University of Iowa
Iowa Research Online
Theses and Dissertations
Summer 2012
Investigating the potential relationship between
skeletal muscle atrophy and obesity
Christopher John Elmore
University of Iowa
Copyright 2012 Christopher John Elmore
This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/3289
Recommended Citation
Elmore, Christopher John. "Investigating the potential relationship between skeletal muscle atrophy and obesity." MS (Master of
Science) thesis, University of Iowa, 2012.
http://ir.uiowa.edu/etd/3289.
Follow this and additional works at: http://ir.uiowa.edu/etd
Part of the Neuroscience and Neurobiology Commons
INVESTIGATING THE POTENTIAL RELATIONSHIP BETWEEN SKELETAL
MUSCLE ATROPHY AND OBESITY
by
Christopher John Elmore
A thesis submitted in partial fulfillment
of the requirements for the Interdisciplinary StudiesMaster of Science degree in Molecular and Cellular Biology
in the Graduate College of
The University of Iowa
July 2012
Thesis Supervisor: Associate Professor Christopher M. Adams
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
MASTER'S THESIS
_______________
This is to certify that the Master's thesis of
Christopher John Elmore
has been approved by the Examining Committee
for the thesis requirement for the Interdisciplinary StudiesMaster of Science degree in Molecular and Cellular Biology
at the July 2012 graduation.
Thesis Committee:__________________________________
Christopher M. Adams, Thesis Supervisor
__________________________________
Christopher Benson
__________________________________
David Motto
To my spouse, who makes my life better than I ever imagined.
To my family, who have way more confidence in my abilities than I do.
To my mentors and educators, for their commitment of time, and ability to inspire
curiosity.
ii
Education is no substitute for intelligence. That elusive quality is defined only in
part by puzzle-solving ability. It is in the creation of new puzzles reflecting what
your senses report that you round out the definition.
Frank Herbert
Dune: Chapterhouse
iii
ACKNOWLEDGMENTS
Thank you to Christopher Adams and all members of the Adams lab for
their advice, support, and most of all laughter.
iv
ABSTRACT
Skeletal muscle atrophy is the most common clinical disorder of skeletal
muscle and typically occurs as a secondary consequence of fasting, disuse,
acute and chronic illness, and aging. It can lead to prolonged recovery and loss
of independent living. Of similar clinical significance, one third of Americans are
obese and at risk for metabolic syndrome. Interestingly recent studies have
demonstrated that both metabolic syndrome and obesity diminish skeletal muscle
strength, power, and endurance. However, there are no effective
pharmacological treatments for these debilitating effects on skeletal muscle. This
is largely due to the fact that the molecular mechanisms underlying its
pathogenesis remain uncharacterized. We have recently identified ursolic acid
(UA) as a small molecule inhibitor of muscle atrophy. In the absence of atrophyinducing stress, UA-supplemented chow elicited muscle hypertrophy with little
adiposity in mice. To further evaluate these data, mice were subjected to a high
fat diet (HFD) with or without UA supplementation, or a standard chow (SC)
control. Our data indicates that UA-supplemented HFD mitigates muscle atrophy
and adiposity, while HFD significantly reduces muscle mass compared to SC.
Furthermore, mice fed a HFD exhibited increased adiposity and reduced muscle
mass, strength, and fiber diameter when compared to SC controls. Molecular
analysis revealed diminished protein content and increased triglycerides. Gene
expression analysis revealed a reduction in Pgc1!, a critical gene that regulates
oxidative metabolism and mitochondrial biogenesis. Additionally, we found
decreased expression of hormonal receptors AR, involved in signaling of
testosterone, and Thr!, involved in signaling of thyroid hormones. Taken
together, these data suggest that alterations in gene expression resulting from
v
diet-induced obesity are an atrophy-inducing stress that may function by
disrupting metabolic and hormonal signaling.
vi
TABLE OF CONTENTS
LIST OF FIGURES .............................................................................................. viii
LIST OF ABBREVIATIONS ................................................................................... x
CHAPTER
I.
INTRODUCTION .................................................................................. 1
Skeletal Muscle Atrophy ....................................................................... 1
Skeletal Muscle Physiology .................................................................. 1
Skeletal Muscle in Obesity ................................................................... 2
ATF4/Gadd45a/Cdkn1a Pathway......................................................... 4
Preliminary Results .............................................................................. 5
II.
MATERIALS AND METHODS ........................................................... 13
Animal Protocols ................................................................................ 13
Skeletal Muscle Histology .................................................................. 13
Skeletal Muscle Composition ............................................................. 14
Skeletal Muscle mRNA Analysis ........................................................ 14
III.
RESULTS ........................................................................................... 16
High Fat Diet-induced Obesity ........................................................... 16
Skeletal Muscle Weights .................................................................... 16
Skeletal Muscle Fibers ....................................................................... 17
Muscle Composition ........................................................................... 17
Gene Expression ................................................................................ 17
IV.
DISCUSSION ..................................................................................... 41
Interpretation Of Results .................................................................... 41
Future Directions ................................................................................ 44
REFERENCES .................................................................................................... 46
vii
LIST OF FIGURES
Figure
1.1 Novel atrophy pathway involving ATF4/Gadd45a/Cdkn1a. ........................... 7
1.2 UA inhibits weight gain from a HFD. ............................................................. 8
1.3 UA reduces fasting blood glucose on a HFD. ............................................... 9
1.4 UA inhibits hepatomegaly from a HFD. ....................................................... 10
1.5 UA inhibits loss of skeletal muscle mass from a HFD. ................................ 11
1.6 HFD increases expression of Cdkn1a. ........................................................ 12
2.1 HFD increases weight gain. ........................................................................ 19
2.2 HFD increases epididymal fat. .................................................................... 20
2.3 HFD increases retroperitoneal fat. .............................................................. 21
2.4 HFD induces hepatomegaly. ....................................................................... 22
2.5 HFD does not effect cardiac muscle weights. ............................................. 23
2.6 HFD has no effect on kidney weights. ......................................................... 24
2.7 HFD reduces weight of tibialis anterior. ....................................................... 25
2.8 HFD reduces weight of gastrocnemius. ...................................................... 26
2.9 HFD reduces weight of quadriceps. ............................................................ 27
2.10 HFD reduces weight of triceps. ................................................................... 28
2.11 HFD increases the weight of soleus. ........................................................... 29
2.12 HFD reduces weight of hindlimb muscles. .................................................. 30
2.13 HFD changes muscle histology. .................................................................. 31
2.14 HFD induces skeletal muscle atrophy. ........................................................ 32
2.15 HFD reduces fiber size distribution of skeletal muscle. ............................... 33
2.16 HFD reduces grip strength. ......................................................................... 34
2.17 Relationship between body weight and skeletal muscle atrophy. ............... 35
2.18 HFD decreases skeletal muscle protein content. ........................................ 36
2.19 HFD increases content of skeletal muscle triglycerides. ............................. 37
viii
2.20 HFD reduces the ratio of skeletal muscle protein to triglycerides. .............. 38
2.21 Relationship between body weight and skeletal muscle triglycerides. ........ 39
2.22 HFD suppresses gene expression of Pgc1!, AR, Thr!............................... 40
ix
LIST OF ABBREVIATIONS
AA, amino acids
AR, androgen receptor
ATF4, activating transcription factor
ANOVA, analysis of variance
Bnip3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3
bZIP, basic leucine zipper domain
Cdkn1a, cycline dependent kinase inhibitor 1a
cDNA, complementary deoxyribonucleic acid
Ctsl, cathepsin L
DNA, deoxyribonucleic acid
Fbox32, F-box only protein 32
Gadd45a, growth arrest and DNA damage inducible 45a
H&E, hematoxylin and eosin
IGF1, insulin-like growth factor 1
HFD, high fat diet
mRNA, messenger ribonucleic acid
Pgc1!, peroxisome proliferator-activated receptor gamma coactivator 1 alpha
qPCR, quantitative real-time polymerase chain reaction
SC, standard chow
Thr!, thyroid receptor alpha
Trim63, tripartite motif containing 63
UA, ursolic acid
x
1
CHAPTER I
INTRODUCTION
Skeletal Muscle Atrophy
Skeletal muscle atrophy is a frequent consequence of prolonged disease
and commonly attributed to illnesses such as cancer, kidney disease, congestive
heart failure, lung disease, sepsis, and chronic infections like tuberculosis and
HIV/AIDS. Atrophy can also be the result of situations fostering muscle disuse,
such as spinal cord injury, prolonged bedrest, uncontrolled diabetes mellitus, and
neurological disorders like multiple sclerosis. Characterized by a wasting of
muscle, it can lead to complications and extended recovery times.
Muscle atrophy also occurs during aging, making the consequences of
atrophy something that may affect everyone at some point during their lifetime.
Ultimately, skeletal muscle atrophy can leave an individual incapable of working,
and result in loss of independent living. Consequently, skeletal muscle atrophy
has an enormous impact on an individual’s quality of life for the patient, as well
as their families. It also places an immense burden on the healthcare system and
society in general. Currently, we lack effective medical treatments to rehabilitate
or prevent skeletal muscle atrophy, largely due to an inadequate understanding
of its molecular pathogenesis for targeted interventions.
Skeletal Muscle Physiology
During times of starvation or fasting, skeletal muscle serves as a reservoir
for amino acids (AA), where proteins are catabolized and exported via atrophy.
These AA serve in important physiological functions, including protein synthesis
and gluconeogenesis. However, this increased catabolism results in an overall
2
reduction of protein content and muscle mass. This transition is histologically
seen as diminished size, rather than a quantitative reduction in myofibers (1).
Normal adults prevent this effect primarily through proper nutrition, muscle use,
and anaboilic signaling of insulin/IGF1. Starvation, disuse, and uncontrolled
diabetes mellitus, are all circumstances resulting in skeletal muscle atrophy. In
opposition to insulin/IGF1 signaling, other hormones can promote skeletal
muscle atrophy, such as glucocorticoids, inflammatory cytokines, and
autocrine/paracrine factors such as myostatin (1-3). Therefore, skeletal muscle
mass is a balance between catabolic proteolysis and anabolic protein synthesis
within the muscle.
Skeletal Muscle In Obesity
One-third of Americans are presently obese (4). A host of comorbidities
are associated with obesity such as diabetes, heart disease, hypertension, and
stroke. Obesity is one of the key diagnostic criteria for metabolic syndrome, a
group of medical disorders including elevations in triglycerides, blood pressure,
and fasting blood glucose (4). These factors in combination increase the
likelihood of developing coronary artery disease, type 2 diabetes, and stroke (4).
The risk factors for obesity and outcomes from metabolic syndrome display
considerable similarity. Ultimately, metabolic syndrome defines the risk factors
that result from chronic obesity.
As obesity quickly becomes a worldwide epidemic, increasing demands
have been placed on the healthcare system to treat obesity-related diseases.
Studies conducted by the Centers for Disease Control estimate the annual
medical spending for obesity, and obesity-related illnesses, at $147 billion in the
United states alone, and may rise to $1.8 trillion by the year 2018 (5, 6). For the
obese individual, the financial burden of long-term therapies, as well as a
3
progressive decline in health and quality of life, makes prevention the best
option.
Obese individuals suffer from muscle weakness and reduced mobility.
Poor diet and sedentary lifestyle are concomitant risk factors for skeletal muscle
atrophy, obesity, and metabolic syndrome. Typically, dietary intervention and
physical activity are initial therapies when treating metabolic syndrome and
obesity. Research suggests that obesity impairs skeletal muscles ability to
hypertrophy under conditions from either external loading or increased body
weight (7). This makes exercise a poor therapy to rehabilitate muscles in the
obese. Studies elucidating the connection between skeletal muscle and
metabolic syndrome show diminished skeletal muscle strength, power, and
endurance in human models (8, 9). Other data has demonstrated that obesity
increases intramyocellular triglycerides in skeletal muscle, impairs insulin
sensitivity, and decreases strength, in mice and human models (10, 11).
Interestingly, studies of ob/ob mice using estimated cross-sectional areas from
single fiber dissections, suggest an obesity-related reduction in myofiber size
(12). Nevertheless, ob/ob mice are an extreme model of obesity. The use of
single fiber dissection imparts mechanical strain on muscle fibers, making the
estimate calculations an inaccurate technique to evaluate normal muscle
histology. Currently, no evidence supports obesity-related muscle atrophy in a
C57BL/6 mouse using less invasive techniques of histological analysis. This
would allow us to determine the potential remodeling of skeletal muscle in a more
applicable translational model of obesity. Taken together, these data lead us to
hypothesize that obesity is an atrophy-inducing stress that impairs skeletal
muscle function.
4
ATF4/Gadd45a/Cdkn1a Pathway
ATF4 (activating transcription factor 4) is a bZIP transcription factor with
an evolutionarily ancient role in cellular stress signaling (13, 14). Diverse
stresses, including fasting, disuse, insulin deficiency, and systemic disease, can
all increase ATF4 expression in skeletal muscle (15-17). To examine the role of
ATF4 in skeletal muscle, we overexpressed ATF4 by transfection in mouse
tibialis anterior muscles and found it sufficient to reduce fiber size, indicating
muscle fiber atrophy. To determine how ATF4 causes atrophy, we used an
unbiased exon expression array and identify Gadd45a (growth arrest and DNA
damage inducible 45a) as an atrophy-associated ATF4 target gene (18).
Previous studies on Gadd45a elucidate its role in cell-type specific stress
responses, such as growth arrest, differentiation, DNA damage repair, and DNA
demethylation, however its function in skeletal muscle is uncharacterized (19-21)
We used qPCR (quantitative real-time polymerase chain reaction) to confirm that
ATF4 overexpression increases Gadd45a mRNA in mouse skeletal muscle. To
determine the role of Gadd45a in atrophy, we reduced its expression by
transfection in mouse tibialis anterior muscles with miRNAs targeting Gadd45a.
This allowed us to identify Gadd45a as the mediator of a molecular pathway that
is activated by skeletal muscle stress and drives skeletal muscle atrophy.
Furthermore, we determined ATF4 induces Gadd45a expression, and Gadd45a
was found to be required for skeletal muscle atrophy under immobilization,
fasting, and denervation stresses (22).
We next sought to determine the mechanism by which Gadd45a induces
skeletal muscle atrophy. Using the exon expression arrays, we compared the
effects of fasting, as well as Gadd45a overexpression, to identify atrophyassociated transcription targets. This identified Cdkn1a (cyclin-dependent kinase
5
inhibitor 1a) as a Gadd45a target, and were consistent with previous findings that
ATF4 increases Cdkn1a (18). Previous studies have shown Cdkn1a, which
encodes the protein p21, is increased under atrophy stimuli such as aging,
disuse, and denervation in muscle (21, 23-25). Further data, in preparation for
publication, determined that Gadd45a increases the expression of Cdkn1a by
demethylation at a specific 5-methylcytosine in the Cdkn1a promoter region
(Figure 1A).
Preliminary Results
Previous studies using the exon expression arrays from fasting and
denervated skeletal muscle in mice and humans identified conserved atrophyassociated mRNA signatures. These signatures were used to query the
Connectivity Map, which characterizes the effects of more than 1,300 small
molecules on global mRNA expression from several cultured human cell lines.
We identified ursolic acid (UA) as having a strong negatively correlated mRNA
signature to skeletal muscle atrophy (26). Experiments utilizing standard chow
(SC) compared to UA supplemented (0.14% by weight) SC demonstrate
induction of skeletal muscle hypertrophy, increased grip strength, as well as
reductions in adiposity and fasting glucose, likely by increasing insulin/IGF1
signaling (26). These data suggests a potential utility for ursolic acid in the
treatment of obesity and diabetes, leading us to investigate its use in combination
with high fat diet (HFD).
To further evaluate UA’s effects, experiments with mice fed HFD with or
without UA supplementation demonstrate UA reduces obesity, glucose
intolerance, and fatty liver disease, possibly by increases in metabolically active
skeletal muscle and brown fat (27). We next sought to determine the efficacy of
UA in obesity reduction with experiments utilizing HFD with or without UA (0.15%
6
by weight) supplementation, as well as a SC control. We find that HFD (55%
calories from fat) leads to increased total body weight, fasting blood glucose, and
hepatomegaly. UA-supplemented HFD was able to attenuate these effects
(Figure 1A-1C). Interestingly, HFD reduces skeletal muscle weights compared to
SC, with UA supplementation normalizing this effect in mice subjected to HFD
(Figure 1D). This suggests that UA may be working as an atrophy inhibitor under
conditions of HFD-fed mice. Furthermore, preliminary data from HFD-fed mice
show a significant increase in Cdkn1a compared to SC (Figure 1E). Taken
together, these data leads us to hypothesize that obesity is an atrophy-inducing
stress that is at least partly mediated by the ATF4/Gadd45a/Cdkn1a pathway.
7
Figure 1.1 Novel atrophy pathway involving ATF4/Gadd45a/Cdkn1a. Cellular
stress activates translation of ATF4, which translocates to the nucleus. ATF4
gene target, Gadd45a, is transcribed and translated. In the nucleus, Gadd45a
demethylates a specific 5-methylcytosine in the promoter region of Cdkn1a. This
increases transcription of gene, resulting in an increase in its encoding protein
p21. Increases in p21 promote skeletal muscle atrophy.
8
Figure 1.2 UA inhibits weight gain from a HFD. Effect of a HFD with or without
0.15% UA compared to SC after 5 weeks with C57BL/6 mice. Each data point
represents one mouse and horizontal bars denote the means. *P<0.05 by
one-way ANOVA with Dunnett’s post-test using SC as control.
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Figure 1.3. UA reduces fasting blood glucose on a HFD. After 16 hours of
fasting, blood glucose was measured. Each data point represents one mouse
and horizontal bars denote the means. *P<0.05 by one-way ANOVA with
Dunnett’s post-test using SC as control.
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10
Figure 1.4. UA inhibits hepatomegaly from a HFD. Effect of a HFD with or
without 0.15% UA compared to SC after 5 weeks with C57BL/6 mice. Each data
point represents one mouse and horizontal bars denote the means. *P<0.05 by
one-way ANOVA with Dunnett’s post-test using SC as control.
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Figure 1.5. UA inhibits loss of skeletal muscle mass from a HFD. Effect of a
HFD with or without 0.15% UA compared to SC after 5 weeks with C57BL/6
mice. The bars represent the means ±SEM. *P<0.05 by one-way ANOVA with
Dunnett’s post-test using SC as control. n!12 mice per diet.
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Figure 1.6. HFD increases expression of Cdkn1a. Effect of HFD on
expression of Cdkn1a mRNA in triceps after 5 weeks on HFD. Tricep mRNA
levels were determined using qPCR. Levels were normalized to the average
levels in SC. *P<0.05 by t-test. n=6 mice per diet.
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13
CHAPTER II
MATERIALS AND METHODS
Animal Protocols
All experiments utilized 6-8 week old male C57BL/6 mice. They were
obtained from NCI, housed at 21° C in colony cages with 12 hour light/ 12 hour
dark cycles. They were housed 4 to a cage and used for experiments within 2
weeks of their arrival. Prior to experimental diets, mice were maintained on
standard chow (Harlan Teklad formula 7013). The HFD was Harlan Teklad
formula TD93075 and contained 55% of calories from fat. Ursolic acid was
obtained from Enzo Life Sciences and incorporated in the HFD. Mice were fasted
for 16 hours by removing food, but not water. Fasting blood glucose levels were
obtained from the tail vein using an Accucheck Aviva glucometer. Skeletal
muscle weights represent the wet weight obtained at time of necropsy using an
A&D GX-400 scale. Grip strength was determined using a grips strength meter
equipped with a triangular pull bar (Columbus Instruments). Each mouse was
subjected to five consecutive grip strength tests to obtain peak value. The
University of Iowa Institutional Animal Care and Use Committee approved all
mouse protocols.
Skeletal Muscle Histology
Skeletal Muscle tissue was fixed in 10% neutral buffered formalin,
embedded in paraffin, and then sectioned at 8µm using Microm HM355 S
motorized microtome (Microm, Waldorf, Germany). Hematoxylin and eosin stains
were performed using the DRS-601 automatic slide stainer (Sakura, Torrance,
California). Slides were examined on as Olympus IX-71 microscope, with images
14
captured with a DP-70 camera. ImageJ (NIH) software was used to perform
image analysis. Muscle fiber diameter was determined using the lesser diameter
method, where the greater diameter of the lesser axis is measured (normalizing
for variations in cross-section angle).
Skeletal Muscle Composition
For analysis of muscle composition, each snap frozen triceps sample was
weighed and placed in 1ml RIPA buffer (10 mM Tris-HCL, pH 7.4, 150 mM NaCl,
0.1% (w/v) Triton X-100, 1% deoxycholate, 5mM EDTA, 1mM NaF, 1mM Na
orthovanadate, 1µg/ml pepstatin A, 2µg/ml aprotonin, 10µg/ml leupeptin, 1:100
dilution of phosphatase inhibitor cocktail 2 (Sigma) and a 1:100 dilution of
phosphatase inhibitor cocktail 3 (Sigma)). After the tissue was homogenized and
centrifuged, the final volume of the supernatant was measured and the protein
concentration was measured using the BCA kit (Thermo Scientific). To determine
triglyceride content of the tissues, Triglyceride Colorimetric Assay Kit (Cayman
Chemical) was used and analyzed on Spectra Max Plus 384 (Molecular Devices)
spectrophotometer. Analysis of triceps composition was calculated using volume
and concentration to determine total protein or triglycerides in each sample.
These values are normalized by sample weight to represent a percent of tricep
mass.
Skeletal Muscle mRNA Analysis
Skeletal muscle was harvested and samples were immediately placed in
RNAlater (Ambion). Total RNA was extracted using TRIzol solution (Invitrogen)
and then purified using the Turbo DNA-free kit (Ambion). Synthesis of cDNA was
accomplished using High Capacity cDNA Reverse Transcription Kit (part no.
4368814; Applied Biosystems) in a 20µl reaction using 2µg RNA per sample. The
qPCR contained a final volume of 20µl, 20 ng of reverse transcribed RNA, 1 µl of
15
20X TaqMan Primer (Applied Biosystems), and 10µl of TaqMan Fast Universal
PCR Master Mix (TaqMan Fast Universal PCR Master Mix (part no. 4352042;
Applied Biosystems). qPCR was carried out using a 7500 Fast Real-Time PCR
System (Applied Biosystems) in 9600 emulation mode. All qPCR reactions were
performed in triplicate to obtain the average cycle threshold (Ct) values for the
final results. The qPCR data was analyzed using the "Ct method, with the level
of Rplp0 mRNA (encoding ribosomal protein 36B4) serving as our invariant
control.
16
CHAPTER III
RESULTS
High Fat Diet-Induced Obesity
Similar to our previous findings, HFD induced significant weight gain in
mice compared to the SC after only one week of dietary intervention (Figure 2.1).
This significance continues throughout the time course of the experiment, with
the increase resulting in an approximately 4g difference at 6 weeks (Figure 2.1).
Upon necropsy, adipose tissue was significantly greater in HFD-fed mice in the
epididymal and retroperitoneal fat pads, compared to the SC (Figure 2.2-2.3).
HFD-fed mice exhibited significant hepatomegaly, which supports our previous
study involving HFD (Figure 2.4) (26). Importantly, control tissues of the heart
and kidneys are not significantly different from each other between the SC- and
HFD-fed mice (Figure 2.5-2.6).
Skeletal Muscle Weights
To determine if HFD induces skeletal muscle atrophy in mice, select
muscles were dissected and weighed prior to storage for later analysis. Among
the skeletal muscles collected, HFD induced a significant reduction in wet weight
of tibialis anterior, gastrocnemius, quadriceps, and triceps when compared to SC
mice (Figure 2.7-2.10). In contrast, the weight of the soleus is significantly
greater in the HFD-fed mice compared to SC (Figure 2.11). Overall, HFD
reduces skeletal muscle weights of the hindlimb, of which the soleus is a
component (Figure 2.12).
17
Skeletal Muscle Fibers
To demonstrate the significant reduction in weight is specific to myofiber
atrophy, we cross-sectioned paraffin embedded quadriceps muscles. Sections
were stained with hematoxylin and eosin (H&E) and measured for fiber diameter.
Accompanying the decrease in individual muscle weights in HFD mice, we find a
significant decrease in quadriceps fiber diameter (Figure 2.13-2.15). Similar to
what we would predict from smaller skeletal muscle fibers, we find a significant
reduction in grip strength in HFD mice (Figure 2.16). Moreover, we find a
negative correlation with body weight and the average fiber diameter of the
quadriceps (Figure 2.17).
Muscle Composition
Next, we sought to determine any difference in skeletal muscle
composition between mice fed SC or HFD. Specifically, we wanted to evaluate
the amount of protein and triglyceride relative to the mass of the muscle. We find
HFD reduces the percentage of protein relative to total muscle mass in the
triceps (Figure 2.18). Contrasting with this decrease, HFD also significantly
increases the percentage of triglyceride content in the triceps, with a significant
decrease in the ratio of protein to triglycerides (Figure 2.19-2.20). Additionally,
we were able to determine a positive correlation with triglyceride content of the
triceps and total body weight (Figure 2.21).
Gene Expression
Using qPCR analysis, we wanted to determine what genes are involved in
obesity-induced atrophy. We find no significant changes in ATF4, Gadd45a,
Cdkn1a, in the triceps (data not shown). Other potential atrophy genes, Trim63,
Fbox32, Bnip3, and Ctsl, also have no significant changes in expression (data
18
not shown). However, HFD mice show significant reductions of mRNA
expression of Pgc1!, AR, and Thr!, compared to the SC mice (Figure 2.22).
19
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Figure 2.1. HFD increases weight gain. Mice were provided ad libitum access
to SC or HFD for 6 weeks. Each data point represents the means ±SEM from 20
mice per diet. *P<0.01 calculated by t-test.
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Figure 2.2. HFD increases epididymal fat. Mice were provided ad libitum
access to SC or HFD for 7 weeks. Each data point represents one mouse and
the horizontal bars denote the means. P-value calculated by t-test.
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Figure 2.3. HFD increases retroperitoneal fat. Mice were provided ad libitum
access to SC or HFD for 7 weeks. Each data point represents one mouse and
the horizontal bars denote the means. P-value calculated by t-test.
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Figure 2.4. HFD induces hepatomegaly. Mice were provided ad libitum access
to SC or HFD for 7 weeks. Each data point represents one mouse and the
horizontal bars denote the means. P-value calculated by t-test.
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Figure 2.5. HFD does not effect cardiac muscle weights. Mice were provided
ad libitum access to SC or HFD for 7 weeks. Each data point represents one
mouse and the horizontal bars denote the means. Calculations by t-test reveal no
statistically significant difference.
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Figure 2.6. HFD has no effect on kidney weights. Mice were provided ad
libitum access to SC or HFD for 7 weeks. Each data point represents one mouse
and the horizontal bars denote the means. Calculations by t-test reveal no
statistically significant difference.
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Figure 2.7. HFD reduces weight of tibialis anterior. Mice were provided ad
libitum access to SC or HFD for 7 weeks. Each data point represents one mouse
and the horizontal bars denote the means. P-value calculated by t-test.
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26
Figure 2.8. HFD reduces weight of gastrocmenius. Mice were provided ad
libitum access to SC or HFD for 7 weeks. Each data point represents one mouse
and the horizontal bars denote the means. P-value calculated by t-test.
!"#$%&'()*+,#-.*/0
=77
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27
Figure 2.9. HFD reduces weight of quadriceps. Mice were provided ad libitum
access to SC or HFD for 7 weeks. Each data point represents one mouse and
the horizontal bars denote the means. P-value calculated by t-test.
&'()*+,-./01234
%#!
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%!!
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89
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28
Figure 2.10. HFD reduces weight of triceps. Mice were provided ad libitum
access to SC or HFD for 7 weeks. Each data point represents one mouse and
the horizontal bars denote the means. P-value calculated by t-test.
$#!
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29
Figure 2.11. HFD increases the weight of soleus. Mice were provided ad
libitum access to SC or HFD for 7 weeks. Each data point represents one mouse
and the horizontal bars denote the means. P-value calculated by t-test.
!"#$%&'()*+
10
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1-
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Figure 2.12. HFD reduces weight of hindlimb muscles. Mice were provided
with ad libitum access to SC or HFD for 7 weeks. The hindlimb muscles
represented are tibialis anterior, gastrocnemius, soleus, and quadriceps. Each
data point represents one mouse and the horizontal bars denote the means.
P-value calculated by t-test.
'()*+(,-./012+3.43(567.8,59
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Figure 2.13. HFD changes muscle histology. Representative H&E stains of
quadriceps muscle cross sections from SC and HFD.
SC
HFD
32
Figure 2.14. HFD induces skeletal muscle atrophy. Mice were provided ad
libitum access to SC or HFD for 7 weeks. Each data point represents the mean
fiber diameter of one mouse determined from >500 quadricep muscle fibers and
the horizontal bars denote the means.
0HDQ)LEHU'LDPHWHUȝP
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33
Figure 2.15. HFD reduces fiber size distribution of skeletal muscle. Mice
were provided with ad libitum access to SC or HFD for 7 weeks. Each distribution
represents the mean fiber diameter of one mouse determined from >500
quadricep muscle fibers from 10 mice. (>500 measurements / animal); p<0.001.
./0123-+45360-789
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34
Figure 2.16. HFD reduces grip strength. Mice were provided ad libitum access
to SC or HFD for 6 weeks. Each data point represents one mouse and the
horizontal bars denote the means. P-value calculated by t-test.
&#!
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,-./0'1-234150647
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Figure 2.17. Relationship between body weight and skeletal muscle
atrophy. Mice were provided ad libitum access to SC or HFD for 7 weeks. Each
data point represents one mouse.
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Figure 2.18. HFD decreases skeletal muscle protein content. Mice were
provided ad libitum access to SC or HFD for 7 weeks. Each data point represents
one mouse and the horizontal bars denote the means. P-value calculated by
t-test.
!"#$%&'()"*+%#,(-.(/0''1
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Figure 2.19. HFD increases content of skeletal muscle triglycerides. Mice
were provided ad libitum access to SC or HFD for 7 weeks. Each data point
represents one mouse and the horizontal bars denote the means. P-value
calculated by t-test.
'()*+,-.'()/01*+()2+-.34.56--7
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Figure 2.20. HFD reduces the ratio of skeletal muscle protein to
triglycerides. Mice were provided ad libitum access to SC or HFD for 7 weeks.
Each data point represents one mouse and the horizontal bars denote the
means. P-value calculated by t-test.
%&'()*)+*,-)'.(/*')*0-(1234.-(5.
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&5*2(G('H8**&44.99*')*<=*)-*>?@*+)-*I*D..J9B*K&4L*
5&'&*E)(/'*-.E-.9./'9*)/.*8)H9.*&/5*'L.*
L)-(M)/'&2*G&-9*5./)'.*'L.*8.&/9B*,NF&2H.*
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39
Figure 2.21. Relationship between body weight and skeletal muscle
triglycerides. Mice were provided ad libitum access to SC or HFD for 7 weeks.
Each data point represents one mouse.
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40
Figure 2.22. HFD suppresses gene expression of Pgc1!, AR, Thr!. Mice
were provided ad libitum access to SC or HFD for 7 weeks. Tricep mRNA levels
were determined using qPCR. Levels were normalized to the average levels in
SC. *P<0.05 by t-test. n=10 mice per diet.
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41
CHAPTER IV
DISCUSSION
Interpretation Of Results
With significant increases in total body weight, along with an increase in
the epididymal and retroperitoneal fat pad weights, we can conclude that the
HFD-fed mice are obese. Moreover, HFD treatment reproduced hepatomegaly
observed in our previous experiments, and is suggestive of fatty liver disease
commonly associated with obesity and metabolic syndrome (28). As we would
expect, we find no significant difference between control tissues of heart and
kidney weights between SC- or HFD-fed mice. These data support the efficacy of
HFD in establishing obesity, with no other observable pathologies.
The obese HFD mice exhibit significant weight reduction from several
skeletal muscles comprising the hindlimb and forelimb. Furthermore, we find a
significant decrease in fiber diameters of the quadriceps, allowing us to
determine that alterations in muscle weight are partially due to atrophy of the
skeletal muscle fiber. These data allow us to conclude the change in weight is a
direct result of skeletal muscle atrophy. Soleus was the only exception, with a
significant increase in muscle weight. Other studies show soleus resistant to HFD
stress, as well as increased storage of triglycerides in oxidative muscle fibers
(29, 30). With soleus composed of predominantly oxidative fibers, these studies
may account for the lack of atrophy, as well as increases in muscle weight from
greater triglyceride storage.
HFD mice have a significant reduction in strength, as demonstrated by
grip strength analysis. This is what we would expect from muscle that has
atrophied, as well as under conditions of obesity. Interestingly, skeletal muscles
42
from HFD mice contained less protein/mg of muscle mass. Moreover, we find a
significant increase in skeletal muscle triglycerides/mg of muscle mass in the
HFD mice, supporting data from other research (11, 28). These data suggest the
atrophy-associated decrease in muscle weights is of even greater impact, with
significant changes in molecular composition of protein and triglycerides in
muscle. Some data suggests increased triglyceride content is related to insulin
resistance in skeletal muscle (28, 31). However, other studies find endurancetrained athletes have similar increases in skeletal muscle triglycerides to obese
subjects, yet have increased insulin sensitivity (11, 32). This research identified a
disruption in oxidative metabolism in skeletal muscle of obese individuals
compared to the endurance-trained athletes to account for the difference in
skeletal muscle function. This suggests a sedentary lifestyle and HFD work in
conjunction for triglyceride content to adversely effect skeletal muscle.
Furthermore, it underscores the importance of exercise for preventing obesity
and skeletal muscle atrophy.
By analyzing the skeletal muscle fiber diameters compared to total body
weight, we find a strong negative correlation with total body weight and skeletal
muscle fiber size, with a positive correlation between triglyceride content in
skeletal muscle and total body weights. Taken together, this study
comprehensively demonstrates via histology, skeletal muscle weight and
composition, and mechanical strength, supporting obesity-induced skeletal
muscle atrophy.
Analysis of qPCR data revealed no significant increase in ATF4,
Gadd45a, or Cdkn1a in the skeletal muscle of mice subjected to the HFD. While
this does not support our hypothesis, the potential exist we missed their
expression during the experiment. In data using immobilization-induced atrophy,
43
we find these mRNAs are initially expressed, with a return to baseline expression
after a week.
Further qPCR analysis exploring other genes known to be involved in
atrophy pathways determined no significant change in expression in Fbox32,
Trim63, Bnip3, and Ctsl. However, we do find significant decreases in Pgc1!,
AR, and Thr!, expression. Interestingly, our previous research determined
Pgc1!, AR, and Thr! are all repressed by overexpression of Gadd45a and
denervation in skeletal muscle (22).
Pgc1! (peroxisome proliferator-activated receptor gamma, coactivator 1
alpha) repression has been identified during various atrophy inducing stresses
like disuse, aging, diabetes, as well as obesity (33-36). Additionally, our own lab
has demonstrated Pgc1! repression in skeletal muscle during both fasting and
spinal cord injury (26). PGC1# and similar coactivators are known to be key
regulators of oxidative metabolism and mitochondrial biogenesis. Their
dysregulation is associated with diseases like diabetes, cardiomyopathy, and
neurodegeneration (37). This identifies Pgc1! as a critical gene likely to be
mediating the skeletal muscle atrophy we find in HFD-fed mice.
A reduction in AR (androgen receptor) would explain downstream events,
such as reductions in lean mass and increased adiposity. Inversely of Gadd45a,
the anabolic hormone testosterone transcriptionally targets expression of its
receptor AR in a feed-forward mechanism. Adiposity would promote aromatase
activity, by enzymatically converting testosterone into estrogen. Subsequently,
transcriptional activation of AR by testosterone would be reduced. Lower
testosterone levels would result in less anabolic activity in skeletal muscle for
growth and maintenance of lean mass. Research overexpressing AR in rat
myocytes support its role in increasing lean mass and decreasing adiposity (38).
44
We find another hormone receptor, Thr# (thyroid receptor alpha), is
significantly reduced in mRNA expression of obese mice. This nuclear receptor
interacts with thyroid hormones, thyroxine and triiodothyroxine, and
transcriptionally activates genes involved in promoting protein synthesis,
mitochondrial biogenesis, and increasing basal metabolism in a variety of
tissues. A reduction in Thr# would have downstream effects in skeletal muscle by
diminishing anabolic activity for skeletal muscle maintenance and growth.
Additionally, skeletal muscle is a metabolically active tissue. A basal decrease in
caloric expenditure would promote adiposity, particularly in hypercaloric
conditions of a HFD.
Future Directions
While these data support our hypothesis of obesity as an atrophy inducing
stress, a clear mechanism has yet to be determined. Although we were unable to
determine involvement of the ATF4/Gadd45a/Cdkn1a pathway, studies involving
several earlier time points would insure we have not missed their role in obesityinduced atrophy. These studies would also determine the involvement of other
potential atrophy pathways, including the potentially Gadd45a-mediated
repression of PGC1!, AR, and Thr!, we find in obesity. A time-course would also
help us determine when obesity-induced atrophy is initiated, and what genes are
expressed at its induction relative to later time points. Ultimately, studies
involving a muscle specific knockout of ATF4 in mice could determine if ATF4 is
necessary for obesity-induced atrophy.
Further histological analysis will help us investigate if fiber type distribution
changes in obesity. This would determine if obesity has any fiber type specific
effects resulting in atrophy or hypertrophy. Staining for triglycerides would
45
support our findings of increased triglycerides in skeletal muscle, as well as
localize where it accumulates e.g., extramyocellular, intramyocellular.
Additionally, it will be important to incorporate sufficient controls to
separate the impact of obesity from diet. Obesity-induced skeletal muscle
atrophy may be a product of a hypercaloric state achieved via alternate types of
diets, regardless of fat content. An additional control diet that is hypercaloric in
carbohydrates would allow us to answer this question. Likewise, an isocaloric
study would determine if skeletal muscle atrophy is specific to HFD in the
absence of obesity. In all these proposed diets, it will be important that the
composition of these diets come from comparable sources of carbohydrates,
protein, and fat, with similar content of vitamin and minerals.
Other potential extraneous variability may still exist in our model. We find
HFD elevates fasting glucose in mice, possibly due to insulin resistance.
Reductions in insulin signaling could mediate the atrophy phenotype we see in
HFD-fed mice. Characterization of a knockout mouse for tumor necrosis factor
alpha exhibit protection from obesity-induced insulin resistance (39). This mouse
strain would allow for experiments with HFD excluding the possibility of insulin
resistance. Furthermore, an evaluation of the activity levels in SC- to HFD-fed
mice will allow us to determine if the pathogenesis of obesity-induced atrophy is
mediated to any degree by disuse. Bringing these comprehensive elements
together, this study will give us a better understanding of the pathogenesis of
obesity and its effect on skeletal muscle atrophy.
46
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