Problem Statement.............................................................................................................. ....12 Variables in Study............................................................................................................. ......12 Hypotheses..............................................................................................................................13 Definition of Terms................................................................................................................13 Limitations/Delimitations/Assumptions................................................................................. 14 Significance of the Study........................................................................................................15 2 LITERATURE REVIEW.......................................................................................................16 Enzymatic Production of Nitric Oxide(NO)...........................................................................16 Nitric Oxide Synthase(NOS) Isoforms............................................................................16 Substrate Availability...................................................................................................... 17 Calcium-Calmodulin....................................................................................................... 17 Physical Activity............................................................................................................. 18 NO Signaling and Skeletal Muscle......................................................................................... 18 Mechanisms of Action..................................................................................................... 18 Acute Effects...................................................................................................................19 Chronic Adaptations........................................................................................................ 20 Adenosine Monophosphate Activated Protei n Kinase(AMP K) is a Key Metabolic Regulator in Skeletal Muscle.............................................................................................. 21 AMPK Structure and Activation..................................................................................... 21 AMPK Signaling Effects.................................................................................................22 Interaction Between NO and AMPK...................................................................................... 25 AMPK-Induced Phosphorylation of nNOS and eNOS...................................................25 NO Facilitates AMPK Activation in Cell Culture........................................................... 25 Conclusions.............................................................................................................................26 3 METHODS.............................................................................................................................29 Experimental Design............................................................................................................ ..29 Animals...................................................................................................................................29

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6 Anatomical Dissection.......................................................................................................... ..29 Experimental Protocol............................................................................................................30 Western Blotting............................................................................................................... ......31 Statistical Analysis........................................................................................................... .......31 4 RESULTS...............................................................................................................................34 Electrical Stimulation......................................................................................................... ....34 Phospho-/Total-AMPK Ratio.......................................................................................... 34 Total-AMPK, Phospho-AMPK, and Phos pho-Acetyl Co-A Carboxylase(ACC) ...........34 5-aminoimidazole4-carboxamide-1-D-ribofuranoside(AIC AR).........................................35 Phospho-/Total-AMPK Ratio.......................................................................................... 35 Total-AMPK, Phospho-AMPK, and Phospho-ACC....................................................... 35 5 DISCUSSION.........................................................................................................................42 Main Findings.........................................................................................................................42 Electrical Stimulation and AICAR Tr eatm ent Induces AMPK Activation............................ 42 NOS Inhibition Decreases Electrical S tim ulationand AICAR-Induced AMPK Activation............................................................................................................................43 ACC Phosphorylation.............................................................................................................45 Limitations and Future Directions.......................................................................................... 45 Conclusions.............................................................................................................................46 LIST OF REFERENCES...............................................................................................................48 BIOGRAPHICAL SKETCH.........................................................................................................54

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7 LIST OF TABLES Table page 4-1 Quantification of phospho-AMPK, total AMPK and phospho-ACC/ actin levels for the EDL and soleus for electrical stimulation experim ental groups.................................. 364-2 Quantification of phospho-AMPK, total AMPK and phospho-ACC/ actin levels for the EDL and soleus for the AICAR experimental groups................................................. 37

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8 LIST OF FIGURES Figure page 2-1 Metabolic processes regulated by AMPK...........................................................................272-2 Exercise-induced signaling cascades................................................................................... 283-1 Experimental design flowchart.32.......................................................................................323-2 Experimental groups chart................................................................................................... 334-1 EDL electrical stimulation phospho-AMPK/ AMPK ratio................................................ 384-2 Soleus electri cal stimulation phospho-AMPK/ AMPK ratio............................................. 394-3 EDL AICAR phospho/total AMPK ratio............................................................................ 404-4 Soleus AICAR phospho/total AMPK ratio......................................................................... 415-1 Proposed model illustrating potential role of nitric oxide in the activation of AMPK....... 47

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science NITRIC OXIDE SYNTHASE ACTIVITY MEDIATES ACTIVATION OF AMPASSOCIATED PROTEIN KI NASE IN ISOLATED MOUSE SKELETAL MUSCLE By Dana Leigh Brown August 2008 Chair: David Criswell Major: Applied Physiology and Kinesiology Nitric oxide (NO) production and signaling has an important role in exercise-induced metabolic and biochemical adaptations. Current re search indicates that low levels of NO produced enzymatically by the nitric oxide synthases (NOS ), regulates 5-AMP-ac tivated protein kinase (AMPK) in cell culture. The phys iological significance of this regulatory mechanism in adult skeletal muscle is unknown. Goal: This research was designed to determine if NOS activity is necessary for induction of AMPK phosphorylation in skeletal muscles isolated from adult mice. Hypotheses: Incubation of isolated extensor digitorum longus (EDL) and soleus muscles with the non-isoform specific NOS inhibitor, L-NMMA, w ill prevent activating phosphorylation of AMPK, and phosphorylation of the AMPK subs trate, acetyl CoA carboxylase (ACC). Approach: EDL and soleus muscles were surgically isolated bilatera lly from young adult mice and incubated in micro tissue baths. AMPK was activated by 10 min of contractile activity or a 20-min incubation with the AMP analog, AICAR. The EDL and soleus muscle from the contralateral limb was treated identically, except for the additi on of L-NMMA to the incubation media throughout the experiment. Results: The main findings of this study are: 1) electrical stimulation or AICAR incubation increases the phospho-AMPK/total AMPK ratio; 2) NOS inhibition prevents th is increase; 3) AMPK activation by electrical stimulation and AICAR treat ment is greater in the fast twitch EDL muscle

PAGE 10

10 than the slow twitch soleus muscle; and 4) L-NMMA treatment tends to increase AMPK phosphorylation status, independe nt of electrical stimulation or AICAR effects. Significance: AMPK is an important regulator of skeletal muscle metabolism and the acute and chronic adaptations to exercise traini ng. Activation of AMPK, pharmacologically or by exercise, increases oxidative capacity and improves in sulin sensitivity leading to a metabolically active phenotype in skeletal muscle that effectively counters th e metabolic syndrome. U nderstanding how AMPK is regulated in adult muscle fibers and the potential role of nitric oxide in mediating AMPK activation is important for development of more potent and efficient treatments for meta bolic disorders.

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11 CHAPTER 1 INTRODUCTION The m etabolic syndrome, which manifests itself in pathophys iological conditions such as insulin resistance, hypertension and dyslipidem ia, is increasing at astounding rates throughout the United States and developing countries due in large part to physical inactivity. These conditions are coupled with increased morbidity and mortality rates and place an excessive burden on healthcare systems. Physical activity has long been hailed as a necessary component for a healthy lifestyle. Skeletal muscle is an exceptionally plastic tissue with a remarkable capacity to adapt to external stresses by making fine metabolic adju stments. Contracting skeletal muscle acutely increases insulin sensitivity in both healthy individuals and in people with Type 2 diabetes, and regular physical exercise is a cornerstone in the treatment of the disease. Exercise training alters the biochemical signaling cascade s that regulate the metabolic properties of skeletal muscle. At present, the exact molecu lar mechanisms for these adaptations are poorly understood. Background During exercise skeletal muscle is p laced under enormous stress in order to maintain energy levels and contractile function. 5-AMP-ac tivated protein kinase (AMPK) is a cellular regulator that responds to change s in the AMP-to-ATP ratio. AMPK is activated allosterically by AMP binding to the enzyme, and by phosphorylation of the catalytic subunit by upstream AMPK kinases (AMPKK). AMPK has an important role in numerous biochemical pathways and is responsible for regulating energy metabolism processes. Chronic AMPK activation is an important signal for upregulation of glucose tr ansporter molecules. Recent research has suggested that AMPK activation may be in fluenced by nitric oxide (NO), produced

PAGE 12

12 enzymatically by the nitric oxide synthases (NOS ). At present, it is not known if NO is necessary for AMPK activation in adult skeletal muscle. Problem Statement The benefits of exercise in metabolic di sease prevention and therapy have been welldocumented in literature but the specific mechanistic signaling pathways that exert the protective effects remain elusive. The aim of this study was to determine whethe r the presence of NO is required for AMPK activation and whether AMPK activation differs between skeletal muscle fiber types. Variables in Study Independent variables : W e will simulate contractile load to isolated extensor digitorum longus (EDL) (primarily fast twitch muscle) and soleus muscles (primarily slow twitch muscle) by means of electrical stimulation in vitro, and by contraction-i ndependent stimulation of AMPK via 5-aminoimidazole-4-carboxamide-1-D-ribonucleoside (AICAR) administration in vitro. NOS activity will be inhibited via N(G)-monome thyl-L-arginine (L-NMMA) administration to determine the necessity of NO production for AMPK activation. Dependent variables : We will measure phosphorylation of metabolic proteins AMPK and acetyl CoA carboxylase (ACC). Control variables : Sex and age were controlled as all animals used within the study were young female adult mice (~4 months old) from th e ICR strain. Animals were brought to the lab at least 12 hours prior to surgery to allow acclima tization to the new environment. All surgeries were conducted at the same time of day. Isolated muscles were set to their optimal length using a micromanipulator and indi vidual isometric twitches.

PAGE 13

13 Extraneous variables : Individual differences in daily activities such as ambulation and feeding were not controlled. Hypotheses We hypothesize that: 1. Ten m inutes of electrical stimulation, in vi tro, will induce phosphorylation of AMPK and ACC in the isolated EDL and soleus muscles. 2. Twenty minutes of AICAR treatment, in vitro, will also induce phosphorylation of AMPK and ACC in these muscles. 3. Addition of L-NMMA to the incubation media will significantly decrease NO biosynthesis in muscles at rest and during electrical stimulat ion, decreasing AMPK phosphorylation. 4. AMPK phosphorylation will be higher in fast twitch muscles than slow twitch muscles. Definition of Terms 5-AMP-activated protein kinase (AMPK). A heterotrimeric protein kinase that regulates cellular ener gy homeostasis and is largely responsible for exercise-induced skeletal muscle adaptations to endurance training. 5-aminoimidazole-4-carboxamide-1-D-ribonucleoside (AICAR). AMP mimetic drug capable of activating AMPK. Acetyl CoA carboxylase (ACC). Rate-limiting enzyme involved in fatty acid synthesis, and a known target of AMPK activity. AMP-activated protein kinase kinase (AMPKK). A collective group of enzymes responsible for phosphorylating AMPK. Electrical stimulation. A technique that uses platinum electrodes to generate a field current in an electrolyte buffer-filled chamber for the purpose of directly activating an isolated mouse skeletal muscle, suspended in the buffer solution.

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14 Optimal length (Lo). The length at which maximum te tanic tension is generated by skeletal muscle. N(G)-monomethyl-L-arginine (L-NMMA). A nonspecific competitive inhibitor of NOS activity. Nitric oxide (NO). A short-lived endogenous mediator th at acts as a signaling molecule and plays a role in a variety of biological reactions in virtua lly all mammalian tissues under certain physiological or pathophysiological conditions. Nitric oxide synthase (NOS). A family of isozymes responsible for NO production. Three forms have been identified in mamma lian skeletal muscle: neuronal NOS (nNOS), endothelial NOS (eNOS) a nd inducible NOS (iNOS). Limitations/Delimitations/Assumptions Limita tions : Due to the invasive nature of this research, the use of humans as experimental subjects was not possible. Inst ead, a mouse model was chosen due to genetic similarity to provide results for the researcher that would mimic thos e seen in humans. The in vitro experimental design allows isolation of intrinsic muscle responses. It is recognized that extrinsic responses to contractile activity, which ar e not considered in our design, likely contribute to skeletal muscle responses in vivo Delimitations: The study is restricted to female mice. Assumptions: 1. L-NMMA, at the dose used, speci fically inhibits NOS activity in skeletal muscle, and has negligible non-specific effects. 2. AMPK activity correlates well with ACC activity.

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15 Significance of the Study Whilst genetic predisposi tion for the metabolic syndrome cannot be modified by behavior, obesity, the largest risk factor, is largel y preventable. Failure to curb increased obesity lies in large part with our lack of understanding of metabolic government in healthy and disease states. To maintain metabolic function, intracel lular ATP concentrations must remain within acceptable limits. AMPK preserves ATP levels by managing energy utilization for metabolic pathways based on necessity. AMPK can also re gulate exercise responsive genes leading to long-term metabolic stability. Therefore, it is clear that a dysfunction in AMPK signaling can induce rapid and contrary responses that directly affect metabolic function. The AMPK pathway is of particular interest within the clinical co mmunity because of its pot ential in treating and preventing the metabolic syndrome. Understanding the mechanism by which muscle contractions induce AMPK activity will aid in development of new strategies to prevent or treat metabolic disorders.

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16 CHAPTER 2 LITERATURE REVIEW Nitric oxide (NO) is a shortlived endogenous mediator that acts as a signaling m olecule and plays a role in a variety of biological reactions in virtually all mammals under certain physiological or pathophysiologi cal conditions. Since its decl aration as a cardiovascular homeostatic mediator, NO has emerged as a fundamental signaling device regulating intracellular processes and cellcell interactions. NO production is primarily dependent on a group of heme-containing proteins known as nitric oxide synt hases (NOS). NO regulates processes in skeletal muscle that include force production, autoregulation of blood flow, mitochondrial biogenesis, mitochondrial respirat ion and glucose transport (6,15, 25, 29, 45, 57). AMPK is a key metabolic enzyme that has emerged as a metabolic master switch. AMPK activity is involved in many of the training adaptations acquired w ith chronic exercise. Recent studies provide evidence that AMPK activation is dependent upon NO (8, 34). At present, a complete understanding and grasp of NO par ticipation in homeostatic functions and pathophysiological conditions has not yet been reached. This chapter will review the recent literature concerning nitric oxide and AMPK effects in skeletal muscle a nd potential interaction between these two important signaling molecules. Enzymatic Production of Nitric Oxide(NO) Nitric Oxide Synthase(NOS) Isoforms Three isoforms of NOS produce NO from the amino acid, L-arginine (L-arg), and require oxygen and NADPH as substrates with citrulline as a by-product. Nitric oxide is synthesized in normal skeletal muscle by neuronal NOS (nNO S) and endothelial NOS (eNOS). NOS is constitutively expressed in rode nt skeletal muscle (56, 66). Lo calization of the nNOS isoform varies greatly among cell types and is directly associated with th e dystrophin complex in skeletal

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17 muscle. It is located in close proximity of the sarcolemma, and primarily in fast-twitch fibers of rodents. nNOS expression in humans appears to be similar, except for a more widespread distribution across fiber types (63). eNOS is uniformly distributed in muscle fibers and localized in the mitochondria and also in vessel walls ( 63). nNOS and eNOS are highly regulated by Ca2+ and calmodulin. The third isoform, inducible NOS (iNOS), is not produced at significant levels in skeletal muscle. Its expr ession is regulated by transcrip tion in response to cytokines and other inflammatory agen ts and is largely Ca2+ independent. Substrate Availability NOS activity varies between m uscles. Genera lly, higher activity of NOS is measured in fast twitch fibers (56). The availability of co factors and co-substrates as well as subcellular localization can influence the iden tity of the enzymatic product ( 57). It has been hypothesized that cellular supply of L-arginine is a ra te-limiting step for NOS activity. Ogonowski and colleagues suggest that Larginine transport by y+ transporters may be the limiting step (41). McConell et al (37) performed a study that infuse d L-arginine into humans while cycling at 72% VO2 Max. They reported improved glucose disposal with arginine infusi on during an exercise protocol. This group theorize d that L-arginine supplemen tation upregulated NO production during skeletal muscle contraction and increased GLUT4 translocation. Calcium-Calmodulin Calm odulin binding is an essential requireme nt for NO production by all NOS isoforms. iNOS is tightly bound to calmodulin in an almost irreversible fashion (21, 56), therefore, the activity of this isoform is e ssentially independent of calcium nNOS and eNOS, however, bind to calcium-calmodulin and, therefore, rely on a calcium signal for upregulation of enzymatic activity. Upon release of calcium from the sarcoplasmic reticu lum during contraction, calcium concentration increases and facilitates the inte raction of nNOS and eNOS with calmodulin.

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18 Jagnandan et al. (21) illustrate d the calcium-dependence of eNOS by limiting access to calcium and measuring NO production. Calcium blocka de essentially eliminated NO production by eNOS but had no significant e ffect on NO production by iNOS. Physical Activity Increasing research findings show that NO is an important m etabolic regulator during exercise. Furthermore, physiological adapta tions such as improved functional ability and cardioprotection result from ch ronic physical activity. At re st skeletal muscle produces relatively small amounts of NO. However, th ere are significant changes in NO production in response to exercise and physical activity. Exercise upregulates NOS activity and nNOS gene expression (3, 29). NO Signaling and Skeletal Muscle Mechanisms of Action Because NO is a short-lived free rad ical with a half life of 2 to 5 s econds (13), regulation of signaling occurs largely at the level of NO biosynthesis. It can act locally on the cell that it is produced within or it can permeate cell membranes of adjacent cells. Research has shown that NO is involved in such diverse activities as vasodila tion, metabolic regula tion, immune function, cellular signaling, contractile re gulation and glucose homeostasis, to name a few (3, 45, 55). There are a number of mechanisms whereby NO effects are mediated. sGC and cGMP. Activation of soluble guanylate cy clase (sGC), a heme-containing heterodimer, is a primary mechanism mediated by NO. NO binds to the heme increasing the catalytic activity of cGC se veral hundredfold increasing the levels of the second messenger cyclic guanylyl monophosphate (cGMP) (54). Th e resulting increase in cGMP concentration serves to signal a multitude of complex physiologi cal operations. cGMP can directly activate its downstream effectors including Pr otein Kinase G (PKG), Cyclic Nucleotide Gated Channels

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19 (CNG) and Cyclic nucleotide phosphodiesterases, all of which govern the operation of many proteins involved in physiological functioning (35). Protein nitrosylation. Following production, NO translocates to nearby cells and interacts with the interior of the cell at metal sites wi thin proteins. Arguably the most influential mechanism of action is nitrosylation, the reaction of NO with cysteine resi dues in proteins or by interactions with heme or non-heme copper a nd iron. A continuum be tween nitrosative and oxidative stress must be maintained to redu ce hazardous levels of stress which has been connected to muscle fati gue and cell injury (57). Acute Effects Glucose transport. Exe rcise acutely increases glucose transport in both healthy individuals and those with Type 2 diabetes. NO signaling markedly increases glucose transport in isolated skeletal muscle fibers (5). Tran slocation of intracellular GLUT4, the primary glucose transporter isoform found in skeletal muscle, to the plasma membrane is the underlying mechanism by which exercise causes increased gl ucose transport (23). Recent studies have shown a link between NO and AMPK responsible for exercise-induced glucose clearance. Contractile function. At present, complete functional significance of the NOS isoforms and skeletal muscle remains elusive. However, NO does play a pivotal role in maintenance of skeletal muscle contractile ability. This re gulation appears to be mediated through cGMPdependent and independent mechanisms. Skelet al muscle fibers exposed to NO have shown reduced actin-myosin cross-bridge cycling in vitr o (18, 26). Lau et al (3 2) provided evidence of the linkage between NO and cGMP. When electri cally stimulated, the cG MP content of wild type mouse extensor digitorum longus muscle cGMP content increased approximately 250%. It has recently been discovered that colocalization of cGMP and nNOS at the sarcolemma inhibits excitation-contraction coupli ng in skeletal muscle by impaired Ca2+ activation of thin

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20 filaments (26). NO also interferes with cont raction by inhibiting cr eatine kinase, and the sarcoplasmic reticulum Ca2+-ATPase in fast-twitch and slow-twitch muscle fibers. Mitochondrial respiration. NOS activity has been associated with inhibition of mitochondrial respiration. Identified resp iration targets of NO include cytochromec oxidase, creatine kinase and Ca2+-ATPase in skeletal muscle (57). NO binds to cytochromec oxidase, inhibiting comples IV of the electron transport chain and m itochondrial oxygen consumption. This disruption in cellula r respiration can re sult in alterations of calcium flux. Disruptions in mitochondrial function can drastically alter ener gy levels. Redox balance is necessary for homeostasis and optimal functioning of nu merous physiological interactions. Satellite cell activation Muscular injury due to exercise, mechanical stretch or blunt force mobilizes satellite cells from quiescence to serve as ante cedents for new muscle formation. At present NO and hepatocyte growth factor (HGF) are the only known act ivators of satellite cells. Satellite cell activation is define d as mobilization and entrance into the G1 interphase of the cell cycle (1). Studies have show n that release of HGF from the extracellular matrix is NOdependent and inhibition of NOS activity reduces HGF release and satellite cell activation (1). Chronic Adaptations Mitochondrial biogenesis. In arterioles, NO vasodilates sm ooth muscle and increases blood flow and O2 delivery to the tissues. NO contribu tes to the regulatio n of mitochondrial respiration by inhibiting cytochrome-c oxidase at complex III of the electr on transport chain. This leads to decreased levels of cellular ATP a nd increase in levels of ADP, AMP, GDP and Pi (13). Adult skeletal muscle experiences a transfor mation from fast to slow fiber type with exercise training. eNOS and neuronal nNOS is oforms may be differen tially involved in the regulation of mitochondrial biogene sis in skeletal muscle (66). Evidence to support this was

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21 presented by Wadley et al. NO donor experiment s in rodents lead to increased expression of peroxisome proliferator-activated receptor coactivator 1 (PGC-1), nucl ear respiratory factor 1 (NRF-1), and mitochondrial transcrip tion factor A (mtTFA), all of which are markers of mitochondrial biogenesis (66). GLUT 4 expression. Regular aerobic exercise is associ ated with biochemical changes to numerous metabolic genes. It is well established that chronic exercise is a cornerstone in the treatment and prevention of Type 2 diabetes due in large part to increased insulin sensitivity. Insulin stimulation leads to recr uitment of the GLUT 4 transporte r to the surface of the cell to transport glucose intracellularly (24). AMPK has recently been linked to upregulation of GLUT4 transporter gene expression. Adenosine Monophosphate Activated Protein Kinase(AM PK) is a Key Metabolic Regulator in Skeletal Muscle AMPK Structure and Activation The AMPK molecule is a hete rotrimeric protein kinase composed of a catalytic subunit and regulatory and subunits (61). The and subunits each have two genes and the subunit has three, yielding at le ast 12 possible heterotrimeric seque nces (17). The presence of AMPK in primitive organisms suggests that this mo lecule has served an important genetic role throughout evolution (12). AMPK is an exercise-responsive gene that acts to preserve cellular energy levels (22, 40, 61). AMPK is activated during times of ce llular energy stress incl uding hypoxia, glucose depletion and exercise. It is we ll established that exercise is characterized by heightened energy turnover. AMPK is activated during times of elevated metabolism and ATP consumption. Once activated, AMPK turns on catabolic processes that generate ATP while concurrently turning off anabolic processes such as cellular growth and proliferation. Two independent articles were

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22 published in 1973 describing protein fragments that had the capac ity to inactivate acetyl-CoA carboxylase (ACC) and 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) reductase, 2 enzymes necessary for lipid synthesis. It was later disc overed that the protein fragments were composed of protein kinases and that ACC kinase and HM G-CoA reductase kinase were activated by 5AMP. In 1987 Carling et al real ized that the same kinase was responsible for ac tivation of both functions (7). This molecule was named AMPK. AMPK is activated by AMP in one of two ways: phosphorylation by upstream kinases or allosteric activation. Phosphorylation of AMPK by upstream kinases occurs within the catalytic subunit at Thr172 (53). LKB1 and Ca2+/calmodulin-dependent protein kinase kinase (CaMKK ) have been identified as we ll-suited upstream kinases capable of activating AMPK (53, 61). Activation of AMPK during ex vivo electrical stimula tion is dependent upon modification of the -Thr172 subunit by AMP phosphorylation making it a desirable substrate for LKB1 (52). Direct activation of AMPK o ccurs allosterically by AMP binding to the subunit. This renders AMPK a more suitab le substrate for upstream kinase s and a less suitable substrate for phosphatases which can deactivate AMPK (61). The level of metabolic stress placed on the body affects the level at which AMPK is activated (17). Interestingly, AM PK is activated during strength training in endurance athletes and during endurance training in st rength athletes. It is reasoned, therefore, that AMPK is upregulated during training at intensity levels greater than the individual is adapted to. AMPK Signaling Effects AMPK was first discovered in the liver and evidence suggested that the enzyme was likely present in other tissues of the body. Prot ein expression analysis found mRNA levels of AMPK expression is greatest within skeletal muscle (64). Over the past years extensive research has been conducted with inconclusive results to decipher which particular subunits are expressed

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23 in animal and human muscle. Within skeletal muscle, AMPK is known to exhibit regulatory effects on glucose transport, fa tty acid oxidation and mitochondrial biogenesis as exhibited in Figure 2-1. Glucose transport. Many of the downstream effects of AMPK homestatic-preservation were first discovered using the AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-Dribofuranoside (AICAR) (17). It was initia lly found by Merrill et al in 1997 that muscle incubated with AICAR exhibited increased gl ucose transport and AMPK activity (38). Ex vivo experiments with AICAR incubation in transg enic animals where AMPK signal is greatly decreased or nonexistent completely diminished glucose transport. The mechanism for exercise-induced skeletal muscle glucose transporter (GLUT4) upregulation appears to be influenced by NO. The interaction between AMPK and NOS is not certain but is very intriguing. Acutely, AMPK influences glucose transport. Long term adaptations show AMPK to be a mediator of exercise-induced glucose transport by increasing GLUT4 concentrations at the cell surface (38) Balon and colleagues demonstrated that incubation of skeletal muscle wi th a NOS inhibitor decreased gluc ose transport (4 ). Recently, Lira et al reported th at NO and cGMP were active partners in inducing GLUT4 expression in skeletal muscle. They also proposed the novel idea that NOS activity is required upstream and downstream of AMPK to induce GLUT4 expression (20). Fatty acid oxidation. Accumulation of fatty acids within the endothelium is a major contributor to atherosclerosis a nd, therefore, the meta bolic syndrome. Fatty acid clearance is necessary for normal endothelial function a nd accumulation can cause excess production of damaging radicals. It has been shown that fa tty acid oxidation accounts for approximately 40% of ATP production by endothelial ce lls (10). Both resting and ac tive skeletal muscle metabolize

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24 fatty acids to produce energy. Several studies have indicated that AMPK activation can augment fatty acid oxidation. AMPK is responsible for inhibitory phosphorylation of acetyl-CoA carboxylase (ACC). ACC is responsible for fatty acid synthesis and when inactivated reduces accumulation of triglycerides by stimulating mitoc hondrial uptake of fatty acids. AMPK activity is markedly reduced in obese animals and activation of AMPK reduces intracellular accumulation of triglyceride levels (40). Mitochondrial biogenesis. Chronic endurance exercise is associated with increased mitochondrial oxidative capacity. One of the mech anisms responsible for mitochondrial enzyme gene transcription involves upregul ation of AMPK during times of skeletal muscle contraction. AMPK promotes mitochondrial biogenesis by increasing PGC-1 and NRF expression. In an experiment by Winder et al. (69) 28 days of chronic AICAR treat ment in rats resulted in increased AMPK activation as well as levels of citrate synthase, succina te dehydrogenase, malate dehydrogenase and cytochrome c, all mitochondrial enzymes. AMPK clearly effects mitochondrial regulation during times of ener gy deprivation by promoting expression of mitochondrial enzymes and biogenesis markers. Inhibition of muscle growth. Mammals have two distinct signaling cascades responsible for exercise-induced adaptations: one which is responsible for increases in cardiovascular endurance and another which signa ls protein synthesis and mu scular growth. These two pathways are exhibited in Figure 2.2. Increa ses in cardiovascular e ndurance are primarily signaled through AMPK. Muscle hypertrophic re sponse to exercise is signaled through the mammalian target of rapamycin (mTOR) pathwa y due to its ability to stimulate protein translation and synthesis. Protein synthesis begins with insulin stimulating the PI 3-kinase/Akt pathway leading to increased mTOR activity ( 11). Inhibition of mTOR immediately before

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25 muscle overload prevents protein synthesis, (31) suggesting that this pathway is necessary for normal muscle hypertrophy. AMPK is a negative regulator of the mTOR pathway. The 2 and 3 subunits of AMPK have been identified as the complexes responsible for inhibiting mTOR activity (11). These two subunits have the ab ility to phosphorylate and thus deactivate the mTOR signaling cascade. Interaction Between NO and AMPK AMPK-Induced Phosphorylation of nNOS and eNOS The interaction between NOS and AMPK is not fully understood. It is known that AMPK has the ability to phosphorylate and activ ate eNOS and nNOS isoforms (8, 9). In addition, the AMP mimetic drug 5aminoimidazole-4carboxamide-1-D-ribofuranoside (AICAR) upregulates NOS activity in H-2Kb muscle cells (14). Alt ogether, the act ivation of AMPK in response to exercise or hypoxia is a critical mechanism required for normal metabolic regulation as well as nNOS and eNOS activity. NO Facilitates AMPK Ac tivation in Cell Cult ure Current research indicates that NO produced en zymatically by the nitric oxide synthases regulates AMPK in cell culture. Lira et al (34) found that NOS activity was necessary for AMPK-induced upregulation of the GLUT4 tran sporter gene expression. L6 myotubes were treated with AICAR or AICAR and the non-specific NOS inhibitor L-NAME. Cotreatment with AICAR and L-NAME ablated 70% of the AICAR effect on GLUT4 mRNA suggesting that NO is necessary downstream of AMPK for normal function. They also suggest that NO is necessary upstream of AMPK. Low concentrati ons of SNAP, an NO donor, at 1 and 10 M increased GLUT4 mRNA expression and AMPK phosphorylation; in contrast high concentrations of SNAP at 100 and 300 M did not affect AMPK or GLUT 4 mRNA expression. Furthermore, they propose the idea of a positiv e feedback system between NOS and AMPK enzyme activity.

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26 Conclusions The metabolic syndrome, characterize d by the clustering of cardiovascular risk factors that leads to Type 2 diabetes is increasing at astounding rate s throughout the United States and developing countries due in larg e part to physical inactivity. NO production and signaling has an important role in exercise-induced metabolic and biochemical adaptations. AMPK is an important regulator of skeletal muscle metabolism and the acute and chronic adaptations to exercise training. Activation of AMPK, pharmacol ogically or by exercise, increases oxidative capacity and improves insulin sensitivity leading to a metabolically active phenotype in skeletal muscle that effectively counters the metabo lic syndrome. Unders tanding how AMPK is regulated in adult muscle fibers and the potenti al role of nitric oxide in mediating AMPK activation is important for development of more potent and efficient treatments for metabolic disorders.

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27 Figure 2-1. Metabolic processes regulated by AMPK. Arrows indicate activation of pathways, whereas lines with a bar at the e nd indicate inhibition of pathways. Glucose Transport AMPK Mitochondrial Biogenesis MTOR Fatty Acid Oxidation Protein Synthesis Fatty Acid Synthesis

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28 Figure 2-2. Exercise-induced signaling cascades. Arro ws indicate the signaling events for adaptations to occur whereas a line with a bar at the end indicates processes inhibited. Cardiovascular/ Endurance Trainin g Ca2+ release Overload/ Resistance Training Akt mTOR Protein Synthesis Hypertrophy AMPK Mitochondrial Biogenesis Increased cardiovascular capacity CaMK

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29 CHAPTER 3 METHODS Experimental Design Extensor digitorum longus (EDL) and soleus muscles were dissected from young mice for in vitro bath manipulation. On e EDL and one soleus muscle were subjected to 10 minutes of electrical stimulation or exposed to 5-aminoimidazole-4-carboxamide-1-D ribonucleoside (AICAR) for 20 minutes. Contralateral EDL and so leus muscles of the mice were treated with N(G)-monomethyl-L-arginine (L-NMMA) to inhi bit NOS activity. The experimental design protocol is depicted in Figure 3-1. Animals The Univers ity of Florida Institutional Animal Care and Use Committee approved the protocol of this study. The subjects were young (~2 months old) female ICR mice purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). All animals were housed in the J. Hillis Miller Animal Science Center and fed the same diet (chow and water ad libitum) throughout the experiment. They were kept on a 12 hr light:d ark photoperiod. Animals were brought to the lab approximately 12 hr prior to surgery to allow acclimatization to their new environment. Animals were divided into 8 treatment gr oups as illustrated in Figure 3-2. Anatomical Dissection Surgical removal of the extensor digitorum longus (EDL) and soleus muscles was necessary for ex vivo manipulation. The mice were anaesthe tized with 2-5% isoflurane with oxygen as the carrier gas. Once anaesthetized, bo th hindlimbs were skinned and the Achilles tendon of the right leg cut. Because of its oxidati ve nature, the soleus was removed first to avoid hypoxia and tissue death. An incision was made along the fascia of the gastrocnemius muscle beginning at the distal end up to the posterior as pect of the fibula. The gastrocnemius/soleus

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30 muscle complex was reflected to expose the deep soleus muscle and the proximal tendon. The proximal tendon was cut and the so leus carefully maneuvered away from the gastrocnemius. The distal tendon was then cut and the muscle i mmediately placed in the oxygenated buffer. The same surgical procedure was repeated on the left leg. The distal ends of the tibialis anterior (T A) and EDL tendons were located and cut from the phalanges. An incision was made along the fascia of the TA up to the proximal end of the tendons at the fibula. The TA/EDL complex was removed as a whole and the EDL carefully maneuvered away from the TA and immediately tran sferred to the oxygenated buffer. Experimental Protocol Soleus and EDL m uscles were surgically removed from fed anaesthetized mice and immediately transferred to a bath containing Kr ebs-Henseleit (KH) buffer supplemented with 25 mM sodium bicarbonate, 5 mM He pes, 2.54 mM calcium chloride and 100 uM L-arginine (pH 7.15) continuously bubbled with 95% O2, 5% CO2. The tendons of each muscle were then clamped in micro tissue clamps and suspended se parately between platin um field electrodes in water-jacketed micro tissue baths containing oxygen ated buffer solution and maintained at 29C. After a 30 min equilibration period, each muscle wa s set to its optimal length (Lo) by repeated isometric twitches while gradually increasing mu scle length. After determining Lo the buffer was washed from the baths and reloaded and equilibrated for an additional 10 min. One EDL and one soleus muscle of each animal was subjected to electrical stimulation or 5aminoimidazole-4-carboxamide-1-D-ribonucleoside (AICAR) treatment. Following the equilibration period, contraction was induced by electrical stimulation delivered at 10 Hz, 13 V for 10 min. For AICAR treatment, 25 mM AICAR solution was added to the baths with buffer for an incubation of 20 min. The contralateral ED L and soleus muscles served as non-contracted or non-AICAR treated controls. Muscles from half of the mice were treated with 1mM N(G)-

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31 monomethyl-L-arginine (L-NMMA) to inhibit NOS activity duri ng electrical stimulation or AICAR treatment. Immediately following the expe rimental treatment, muscles were blotted and frozen for subsequent protein isolation and quantification. Western Blotting Protein levels of total AMPK, phosphoryl ated AMPK, and phosphorylated acetyl CoA carboxylase (ACC) were determined by standard immunoblotting technique. All muscles were homogenized using glass-on-glass technique in 2X homogenizing buffer: 20 mM Tris (pH7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% N onidet P-40, 2.5 mM s odium pyrophosphate, 1 mM -glycerol phosphate, 1 mM sodium orthovanadate, 1 g/ml leupeptin, 1 mM PMSF, and 10 g/ml aprotinin containing 1% vol/vol phosphata se inhibitor (p-5726) from Sigma. Protein concentrations were measured using the DC Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA). Aliquots of muscle homogenates (16 g) were run in SDS-PAGE gels for phospho-ACC, phosphoand total AMPK blots. The primary antibodies used are as follows: rabbit antiAMPK and anti-phosphoAMPK (1:1000 dilution; Cell Signaling), rabbit antiphosphoACC (1:500 dilution; Up state). Ponceau stain and -actin blots were used to control for loading. Reactions were develope d by using the enhanced chemilu minescence 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). Statistical Analysis Treatm ent main effects were analyzed by 2-way ANOVA (E-stim or AICAR vs. LNMMA) with repeated measure on E-stim or AICAR. Individual group differences were assessed by paired t-tests with Bonferronis correction for multiple tests applied.

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32 Figure 3-1. Experimental design flowchart. Electrical stimulation muscles were subjected to 10 minutes of stimulation; half of the muscles were treated with N(G)-monomethyl-L-arginine (L -NMMA) to inhibit NOS activity. 5-aminoimidazole-4carboxamide-1-D ribonucleoside (AICAR) muscles were subjected to 20 minutes of AICAR treatment; half of the muscles were co-treated with L-NMMA to inhibit NOS activity. 30 minute equilibration 10 minute incubation Find Lo; wash baths add buffer and/or LN MMA Hang muscles in bath 10 minute contraction Electrical stimulation Freeze muscle Electrical Stimulation Protocol 30 minute equilibration 10 minute incubation Find Lo; wash baths, add buffer and/or LNMMA Hang muscles in bath 20 minute incubation Wash baths, add buffer, L-NMMA and/or AICAR Freeze muscle AICAR Incubation Protocol

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33 Experiment #1 Experiment #2 Electrical Stimulation AICAR Incubation Figure 3-2. Experimental groups chart. Animals were divided based on electrical stimulation, AICAR treatment or L-NMMA treatment. L-NMMA + EDL (n=5) Soleus (n=5) EDL (n=5) Soleus (n=5) E-Stim + EDL (n=5) Soleus (n=5) EDL (n=5) Soleus (n=5) L-NMMA + EDL (n=7) Soleus (n=6) EDL (n=7) Soleus (n=6) AICAR + EDL (n=7) Soleus (n=6) EDL (n=7) Soleus (n=6)

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34 CHAPTER 4 RESULTS Electrical Stimulation Phospho-/Total-AMPK Ratio EDL. To exam ine the effects of electrical stimulation and NOS inhibition on AMPK activation, a 2-way repeated m easures ANOVA was conducted. The main effect of electrical stimulation on the ratio of phospho-to-t otal AMPK was near significant ( p = .082) in the EDL. Further, we observed a trend of L-NMMA in creasing the phospho-AMPK/total AMPK ratio at rest in the EDL though there was not a significant main effect of NOS inhibition. Comparison of individual groups revealed that electrical stimulation caused a si gnificant, ~ 4 fold, increase in the phospho-AMPK/total AMPK ratio, while NOS inhibition by L-NMMA abrogated the electrical stimulation effect in the muscle (Figure 4-1). Soleus. Analysis of the soleus reveal ed a significant main effect of electrical stimulation on phospho-AMPK/total AMPK ratio; however, a mi nimal increase in AMPK activation with electrical stimulation alone was obs erved (Figure 4-2). There was not a statistical main effect of L-NMMA on AMPK activation ( p = .065). Interestingly, indivi dual group comparison revealed that L-NMMA treatment during electrical stimulation increa sed AMPK activation above LNMMA treatment alone. Total-AMPK, Phospho-AMPK, and Phospho-Acetyl Co-A Carboxylase(ACC) Quantification of phospho-AMPK, total AMPK and phospho-ACC data (m eans SEM) normalized to the control mean for the EDL and soleus is provided in Table 4-1. Electrical stimulation and NOS inhibition of the EDL did not produce significant main effects on these proteins, although phospho-AMPK wa s elevated in the el ectrical stimulation, LNMMA, and electrical stimulation plus L-NM MA groups, compared to control levels.

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35 In the soleus, electrical stim ulation and NOS inhibition did not have a significant effect on total AMPK levels. In contrast, there was a significant effect of electrical stimulation and NOS inhibition on phospho-AMPK. Although no cha nges in phospho-ACC reached statistical significance in the electrical s timulation study, the electrical s timulation main effect on phosphoACC in the soleus was nearing significance ( p = .051). 5-aminoimidazole4-carboxamide-1-D-ribofuranoside(AICAR) Phospho-/Total-AMPK Ratio EDL. To exam ine the effects of AICAR tr eatment and NOS inhibition on AMPK activation a 2-way repeated m easures ANOVA was conducted. There was a significant main effect of AICAR treatment on phospho-AMPK/total-AMPK ratio ( p = .013) in the EDL. There was not a significant main effect of L-NMMA on AMPK activation. AICAR treatment caused a significant, ~ 2 fold increase in the phospho-AM PK/total AMPK ratio. However, NOS inhibition by L-NMMA incubation blunted the AICAR effect (Figure 4-3). Soleus. Analysis of the soleus revealed no signi ficant main effects of AICAR treatment or NOS inhibition on AMPK phosphorylation status. However, it is important to note that the AICAR main effect was nearing significance ( p = .107). (Figure 4-4). Total-AMPK, Phospho-AMPK, and Phospho-ACC Quantification of phospho-AMPK, total AMPK and phospho-ACC data (m eans SEM) normalized to the control mean is provided in Table 4-2 for the EDL and soleus. AICAR or LNMMA treatment did not have a significant effect on these protei ns in the EDL or soleus.

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36 Table 4-1. Quantification of phospho-AMPK, total AMPK and phospho-ACC/ actin levels for the EDL and soleus for electrical stimula tion experimental groups. Data were normalized to the mean of the contro l group. Values represent mean SEM. EDL=extensor digitorum longus, E-stim=10min of in vitro electrical stimulation (see text for details), L-NMMA=L-NG-monomethyl Arginine citrate. *Significantly different from control. Control E-stim L-NMMA E-stim + LNMMA EDL p-AMPK 1.000 .754 5.877 1.939* 2.566 .754 3.169 1.939 t-AMPK 1.000 .201 1.054 .250 1.108 .201 1.334 .250 p-ACC 1.217 .409 1.113 .44 .841 .366 1.534 .393 Soleus p-AMPK 1.000 .305 .899 .192 1.328 .341 2.162 .215 t-AMPK 1.000 .295 .965 .300 1.376 .330 1.388 .335 p-ACC 1.000 .253 .844 .370 .659 .326 2.322 .478*

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37 Table 4-2. Quantification of phospho-AMPK, total AMPK and phospho-ACC/ actin levels for the EDL and soleus for the AICAR experime ntal groups. Data were normalized to the mean of the control group. Values represent mean SEM. AICAR = 5aminoimidazole-4-carboxamide-1 -D-ribofuranoside (1 mM). Control AICAR L-NMMA AICAR + LNMMA EDL p-AMPK 1.009 .318 1.289 .269 1.668 .318 1.673 .269 t-AMPK 1.000 .105 .862 .114 .935 .105 .897 .114 p-ACC 1.000 .273 1.741 .392 1.319 .273 1.193 .392 Soleus p-AMPK 1.000 .827 3.176 1.193 2.547 .906 3.117 1.307 t-AMPK 1.000 .474 1.804 .517 1.588 .520 1.482 .567 p-ACC .965 .555 2.826 1.634 2.076 .555 3.661 1.634

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38 phospho-AMPK AMPK Arbitrary Units (Normalized to control mean)A. B. Figure 4-1. EDL phospho-AMPK/ AMPK ratio for electrical stimulation experiment. (A) Representative immunoblot from control, electrical stimul ation, L-NMMA and LNMMA + electrical stimulati on for phospho-AMPK and total( ) AMPK. (B) Quantification of immunoblots for phos pho-AMPK/total AMPK ratio. Values represent mean SEM. L-NMMA = L-mono methyl Arginine citrate. E-stim = electrical stimulation. *Significan tly different from control.

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39 Arbitrary Units (Normalized to control mean) phospho-AMPK AMPK B. A. Figure 4-2. Soleus phospho-AMPK/ AMPK ratio for electrical st imulation experiment. (A) Representative immunoblot from electrical stimulation, LNMMA and L-NMMA + electrical stimulation for phospho-AMPK a nd totalAMPK. (B) Quantification of immunoblots for phospho-AMPK/total AMPK rati o. Values represent mean SEM. *Significantly different from control.

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40 Arbitrary Units (Normalized to control mean) phospho-AMPK AMPK A. B. Figure 4-3. EDL phospho/total AMPK ratio fo r AICAR experiment. (A) Representative immunoblot from control, AICAR, L-NMMA and L-NMMA + AICAR for phosphoAMPK and total AMPK. (B) Quantification of immunoblots for phosphoAMPK/total AMPK ratio. Values represent mean SEM. AICAR =5aminoimidazole-4-carboxamide-1-D ribonucleoside (1 mM). *Significantly different from control.

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41 Arbitrary Units (Normalized to control mean)phospho-AMPK AMPK A. B. Figure 4-4. Soleus phospho/total AMPK ratio fo r AICAR experiment. (A) Representative immunoblot from control, AICAR, LNMMA and L-NMMA + AICAR for phosphoAMPK and total( ). (B) Quantification of im munoblots for phospho-AMPK/total AMPK ratio. Values represent mean SEM. AICAR =5-aminoimidazole-4carboxamide-1-D ribonucleoside (1 mM). *Signifi cantly different from control.

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42 CHAPTER 5 DISCUSSION Main Findings Our observations are based on relatively short-te rm exposure of isolated skeletal m uscles to AMPK-activating stimuli. The data support our hypotheses that electrical stimulation or AICAR treatment is sufficient to increase AMPK activation, in vitro, in the fast-twitch EDL muscle, and that NOS activity is necessary for this affect. Inhibi tion of NOS abrogated electrical stimulationand AICAR-induced activation of AMPK in the EDL; however interpretation is complicated by a trend for L-NMMA treatment to induce phosphorylation of AMPK. The main findings of this study are: 1) electrical stimul ation or AICAR incubation increases the phosphoAMPK/total AMPK ratio; 2) NOS inhibition prevents this increase; 3) AMPK activation by electrical stimulation and AICAR treatment is gr eater in the fast twitch EDL muscle than the slow twitch soleus muscle; and 4) L-NMMA treatment tends to increase AMPK phosphorylation status, independent of electrical stimulation or AICAR effects. Electrical Stimulation and AICAR Treatment Induces AMPK Activation Activation of AMPK in response to exercise or hypoxia is a critical m echanism required for normal metabolic regulation as well as nNOS and eNOS activity (8, 9, 14). Our data, in agreement with several studies (14, 22, 38, 60) demonstrates that AMPK is an exerciseresponsive gene, activated by increases in intra cellular AMP/ATP concentr ations. The greater the metabolic stress placed on skeletal muscle fibe rs, the greater the activation of AMPK (17). Our stimulation protocol induced greater AMPK ac tivation in the EDL compared to the soleus. The level of metabolic stress pl aced on the oxidative fibers of the soleus was not great enough to induce a significant increas e in AMPK activation.

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43 NOS Inhibition Decreases Electrical Stimulationand AI CAR-Induced AMPK Activation NOS inhibition decreased the level of AMPK activation in the EDL following electrical stimulation and AICAR treatment. We recently reported experiments in cultured L6 myotubes demonstrating that AICAR-induced AMPK phos phorylation is nitric oxide-dependent (34). Further, we found that nitric oxide donors are sufficient to induce AM PK phosphorylation (34). Our working hypothesis to explain these data involves the convergence of AMP binding to AMPK (induced experimentally be the AMP mimetic, AICAR) and NO-dependent activation of AMPK kinases. Both of these events seem to be required to induce AMPK phosphorylation and downstream signaling (Figure 5-1). The current experiments were designed to confirm that these mechanisms are functional in adult skeletal muscle. We predicted that L-NMMA treatment would prevent AMPK phosphorylation induced by either contractile activity or AICAR treatment in adult skeletal muscle, in vitro. The current data indicate that NO plays an important role in the regulation of AMPK phosphorylation in contracting or metabolically active muscle fibers. It appears that NOS inhibition caused a nonspecific eff ect on the phospho-AMPK/total AMPK ratio in the soleus. Understanding this in teraction is complicated as NO is intricately involved in numerous physiological processes. The exact mechanisms whereby NO exerts its effects on skeletal muscle contractility and meta bolism remain uncertain. It is well established that NOS inhibition increases force production by slowing cro ss bridge cycling, decreasing ATPase activity and AMP levels and increasing oxidative phosphorylation. Logically, this would suggest that basal AMPK activation would be re duced following treatment with a NOS inhibitor. Nevertheless, our results suggest the opposite effect. Interest ingly, NOS inhibition decreases maximum shortening velocity in mixed fiber type s but has no affect on the velocity of unloaded shortening (determined by fast fibers) suggestin g the possibility of a fiber-type-specific, NO-

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44 dependent augmentation of ATPase activity (2, 3 6, 39) In contrast, NO thiol nitrosylation can reduce ATPase activity and fo rce production (28, 62). Redox balance is necessary for homeostasis and optimal functioning of numerous physiological interactions and may be an underlying condition affecting our results. The role of NO as a signaling molecule has been well descri bed in skeletal muscle (57). Most of the physiological actions of NO are the result of its ab ility to stimulate gua nylate cyclase, thereby increasing the production of cGMP (57). Nevertheless, NO is a fr ee radical capable of inducing oxidative stress. Although it is not highly reactiv e itself, it can react with other compounds to produce more toxic species (46, 70). Most notab ly, NO reacts with superoxide to produce peroxynitrite, a highly reactive spec ies that can oxidize lipids, proteins and nucleic acids (46). Paradoxically, due to NOs low redox potential a nd its ability to combin e with more reactive species and convert them to less reactive products, it is capable of acting as an antioxidant as well as a pro-oxidant (46, 70). The disparate effects of NO in tissue (i.e. positive signaling and antioxidant effects vs. negative pro-oxidant effects) appear to be primarily determined by concentration, and perhaps localization of the NO signal. The constitutive, calcium-dependent NOS isoforms (nNOS and eNOS) produce nanomolar concentrations of NO, which are sufficient for activation of guanylate cyclase, and produ ce antioxidant effects (46, 57, 70). Conversely, iNOS which is expressed by neutrophils or macr ophages, or by muscle cells in response to oxidative stress, endotoxin, or inflammatory cytokine signaling, produces 100 to 1000 fold higher concentrations of NO leading to nitrosylat ion and oxidant stress (50, 71). We believe that NO concentration regulates the equilibrium betw een reduced and oxidized states within cells, similar to that proposed by other research teams in relation to satellite cell activation and NO concentration (1). At low and hi gh concentrations of NO, the cell is placed in a state of oxidized

PAGE 45

45 stress. An optimal concentration of NO exists creating an environment wherein skeletal muscle metabolism can transpire without placing excess stress on the cell. This idea could explain why lowering NO concentration by treatment with LNMMA (a non-isoform-sp ecific NOS inhibitor) tended to increase basal me tabolic stress and induce AMPK phosphorylation. Meanwhile, activation of AMPK during contractile activity or elevated AMP concentration (mimicked by AICAR) requires NO. ACC Phosphorylation Our electrical stimulation and AICAR treatment protocols were not sufficient to induce significant main effects on phosphoryl ation of the AMPK target, ACC, in the soleus or EDL, most likely due to the relatively short inc ubation times (10 min for E-stim and 20 min for AICAR). The only significant group difference was observed between c ontrol and electrical stimulation groups in the L-NMMA -treated soleus muscles. Although contractile performance data was not recorded for this study, we consis tently observed an increased force production during electrical stimulation plus L-NMMA treatment in the soleus (~50% increase) compared to electrical stimulation alone. Consistent with this greater force produc tion, there were strong trends for AMPK and ACC phosphorylation to be elevated in these samples above L-NMMA alone ( p = .051). Apparently, there is a fiber type-specific eff ect of NOS inhibition during electrical stimulation since only the soleus demons trated this synergistic effect of contractile activity and NOS inhibition on AMPK activation. This could be due to a general mitochondrial dysfunction in the mitochondria-rich slow-twitch fibers of the so leus leading to exaggerated metabolic stress during contractile activity. Limitations and Future Directions The electrical stimulation protocol was not sufficient to cause significant changes in AMPK activation in the soleus most likely because of its oxidative phenotype. Stimulation for a

PAGE 46

46 longer period of time most likely would have activated AMPK to a higher degree. Future studies need to increase the time of stim ulation to see these changes. Our limited ability to probe for oxidative enzy mes and other proteins made it difficult to interpret our results. Data on ATPase, total ACC and cytochrome-c oxidase may have given additional insight as to wh at changes occurred as a result of our protocol. A dose-response experiment needs to be performed to test our theory that NO concentration regulates a balance between reducti on and oxidation in healthy cells. In addition, future studies should examine the effects of a high-intensity electrica l stimulation protocol imitating a strength or power exercise to de termine how exercise intensity affects AMPK activation. Conclusions NOS activity is involved in activation of AM PK during chronic elect rical stimulation or AICAR treatment. To date, the full extent of NOS involvement in AMPK activation and exercise-induced metabolic adap tations is not known. Further studies concentrated on NOS activity and AMPK are needed to investigate this relationship.

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47 Figure 5-1. Proposed model illustra ting potential role of nitric oxi de in the activation of AMPK. NO may facilitate activation of AMPK by increasing AMPK kinases and/or decreasing phosphatases. I nhibiting NOs metabolic signa l regulation may have affected our results. + + + NOS Mitochondrial biogenesis, GLUT 4 upregulation AMPK AICAR AMP Electrical Stimulation + AMPKK Phosphatases Contractile Activity (ATP NO ?

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48 LIST OF REFERENCES 1. Anderson JE A role f or nitric oxide in muscle repair: NO-mediated satellite cell activation. Mol Biol Cell 11: 1859-1874, 2000. 2. Andrade FH, Reid MB, Allen DG, Westerblad H Effect of nitric oxide on single skeletal muscle fibres from the mouse. J Physiol 509: 577-586, 1998. 3. Balon TW, and Nadler JL Nitric oxide release is present from incubated skeletal muscle preparations. J Appl Physiol 77: 2519-2521, 1994. 4. Balon TW, Nadler JL Evidence that nitric oxide increases glucose transport in skeletal muscle. J Appl Physiol 82: 359-363, 1997. 5. Bergandi L, Silvagno F, Russo I, Riganti C, Anfossi G, Aldieri E, Ghigo D, Trovati M, Bosia A Insulin stimulates glucose transport via nitric oxide/cyclic GMP pathway in human vascular smooth muscle cells. Athersclerosis, Thrombosis, and Vascular Biology 23: 2215, 2003. 6. Brooks SV, Faulkner JA Contractile properties of skeletal muscles from young, adult and aged mice. J Physiol 404: 71-81, 1987. 7. Carling D, Zammit VA, Hardie DG A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett 223: 217, 1987. 8. Chen ZP, McConell GK, Michell BJ, Snow RJ, Canny BJ, Kemp BE AMPK signaling in contracting hu man skeletal muscle: a cetyl-CoA carboxylase and NO synthase phosphorylation. Am J Physiol Endocrinol Metab 279: E1202E1206, 2000. 9. Chen ZP, Mitchelhill KI, Mich ell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285, 1999. 10. Dagher Z, Ruderman N, Tornheim K, Ido Y Acute regulation of fatty acid oxidation and AMP -activated protein kinase in human umbilical vein endothelial cells. Circ Res 88: 1276-1282, 2001. 11. Deshmukh AS, Treebak JT, Long YC, Vi ollet B, Wojtaszewski JFP, Zierath JR Role of AMPK subunits in sk eletal muscle mTOR signaling. Mol Endocrinol [Epub ahead of print], 2008. 12. Dyck JR, Kudo N, Barr AJ, Davi es SP, Hardie DG, Lopaschuk GD Phosphorylation control of cardiac acetyl Co-A carboxylase by cAMP-dependent protein kinase and 5AMP activated protein kinase. Eur J Biochem 262: 184-90, 1999. 13. Frstermann U, Boissel J, Kleinert H Expressional control of the constitutive isoforms of nitric oxide s ynthase (NOS I and NOS III). FASEB J 12: 773-790, 1998.

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50 26. Katsuki S, Arnold W, Mittal C, Murad F Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine. J Cyclic Nucleotide Res 3: 23-35, 1977. 27. King TJ, Song L, Jope RS. AMP-activated protein kinase (AMPK) activating agents cause dephosphorylation of Akt a nd glycogen synthase kinase-3. Biochem Pharmacol 71: 1637-1647, 2006. 28. Klebl BM, Ayoub AT, Pette D Protein oxidation, tyrosine nitration, and inactivation of sarcoplasmic reticulul Ca2+-ATPase in low-frequency stimulated rabbit muscle. FEBS Lett 422(3): 381-384, 1998. 29. Kobzik L, Reid MB, Bredt DS, Stamler JS Nitric oxide in skeletal muscle. Nature 372: 546-548, 1994. 30. Kramer H, Goodyear L Exercise, MAPK, and NF b signaling in skeletal muscle. J Appl Physiol 103: 388-395, 2007. 31. Kubica N, Bolster DR, Farrell PA, Kimball SR, Jefferson LS Resistance exercise increases muscle protein synthesis and tran slocation of eukaryo tic initiation factor 2Bepsilon mRNA in a mammalian target of rapamycin-dependent manner. J Biol Chem 280: 7570-7580, 2005. 32. Lau KS, Grange RW, Chang WJ, Kamm KE, Sarelius I, Stull JT. Skeletal muscle contractions stimulate cGMP formation and attenuates vascular smooth muscle myosin phosphorylation via nitric oxide. FEBS Lett 431: 71-74, 1998. 33. Li J, Hu X, Selvakumar P, Russell III RR, Cushman SW, Holman GD, Young LH Role of nitric oxide pathway in AM PK-mediated glucose uptake and GLUT4 translocation in heart muscle. Am J Physiol Endocrinol Metab 287: E834-E841, 2004. 34. Lira VA, Soltow QA, Long JHD, Betters JL, Sellman JE, Criswell DS Nitric oxide increases GLUT4 expression and regulates AMPK signaling in skeletal muscle. Am J Physiol Endocrinol Metab 293: E1062-E1068, 2007. 35. Madhusoodanan KS, Murad F. NO-cGMP signaling and regenerative medicine involving stem cells. Neurochemical Research 32: 681-694, 2006. 36. Marchal G, Beckers-Bleukx G Effect of nitric oxide on the maximal velocity of shortening of a mouse skeletal muscle. Pflgers Arch 436: 906-913, 1998. 37. McConell GK, Huynh NN, Lee-Yo ung RS, Canny BJ, Wadley GD L-Arginine infusion increases glucose clearance dur ing prolonged exercise in humans. Am J Physiol Endocrinol Metab 290: E60-E66, 2006.

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51 38. Merrill GF, Kurth EJ, Hardie DG, Winder WW AICA riboside increases AMPactivated protein kinase, fatty acid oxidat ion and glucose uptake in rat muscle. AM J Physiol 273: E1101-E1112, 1997. 39. Morrison RJ, Miller CC, Reid MB Nitric oxide effects on shortening velocity and power production in the rat diaphragm. J Appl Physiol 80:1065-1069, 1996. 40. Mount PF, Lane N, Venkatesan S, Stei nberg GR, Fraser SA, Kemp BE, Power DA Bradykinin stimulates endothelial cell fa tty acid oxidation by CaMKK-dependent activation of AMPK. Atherosclerosis [Epub ahead of print], 2008. 41. Ogonowski AA, Kaesemeyer WH, Jin L, Ganapathy V, Leibach FH, Caldwell RW Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production. Am J Physiol Cell Physiol 278: C136-C143, 2000. 42. Patwell DM, McArdle A, Morgan JE, Patridge TA, Jackson MJ Release of reactive oxygen and nitrogen species from contracting skeletal muscle cells. Free Radical Biology & Medicine 37: 1064-1072, 2004. 43. Perkins W, Han Y, Sieck G Skeletal muscle force and actomyosin ATPase activity reduced by nitric oxide donor. J Appl Physiol 83: 1326-1332, 1997. 44. Pilon G, Dallaire P, Marette A. Inhibition of inducible nitric oxide synthase by activators of AMP-activated protein kinase: a new mechanism of action of insulinsensitizing drugs. J Biol Chem 279: 20767, 2004 45. Pye D, Palomero J, Kabayo T, Jackson MJ Real-time measurement of nitric oxide in single mature mouse skeletal muscle fibres during contractions. J Physiol 581: 309-318, 2007. 46. Rando TA Role of nitric oxide in the pathogenesi s of muscular dystrophies: a "two hit" hypothesis of the cause of muscle necrosis. Microsc Res Tech 55(4):223-35, 2001. 47. Reid M Plasticity in skeletal, cardiac a nd smooth muscle invited review: redox modulation of skeletal muscle contract ion: what we know and what we dont. J Appl Physiol 90:724-731, 2001. 48. Reiser PJ, Kline WO, Vaghy PL Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic el ectrical stimulation in vivo. J Appl Physiol 82: 1250-1255, 1997. 49. Rez nick RM, Zong H, Morino K, Moore IK, Yu HJ, Liu Z, Dong J, Mustard KJ, Hawley SA, Befroy D, Pypaert M, Hardie DG, Young LH, Shulman GI Agingassociated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis. Cell Metab 5: 151-156, 2007.

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52 50. Ridnour LA, Thomas DD, Donzelli S, Es pey MG, Roberts DD, Wink DA, Isenberg JS The biphasic nature of nitric oxide responses in tumor biology. Antioxid Redox Signal 8(7-8):1329-37, 2006. 51. Sakamoto K, Goodyear L Exercise effects on muscle insulin signaling and action invited review: intracellular signaling in contracting skeletal muscle. J Appl Physiol 93: 369-383, 2002. 52. Sakamoto K, McCarthy A, Smith D, Green KA, Hardie DG, Ashworth A, Alessi DR. Deficiency of LKB1 in skeletal musc le prevents AMPK activation and glucose uptake during contraction. EMBO J 24: 1810-1820, 2005. 53. Sanders MJ, Grondin PO, Hegarty BD, Snowden MA, Carling D. Investigating the mechanism for AMP activation and the AM P-activated protein kinase cascade. Biochem J 403: 139-148, 2007. 54. Sayed N, Baskaran P, Ma X, Al Beuve F Desenstization of soluble guanylyl cyclase, the NO receptor nitrosylation. Proc Natl Acad Sci USA 104: 12312-12317, 2007. 55. Silveira LR, Pereira-Da-Silva L, Juel C, Hellsten Y Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells dur ing contraction. Free Radical Biology & Medicine 35: 455-464, 2003. 56. Smith LW, Smith JD, Criswell DS Involvement of nitric oxide synthase in skeletal muscle adaptation to chronic overload. J Appl Physiol 92: 2005-2011, 2002. 57. Stamler JS, Meissner G. Physiology of nitric oxi de in skeletal muscle. Physiological Reviews 81: 209-237, 2001. 58. Suter M, Riek U, Tuerk R, Schlat tner U, Wallimann T, Neumann D Dissecting the role of 5-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase. J Biol Chem 281: 32207-32216, 2006. 59. Suzuki J. Microvascular angioadaptation afte r endurance training with L-arginine supplementation in rat heart and hindleg muscles. Exp Physiol 90: 763-771, 2005. 60. Thomson DM, Fick CA, Gordon SE AMPK activation attenuates S6K1, 4E-BP1, and eEF2 signaling responses to high-frequency el ectrically stimulated skeletal muscle contractions. J Appl Physiol 104: 625-632, 2008. 61. Tow ler MC, Hardie DG AMP-activated protein kinase in metabolic control and insulin signaling. Circ Res 100: 328-341, 2007. 62. Viner RI, Williams TD, Schneich C. Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functi onally important cysteine residue(s) in the sarcoplas mic reticulum Ca-ATPase. Biochemistry 38: 12408-12415, 1999.

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53 63. Vassilakopoulos T, Hussain SNA Ventilatory muscle activation and inflammation: cytokines, reactive oxygen sp ecies and nitric oxide. J Appl Physiol 1273, 2006. 64. Verhoeven AJ, Woods A, Brennan CH, Hawl ey SA, Hardie DG, Scott J, Beri RK, Carling D The AMP-activated protein kinase gene is highly expresse d in rat skeletal muscle. Alternative splicing and tissue distribution of the mRNA. Eur J Biochem 228: C283-C292, 1995. 65. Wadley GD, Choate J, McConnell GK NOS isoform-specific regulation of basal but not exercise-induced mitochondrial bioge nesis in mouse skeletal muscle. J Physiol 585:253-262, 2007. 66. Wadley GD, McConnell GK Effect of nitric oxide syntha se inhibition on mitochondrial biogenesis in rat skeletal muscle. J Appl Physiol 102: 314-320, 2007. 67. Williams G, Brown T, Becker L, Prager M., Giroir BP Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes. Am J Regul Integr Comp Physiol 267: R1020 R1-25, 1994. 68. Williamson DL, Bolster DR, Kimball SR, Jefferson LS. Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR. Am J Physiol Endocrinol Metab 291: 80-89, 2006. 69. Winder WW, Holmes BF, Rubink DS, Jensen EB, Chen M, Holloszy JO Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle. J Appl Physiol 88: 2219-2226, 2000. 70. Wink DA, Mitchell JB Chemical biology of nitric ox ide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25:434 456, 1998. 71. Xie Q, Nathan C The high-output nitric oxide pathway: role and regulation. J Leukoc Biol 56(5):576-82, 1994.

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54 BIOGRAPHICAL SKETCH Dana Brown was born in Fort W alton Beach, Fl orida, in 1984. She graduated in the top 3% of her high school in 2002. She received a Bach elor of Science degree in exercise and sport sciences in 2006 from the Univer sity of Florida, where she was a member of the Golden Key Honor Society and Social Entrepreneurship and an employee of UFs Department of Recreational Sports. Dana will begin an additi onal Master of Science degree in business entrepreneurship at the Univers ity of Florida beginning in the summer of 2008. After finishing school Dana plans to conti nue working in the field of health and fitness.


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