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EFFECTS OF AGE AND CALORIE RESTRICTION
ON TUMOR NECROSIS FACTOR-ALPHA SIGNALING IN SKELETAL MUSCLE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
This work is dedicated to my dad, Norman Phillips, 1940-2001. Love always.
I would like to extend my thanks to my committee chair (Dr. Christiaan
Leeuwenburgh) and committee members (Dr. Scott Powers, Dr Steve Dodd and Dr
Michael Clare-Salzler) for their valuable guidance and expertise, contributing to the
completion of this work. I am also indebted to Jessica Staib for her assistance in tissue
acquisition, technical input and overall support throughout this project.
Most critically, the conviction with which to pursue and realize my doctorate was
made a reality only through the unwavering love, support, and belief of my mum and
dad; and the inspirational work ethic and encouragement afforded me by my brother.
TABLE OF CONTENTS
A C K N O W L E D G E M E N T S ......... .................................................................................... iv
LIST OF TABLES .................. .............. ................. ............ .............. .. vii
LIST OF FIGURES .................. ............ ................ .......... ................ viii
A B ST R A C T .......... ..... ...................................................................................... x
1 IN TR OD U CTION ............................................... .. ......................... ..
S ig n ifi c a n c e .......................................................... ................. .
Q questions and H ypotheses ........................................................... .............3
2 REVIEW OF LITERATURE ......................................................... .............. 7
Aging and Inflammation: TNF-a / NF-KB Signaling Pathway............... ...............7
Overview of Tumor Necrosis Factor-Alpha (TNF-c) ............... .................... 8
Nuclear Factor-KB (NF-KB): An Overview ................................... ...............12
S u m m ary .................................... .................. ......... ......... ................. 13
Aging, Calorie Restriction and TNF-ac /NF-KB Signaling..............................14
Aging and Inflammation: Significance in Skeletal Muscle.............................17
Sum m ary .............................18.............................................
3 M E T H O D S ...................................................................................................19
A n im a ls ................................................................................................................. 1 9
Tissue Harvesting .................................... .. ......... ............... 19
B iochem ical A ssay s............. .................................................... .. .... ...... 20
Cellular fractionation..................... ...... ............................. 20
W western blotting ........................................... .......... .. ........ .. ........ .... 2 1
H istochem istries ........................................... .. ........ ................. 22
DNA fragmentation..................... ........ ............................. 24
A ntioxidant enzym e activity ............................................. ............... 25
Protein concentration..................... ...... ............................. 25
Statistical analysis ............................ ..... ...... .... ............... .25
4 R E SU L T S ....................................................... 26
M orphological M easurem ents .............................................................................26
A nim al B ody W eights ........................................ ...................... .....................26
M u scle M ass ................................................................... ............ 26
M uscle Cross Sectional A rea........................................................... ............... 28
B iochem ical A ssay s............. .................................................... .. .... ...... 32
A antioxidant Enzym e A activity .................................... ............................. ....... 32
Determination of Plasma TNF- ........................... ......... ..... ................. 33
Western Blotting Detection of TNF-a / NF-KB Signaling Proteins..................34
Nuclear Binding Activity of NF-KB..............................................................40
The TNF-a receptor-mediated pathway of apoptosis ......................................41
FADD content elevated with age ...................................... ............... 42
Caspase-8: zymogen and cleaved product.................................................43
DNA fragmentation...................... ....... ............................. 45
5 DISCUSSION .................. ................................... ........... ............... 47
Aging and Sarcopenia......................... ......... ....... .............. ........ .. 47
TN F-c A going and Skeletal M uscle ............................................ ............... .... 47
Age Decline in Muscle Cell Area and Increased TNF-a Expression .................49
Fiber Type Variations in NF-KB Signaling ................................... .................. 50
Fiber Type Variations in NF-KB Subunits and Binding Activities ...................52
Fiber Type Differences in Apoptotic Signaling ....................... ...............54
Final Conclusions: TNF-a Promoting Skeletal Muscle Cell Survival or Cell
D death? .......................................... ............................ 55
LIST OF REFEREN CE S .. ....... ................................ ........................... ............... 59
B IO G R A PH IC A L SK E TCH ..................................................................... ..................69
LIST OF TABLES
1 Body mass and muscle mass of 6-month- and 26-month-old ad libitum and 26-
month-old calorie restricted male Fischer 344 rats. ............................................27
2 Soleus and Superficial vastus lateralis cross sectional areas of 6-month- and 26-
month-old ad libitum and 26-month-old calorie restricted male F344 rats..............29
3 Antioxidant enzyme activity in cytosol from 6-month- and 26-month-old ad libitum
and 26-month-old calorie restricted male F344 rats..........................................32
LIST OF FIGURES
1 The TNF-a signal transduction pathw ay. ........................................ .....................9
2 Soleus and superficial vastus lateralis muscle wet weights. ...................................28
3 H&E staining in soleus and superficial vastus lateralis sections from 6AL (A,D),
26AL (B,E) and 26CR (C,F) Fischer-344 rats.. ................................... ............... 30
4 TNF-a expression stemming from myocytes................................ ............... 31
5 TNF-a immunohistochemical staining in superficial vastus lateralis sections from
6AL (A), 26AL (B) and 26CR (C) Fischer-344 male rats. ...................................32
6 Plasma TNF-a levels in 6-month-old ad libitum, 26-month-old ad libitum and 26-
month-old calorie restricted F344 rats. ........................................ ............... 33
7 TNF-R1 in soleus and superficial vastus lateralis muscle................... ............ 34
8 Western blot analysis of IKKP content in soleus and superficial vastus lateralis
m u sc le s ...........................................................................................3 6
9 IKKy content in soleus and SVL muscle from 6AL, 26AL and 26CR male F344
rats ...................... .................................... 37
10 Western blot analysis of IKBa in soleus and SVL (superficial vastus lateralis)
muscle of 6-month-old- (6AL), 26-month-old ad libitum (26AL) and
26-month-old calorie restriction (26CR) F344 rats.............. ................................. 38
11 p65 protein content in soleus muscle of 6AL, 26AL and 26CR male F344 rats. ....39
12 NF-KB binding activity in soleus and superficial vastus lateralis nuclear
e x tra c ts ........................................................................... 4 1
13 FADD protein content in soleus and SVL muscles................... ...............42
14 Procaspase-8 protein content in soleus and superficial vastus lateralis muscles .....44
15 Caspase-8 protein content in soleus and SVL muscles ............................... 45
16 DNA fragmentation in superficial vastus lateralis and soleus .............................46
17 Proposed model of TNF-ac signaling with age in soleus and superficial vastus
lateralis (SVL) skeletal m uscles ......................................................................... 57
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EFFECTS OF AGE AND CALORIE RESTRICTION
ON TUMOR NECROSIS FACTOR-ALPHA SIGNALING IN SKELETAL MUSCLE
Chair: Christiaan Leeuwenburgh
Major Department: Exercise and Sport Sciences
The stark reduction in muscle mass and function noted with age, termed
sarcopenia, is a normal yet debilitative feature of aging. Stemming from the lack of
clarity surrounding the role of TNF-a in age-associated muscle loss, we investigated the
involvement of this cytokine in promoting reductions in muscle mass, and the cellular
signaling pathways through which these effects were executed. We studied the
inflammatory and apoptotic pathways emanating from TNF-a stimulation in the muscle
cells, and also compared the responses to TNF-a in two different muscles, the soleus and
the superficial vastus lateralis. These muscles were selected as they contained different
types of muscle fibers known to exhibit the effects of aging (i.e., muscle loss) to differing
degrees. We found that aging was paralleled with increased TNF-c, and that the
inflammatory and apoptotic signaling capability of TNF-a was dependent on the muscle
being examined. In the soleus with age, we report a greater capacity to cultivate
inflammatory signaling through the transcription factor, NF-KB, compared to that
detected in the superficial vastus lateralis. Alternatively, in the superficial vastus
lateralis, TNF-a stimulated apoptotic signaling with age to a much higher extent than was
observed in the soleus. Moreover, a reduction in muscle cell area in the superficial vastus
lateralis coincided with this age-linked elevation in apoptosis.
Application of the life-extending intervention, calorie restriction, was also relied
upon to provide further elucidation of the contribution of TNF-a to skeletal muscle loss
with age. Calorie restriction is the only robust intervention shown to repeatedly evade
the physiological declines associated with aging, and in agreement with this ability,
TNF-a stimulation of both inflammatory and apoptotic pathways were abrogated when
calorie restriction was applied. Our results suggest that specific fiber types may play a
regulatory role in determining the nature of the TNF-a signal transmitted at the cellular
level, with the decision of selecting life or death signals intimately tied to the extent of
fiber loss experienced in the muscle; such a potential may constitute a major proponent in
the pathogenesis of sarcopenia.
Aging, identified by a progressive and irreversible decline in cellular structure and
function, is exhibited in most multicellular organisms, with the molecular basis for such
depreciation yet undefined. In mammals, the only intervention repeatedly shown to
retard the onset of age-associated cellular deficits is calorie restriction (CR) in the
absence of malnourishment (1). Most CR studies have involved laboratory rodents
which, when subjected to 25 to 50% reduction in caloric intake displayed a delay in the
onset of age-associated pathological and physiological changes and an extension of
median and maximum life span (2-5). Simply put, calorie restriction imparts aging
resistant qualities to the organism (that serve to stall cellular defects attributable to age,
lessen the severity and prevalence of age-related pathologies, and permit a lengthier
median and maximum life span). Attempting to establish the mechanisms behind CR's
anti-aging effects has and continues to be fervently, yet elusively, investigated. However,
the effects of CR provide a base from which several theories have emerged to explain the
Of the multiple hypotheses proposed (6), the oxidative-stress hypothesis of aging
(7) (which arguably provides the most comprehensive elucidation of the aging process
and other age-related phenomena) points toward a reduction/oxidation redoxx) imbalance
as founding the characteristic changes witnessed with aging (7-9). This redox imbalance
describes a disparity between oxidative stress and antioxidant buffering capabilities
culminating in damage to cellular structures (e.g., protein, lipid and DNA) (10-12). In
terms of aging, the frequency and presence of cell structural damage is thought to
accumulate. One of calorie restrictions' anti-aging mechanisms is believed to be targeted
against the redox inequality, manifest as an improved redox-balancing capability
exhibited by the organism, and a reduction in oxidative stress-induced damage (1).
An additional area closely implicated with redox flux explored in conjunction with
aging is inflammation. Specifically, key mediators of inflammatory pathways, (i.e.,
tumor necrosis factor-alpha (TNF-a) and nuclear factor-kappa B (NF-KB)) are under
investigation with regard to a possible role in aging (13-15). Inflammatory reactions are
known to be an extremely complex series of physiological reactionary events prompted to
minimize cellular trauma and promote repair. Critically, TNF-a and NF-KB, responsive
to multiple stimuli including oxidative stress, have the ability to rapidly direct cellular
remodeling. However, persistent, unwarranted immunological episodes implicating these
and other pivotal inflammatory agents (e.g., inhibitor of KB kinase (IKK) complex and
inhibitor of KB (IKB) proteins) have been observed with increased frequency in aged
tissues (13, 15-17). Given that many of the inflammatory cell types are sensitive to
reactive oxygen species (ROS) and regulate their function based on ROS presence (18,
19), the possibility exists of a pro-inflammatory state becoming more chronic concurrent
with advancing age. What's more, few studies have examined the anti-aging effects of
CR from the perspective of the inflammatory response in post-mitotic tissue.
In the case of tissue-specific responses to aging processes, skeletal muscle, (which
accounts for approximately 40% of the total body mass and 75% of the body's cell mass)
(20), provides a substantial target for age-associated degeneration. Furthermore, the
different skeletal muscle fiber types have been reported to experience the effects of aging
to varying degrees, with the fastest fibers (i.e., type II) argued to suffer the most
deleterious effects (21-27). The stark reduction in muscle mass and function noted with
age (sarcopenia) has been well characterized (24, 28). However, the impact of sarcopenia
is not clearly established because of the lack of suitable approaches for estimating its
prevalence and incidence in elderly populations (29). From a functional perspective,
weakness of the lower extremities has been implicated with difficulty in balance
problems and falls (30) and is thus recognized as holding great public health significance
(31) both economically and socially. Furthermore, other age-related diseases with an
inflammatory flavor (e.g., congestive heart failure) often impair skeletal muscle. The loss
of muscle mass and function contribute substantially to morbidity and mortality in
sufferers of such conditions (29, 32).
In summary, given the proposed involvement of TNF-a and NF-KB in aging and in
age-linked disorders, and given the paucity of studies examining this area, we intended to
investigate the TNF-a / NF-KB signaling pathway concurrently with aging in skeletal
muscle. Additionally, in view of the ability of CR to retard the debilitative changes seen
with aging, we proposed to apply this intervention to evaluate whether CR exerted its
anti-aging effect by counteracting inflammatory mediators involved in TNF-a / NF-KB
signaling. Accordingly, the following questions were devised.
Questions and Hypotheses
Question 1. How do age and calorie restriction affect the inflammatory cytokine
TNF-a and its receptor TNF-R1 in skeletal muscle?
Hypothesis 1. Skeletal muscle will display an increased presence of TNF-a and
TNF-R1 with age, and calorie restriction will attenuate this elevation.
An increase in TNF-c, particularly in skeletal muscle, could play a role in the
processes contributing to aging, and to many age-linked disorders with inflammatory and
catabolic elements (e.g., sarcopenia, rheumatoid arthritis, and cachexia). Protein and
mRNA of TNF-a have been detected in biopsy samples from human skeletal muscle (33)
and adherence to an exercise regimen was found to reduce TNF-a in elderly humans
(14). However, no studies have explored the effects of age and calorie restriction on
TNF-a in skeletal muscle.
Question 2. What effects do age and calorie restriction have on the transcription
factor NF-KB in skeletal muscle?
Hypothesis 2. We anticipate an age-associated increase in NF-KB and an
attenuation of this elevation by calorie restriction.
Aging was found to induce an increase in NF-KB nuclear binding activities in
cardiac muscle and liver (16, 34). Others have reported a decline in NF-KB activity when
CR was used as an intervention in a variety of models (35-37). To our knowledge, no
studies have assessed the combined effect of age and calorie restriction on NF-KB in
Question 3. How are the IKK and IKB regulatory proteins altered by age and
calorie restriction in skeletal muscle?
Hypothesis 3. Age will result in an heightened stimulation of IKK and IKB
regulatory proteins, and calorie restriction will reduce this stimulus.
Several studies examining mitotic tissue (i.e., liver) (13, 15) reported an increase
with age and a downregulation by calorie restriction of the NF-KB regulatory proteins
(inhibitor of KB kinase (IKK) and inhibitor of KB (IKB) proteins). Whether this same
phenomenon occurred in post-mitotic tissue (such as skeletal muscle) was addressed in
these proposed experiments.
Question 4. Does oxidative stress act as an endogenous mediator of the TNF-a /
NF-KB signaling pathway in skeletal muscle, and what is the effect of age and calorie
Hypothesis 4. Oxidative stress will participate in mediating the TNF-a / NF-KB
signaling pathway in skeletal muscle; this involvement will be heightened with age and
reduced by calorie restriction.
Reactive oxygen-mediated NF-KB activation in response to TNF-a has been
reported in skeletal-muscle myocytes (38). This association was confirmed by adding an
antioxidant enzyme (catalase) that served to inhibit NF-KB activation, and also adding a
known oxidant (hydrogen peroxide) that increased activation.
We endeavored to investigate oxidative-stress involvement in TNF-a induced NF-
KB activation in aged skeletal muscle, and assessed the affects of age and CR on
Question 5. Will type I and type II skeletal muscle fibers display differences in the
TNF-a / NF-KB signaling mediators (TNF-c, TNF-R1, IKK, IKB, and NF-KB), and how
will age and calorie restriction affect the fiber-type responses?
Hypothesis 5. Type II fibers will display a greater presence of TNF-c, TNF-R1,
IKK, IKB and NF-KB compared to type I fibers, and this will be accentuated with age and
alleviated by calorie restriction.
Type II (fast-glycolytic) fibers have been argued to be more susceptible than type I
(slow-oxidative) fibers to age-related fiber atrophy and fiber loss (21-24, 26, 27, 39, 40).
Several explanations (including a preferential loss of larger motoneurons, recruitment of
motor units, and integrity of oxidative metabolic pathways) reflect but a few of the ideas
put forth to explain this occurrence (5, 23-26). We set out to determine whether a
difference in TNF-a / NF-KB signaling was connected to this fiber type-specific aging
response, and how it may have been affected by age and calorie restriction.
REVIEW OF LITERATURE
Aging is characterized by a progressive decline in physiological function in the
absence of disease and malnutrition. The underlying causes for the functional
deterioration of aging have been extensively but inconclusively investigated.
Accordingly, our focus was the role of inflammation in aging and age-associated
conditions, and the means by which calorie restriction affords considerable resistance to
the physiological declines of aging.
Aging and Inflammation: TNF-a / NF-KB Signaling Pathway
The inflammatory response performs an essential role in detecting and preventing
prolonged cellular insult caused by different stressors including physical agents (UV and
gamma radiation); chemical agents (components of the body and products of metabolism
such as free radicals); and biological agents (viruses, bacteria). A complex circuitry of
inflammatory mediators works in concert to sense, apprehend, and rectify the ensuing
cellular damage. Such capabilities are critical for survival. Yet with age, the very
responses needed to ward off stressors are thought to become the players contributing, in
part, to the functional declines noted with aging (41-44). Among the candidates argued
to instigate an inflammatory aspect of aging are tumor necrosis factor-a (TNF-u) and
nuclear factor KB (NF-KB). Thus, the TNF-a / NF-KB signaling pathway in association
with aging and CR is considered in the following paragraphs.
Overview of Tumor Necrosis Factor-Alpha (TNF-a)
The cytokine, tumor necrosis factor-alpha (TNF-u), originally named cachectin due
to its catabolic action (45), is a homotrimer of 157 amino-acid subunits that promotes
antitumor and immune responses (46). The catabolic activity of TNF-a has afforded the
cytokine an association with muscle pathology (47), but has also been implicated in a
wide variety of functions in many other cell types (48). Thought to contribute to
age-related skeletal-muscle degeneration (13), TNF-a is primarily produced by activated
macrophages and has been evidenced in patients with heart failure (49), sepsis (50), and
other inflammatory diseases that result in secondary muscle weakness (51-53). Further,
elevated TNF-a levels have been reported with age in heart, liver, kidney and brain
tissues (16, 17).
TNF- a signaling pathways. Cellular responses to TNF-a are varied and
dependent on cell type. A post-receptor, intricate cascade of signaling events holds the
key to a host of responses attributed to this pleiotropic cytokine (46). Three major
pathways that transduce the TNF-a signal have been identified (54, 55). These include a
proapoptotic pathway regulated by interaction of the TNF-a-receptor complex with the
Fas-associated death domain. A second pathway activates the transcription factor
activator protein-1 via Jun-NH2-terminal kinases. The third means of signaling activates
NF-KB. This last pathway, known to occur in skeletal muscle (19, 38, 47), represents a
major mechanism of transcriptional control by TNF-a particularly in terms of promoting
the inflammatory response.
Signaling through TNF-R1. The biological and signaling activity of TNF-a is
exerted through binding to two types of receptors: TNF-R1 and TNF-R2. These
:-L --*---- Apoptosis
------------- JNK activation
S -- ------- -.Anti-apoptosis
jCdc37 i H Cdc3i1
'"~--^^ -- 1*
Figure 1. The TNF-a signal transduction pathway.
receptors belong to a family of type I transmembrane receptors, with one to five cysteine-
rich repeats in their extracellular domain, and a common death domain in their
cytoplasmic tail (56). TNF-R1 contains the death domain, whereas TNF-R2 does not.
Consequently, TNF-R1 has the ability to direct both cell-survival and cell-death signals,
whereas TNF-R2 primarily conveys cell-survival signals (56). Despite the presence of
two receptors, multiple studies show that TNF-R1 initiates most of TNF-a's biological
activities (57) (Fig 1). In an analysis of lymphocyte responses to aging, concurrent with
elevated serum TNF-a levels, increased expression of TNF-R1 was implicated with
T-cell dysfunction and lymphopenia in aging populations (58).
TNF-R1 adaptor proteins. The absence of intrinsic enzymatic activity in the
receptor death domains requires multiple adaptor proteins to be recruited for signal
conductance to occur. A highly coordinated amalgamation of signaling intermediaries
then follows. The extracellular binding of the TNF-a trimer to TNF-R1 releases the
inhibitory protein, silencer of death domains (SODD), from TNF-R1's intracellular
domain. When exposed, the TNF-R1 intracellular domain is identified by the adaptor
protein TNF receptor-associated death domain (TRADD), which then recruits additional
adaptor proteins: receptor-interacting protein (RIP), TNF-R-associated factor 2
(TRAF2), and Fas-associated death domain (FADD) (57). These latter adaptor proteins
recruit key enzymes to TNF-R1 that, are responsible for initiating the various signaling
TNF-a signaling to NF-KB. Knowledge of the signaling intermediaries that
facilitate TNF-a induced activation ofNF-KB has progressed substantially in recent
times. The crux of this activation relies on phosphorylation-dependent ubiquitination and
degradation of the inhibitor of KB (IKB) proteins, which usually retain NF-KB within the
cytoplasm of unstimulated cells (59). The phosphorylating agent that assists in the
release of this inhibition arises from a multiprotein IKB kinase (IKK) complex (60). Two
catalytic subunits, IKKc and IKK3, and a regulatory subunit, NF-KB essential modulator
(NEMO or IKKy) comprise the core of the IKK complex. Further, the IKK complex
contains a specific chaperone consisting of Cdc37 and Hsp90 that serves to shuttle the
complex from cytoplasm to membrane.
Upon TNF-a binding to TNF-R1, the IKK complex is recruited in sync with the
adaptor proteins. Binding is dependent on RIP, and once IKK interacts with RIP or an
RIP-dependent kinase, the complex becomes activated. Investigators have shown that
phosphorylation of two sites at the activation loop of IKK3, but not IKKc, are essential
for the activation of IKK by TNF-a and other proinflammatory cytokines (61). Also, the
existence of an autophosphorylation capability of IKKP is postulated to endow a negative
regulatory constraint on IKK activation (which contributes to the transient nature of its
activation). This holds importance as prolonged activation of the NF-KB signaling
pathway carries with it the potential for toxicity and pathophysiology (62); thus a rapid
termination to IKK activation is critical for attainment of an appropriately weighed NF-
The noncatalytic component of the IKK complex (IKKy) has also been keenly
investigated with efforts geared toward delineating its role. Early studies noted the
essential requisite of the IKKy subunit in order for NF-KB activity to be achieved by a
variety of stimuli (63, 64). More recent work substantiated these findings and further
presented the notion that the oligomeric organization of IKKy enforces a spatial
positioning of the two kinases, IKKc and IKK3, in the IKK complex serving to facilitate
transautophosphorylation, activation, and subsequent NF-KB activation (65).
The specific role of IKKc remains a subject of debate; experiments using
IKKa-knockout mice point toward an involvement of IKKc in keratinocyte
differentiation in the epidermis independent of its kinase activity and independent of
NF-KB (66). Studies using gene ablation have also ascertained that IKK3 and IKKy are
required for NF-KB activation by proinflammatory stimuli, while IKKu is essential for
morphogenic signals (67-69). Once activated, IKK proceeds to phosphorylate the IKBs
(which in the case of IKBa occurs at two conserved series, S32 and S36) in the
N-terminal regulatory region (70), targeting them for ubiquitination and proteasomal
degradation; and liberating NF-KB for nuclear translocation (65).
Although most of the involved parties in this cascade have been validated by both
biochemical and genetic means (57), several unresolved interactions and functions linger:
for example, the possibility of there being an intermediate factor or kinase operating
between RIP and IKK. Such unknowns should not be overlooked when considering the
Nuclear Factor-KB (NF-KB): An Overview
The transcription factor NF-KB is a central regulator of the immune response whose
actions are rapidly induced by proinflammatory stimuli such as TNF-a (Pahl (71) lists
known inducers). Further, NF-KB acts to promote cellular growth, staving off
programmed cell death by pro-apoptotic stimuli such as TNF-a (54, 72). The NF-KB
exists as either heterodimers or homodimers of the Rel family of proteins. The
predominant form of NF-KB is a heterodimer consisting of p50/p 105 and p65 (RelA).
Other forms contain RelB, c-Rel and p52/pl00 subunits (73). These dimers bind to a set
often base pair DNA sites (recognized as the KB sites) to regulate gene expression. In
latent cells, the NF-KB dimers are retained in the cytoplasm bound to the IKB proteins.
When stimulated, however, the IKB proteins undergo proteasomal degradation, which
allows liberated NF-KB to translocate to the nuclear binding site and facilitate gene
NF-KB regulated gene expression. The active NF-KB transcription factor
promotes the expression of over 150 target genes (71). Among these are members of the
Rel / NF-KB / IKB family. As a result, NF-KB limits its own activation by prompting the
new synthesis of IKB. Newly-synthesized IKB can enter the nucleus and dislodge active
NF-KB from its DNA binding site; thus, in most cell types, NF-KB activation is fleeting
(74, 75). In addition, a substantial number of the proteins encoded by the NF-KB target
genes participate in host immune response, giving rise to the designation of NF-KB as a
central mediator of immune response. However, NF-KB is also involved in
transcriptional control of many genes whose functions reach beyond an immediate
immune response and are more associated with stress responses. Accordingly, rather
than being labeled a central mediator of the immune response, it has been proposed that
NF-KB is more global, and represents a regulator of stress responses (71).
Using knockout mice models, the importance of NF-KB has been shown for normal
physiological function. Knockout mice created for p65/RelA, IKBU, p50, c-Rel, and
RelB have been examined (76-78). Deficiencies of p50, c-Rel, or RelB were reported to
result in developmentally normal mice; and a RelA deficiency resulted in embryonic
lethality from liver apoptosis (77). Furthermore, IKBu-deficient mice appeared normal at
birth; but in the postnatal period, their growth ceased and death occurred within 7 to 10
days of age (77, 78).
The inflammatory response is governed and executed by a multifaceted circuitry of
inflammatory cell types. While essential for survival against cellular insult, inflammation
paradoxically may be implicated in aging and age-related functional declines (47, 79).
The TNF-a and NF-KB influential inflammatory mediators, have been considered in
terms of contributing to aging processes.
TNF-a has been found to signal through three major pathways (including the
NF-KB pathway responsible for transcriptional activation of many genes linked with the
inflammatory response). Despite the prevalence of two TNF-a receptors, most of
TNF-a's activities are initiated through TNF-R1 (57). The TNF-a / NF-KB signaling
pathway comprises a finely integrated cascade of events (encompassing initial ligand-
receptor interaction, adaptor protein recruitment, and inhibitory protein ubiquitination-
proteasomal degradation), permitting translocation of NF-KB from cytoplasm to nucleus.
Although a complete understanding of all the involved proteins and the extent of their
contributions to signal transduction remains equivocal, key intermediaries have been
identified. With respect to proinflammatory (e.g., TNF-c) induced stimulation of
NF-KB, IKKP and IKKy (kinase and regulatory components) in the IKK complex,
respectively, have been shown to be essential for NF-KB activation (61, 63-65).
Once liberated, NF-KB has the ability to translocate to the nucleus where it can
promote the expression of over 150 target genes (71). The breadth and diversity of
proteins whose genes contain NF-KB binding sites provides some illustration of the
global responses (e.g., immune and stress-related) that are enacted through NF-KB
transcriptional control, and exemplifies the wide-reaching impact of this transcription
Aging, Calorie Restriction and TNF-a / NF-KB Signaling
Immunosenescence, has been used to describe the proposition that a global
reduction in the capacity to cope with various stressors and a concurrent progressive
increase in proinflammatory status are major characteristics of the aging process (41).
What determines the propensity of an individual to be more susceptible to inflammatory
dysregulation is argued to stem from environmental and genetic factors. The genetic
component of this postulate identifies the absence of robust gene variants or the presence
of frail gene variants (or some combination thereof) as fuelling the likelihood of
age-related diseases occurring that contain an inflammatory element. These include
Alzheimer's dementia, atherosclerosis, and maturity-onset diabetes mellitus (80, 81).
The environmental contribution to this status describes the lifelong series of
inflammatory stimuli that persist and inflammatory reactions that accumulate over time,
forming the inflammatory background. Susceptibility to disease occurrence is deemed to
be dependent on the nature of the inflammatory background, which is fashioned by
individual responses to stressors at the cellular level. To offer further delineation, the
responses of influential inflammatory pathways (e.g., TNF-ac /NF-KB signaling) to
oxidative stress have been examined because of the intimate link thought to exist between
oxidant production and aging processes (7, 8).
Oxidative stress and the molecular inflammatory theory of aging. Free
radicals; molecules (typically oxygen or nitrogen derivatives) that contain one or more
unpaired electrons in their outer orbit (9), and also nonradical oxidants (e.g., hydrogen
peroxide and peroxynitrite) have the propensity to act as signaling agents and modulators
of cellular function that can equate to cellular damage. Buffering systems persist that act
to protect cellular structures from excessive radical assault and maintain a redox
equilibrium. If a disparity exists between the amount of oxidative stress prevalent and
the antioxidative force buffering capability, then the net result equates to oxidant stress.
Further, studies have reported the sensitivity of NF-KB to oxidative stress and the
presence of NF-KB binding sites in the promoter regions of antioxidant genes (40, 82-84).
On a related track, a molecular inflammatory theory of aging has been proposed
implicating reactive oxygen and nitrogen species and proinflammatory molecules as key
players in the aging process (13). In an examination of rat kidney from young and old
animals, signaling molecules of the NF-KB family were shown to be redox sensitive, a
condition that was amplified with age (15). Specifically, an increase in the nuclear
binding activity ofNF-KB was found to occur with advancing age. This was
accompanied by a decrease of IKBa and IKBP, the regulatory proteins that prevent
nuclear translocation ofNF-KB and subsequent transcription-factor activities. Further,
the IKK complex responded in agreement with the increased NF-KB activity indicated by
elevated phosphorylation of this complex with age.
Calorie restriction and inflammation. In the aforementioned study, conclusions
drawn from the age-associated elevation of inflammatory mediators were made even
more robust by including a calorie restriction intervention (15). The CR animals showed
reduced IKK activation, downregulating the NF-KB activation reportedly through
increased bound IKBa and IKB3 proteins in the cytoplasm (15). These reductions with
CR fell concomitantly with lowered reactive oxygen species levels compared to ad
libitum fed groups at all ages studied, supporting the notion of oxidant stress having some
involvement in promoting the inflammatory state found with age. The responsiveness of
NF-KB to oxidative stress has been well documented (18, 85); however, the idea of aging
being molded by an interplay between oxidative stress and inflammatory mediators has
received much less attention.
Aging and Inflammation: Significance in Skeletal Muscle
Age-associated reductions in skeletal muscle size, mass, and function have been
found to persist in various mammalian species (86). Further, the observation of specific
skeletal muscles in humans undergoing -40% decline in muscle mass between the ages
of 20 and 80 years (25) imparts significant concern for public health and also impinges
on the issue of independence in the elderly (87).
The atrophy and dysfunction of skeletal muscle associated with aging has been
investigated from several avenues to determine the underlying causes) (29). Use of
calorie restriction to further unveil contributing mechanisms has unearthed a preventive
intervention against age-related muscle degeneration (88, 89). Work that analyzed the
involvement of mitochondrial DNA deletions in muscle fiber loss reported a significantly
blunted progression of age-dependent fiber and mitochondrial abnormalities present in
CR compared to ad libitum fed animals (5). Aspnes et al., hypothesized that CR's ability
to attenuate accumulation of oxidative damage in skeletal muscle was responsible for this
outcome. Whether a similar effect of CR occurs on age-dependent elevated
inflammatory pathways (TNF-a / NF-KB signaling) while, in turn, maintaining muscle
integrity remains to be determined, but this possibility has been reported in other tissues
Alternatively, the influence of inflammatory cytokines such as TNF-a on muscle
biology remains interesting given the broad range of biological events this pleiotropic
factor can direct, many of which remain to be fully understood. Accordingly, elucidating
skeletal muscle responses to TNF-a in the backdrop of normal, progressive aging may
help to shed light on age-dependent alterations in signaling patterns that compromise
muscle homeostasis, and ultimately hinder function. In turn, signaling patterns gone
awry may hold important clues to the more severe destructive effects of this cytokine on
muscle biology reported to accompany inflammatory disorders including cancer (90, 91),
chronic obstructive pulmonary disease (92), congestive heart failure (32), and sarcopenia
Inflammatory response as a potential contributor to aging processes in skeletal
muscle may help explain the dramatic degeneration observed in this tissue with age. The
inflammatory cytokine, TNF-u, attracts particular interest as it has the ability to affect a
multitude of cellular responses and drive gene expression through activation of the
transcription factor NF-KB. Further, TNF-a has been linked to many pathologies (some
of which are age-related) where muscle degeneration is apparent (29, 32, 91-93), and has
also been shown to be elevated with age (16, 17). Moreover, applying the intervention of
calorie restriction may help to curb age-dependent deterioration of skeletal muscle
induced by inflammation, as proposed by others regarding nonmuscle tissue (13), and
supply additional information regarding the mechanisms at play.
Undoubtedly, many other factors must come under scrutiny before we can
substantiate the involvement of inflammation in aging. However, the TNF-a / NF-KB
signaling pathway provides an attractive avenue for exploring this potential, and
ultimately may assist in further unraveling the mechanisms behind aging and age-related
Eight 6-month-old ad libitum fed (6AL), eight 26-month-old ad libitum fed (26AL)
and eight 26-month-old calorie restricted (26CR) male Fischer 344 rats (National
Institutes of Aging Colony, Harlan Sprague Dawley, Indianapolis, IN) were used. The
26CR animals had been subjected to calorie restriction starting at 3.5 months of age (10%
restriction), increased to 25% restriction at 3.75 months, and maintained at 40%
restriction from 4 months throughout the animal's life (until 26-months of age). Fischer
344 rats were used as there was extensive background data available on these animals and
they were the species we have used in previous projects. Males were used to avoid
possible skeletal muscle protective effects of estrogen which may have functioned as an
antioxidant (94). The rats were individually housed in a temperature (18-220C) and light-
controlled environment with a 12-hour light/dark cycle. After one week of acclimation
the animals were randomly sacrificed (5 per day) on consecutive days. All treatment of
animals throughout this study conformed fully with the Guiding Principles for Research
Involving Animals and Human Beings of the American Physiological Society (95), and,
received University of Florida institutional animal care and use committee approval.
Animals were anesthetized with an intraperitoneal injection of sodium
pentobarbital (Abbott Laboratories, Chicago, IL) (5 mg/100 g). The superficial vastus
lateralis (SVL) and all hind limb muscles from both legs were removed. Soleus (type I)
and SVL (type lib) (40) from the left leg were weighed, frozen in liquid nitrogen-cooled
isopentane and stored at -800C for use in histochemical and immunohistochemical
analyses. The right leg soleus and SVL were used for isolation of the nuclear and
cytosolic fractions. Lastly, the chest was opened and blood removed by cardiac puncture
drawn directly into Vacutainer tubes containing ethylenediaminetetraacetic acid
(K3EDTA; 8.4mg/ Vacutainer) for plasma acquisition. The blood aliquots were
centrifuged at 40C at 1500 x g for ten minutes and plasma stored at -800C until use.
Isolation of cytosolic fraction. Soleus and SVL muscles were homogenized in
isolation buffer (0.225 M mannitol, 0.075 M sucrose, 0.2 % BSA, ImM EDTA, pH 7.4)
with a dilution of 1:10 using a Potter-Elvehjem glass homogenizer. Homogenate was
centrifuged at 1,000 x g for 10 minutes. The supernatant was centrifuged at 14,000 x g
for 10 minutes and subsequently used for Western blotting and antioxidant enzyme
analyses in the cytosolic fraction.
Isolation of nuclear extracts. Nuclear extracts were isolated from the soleus and
SVL muscle using the protocol described by Blough et al (96). Briefly, 50 mg of tissue
was homogenized in 35 ml of Buffer 1 (10 mM HEPES, pH 7.5, 10mM MgC12, 5mM
KCL, 0.1 mM EDTA, pH 8.0, 0.1% Triton X-100, 1 mM dithiothreitol, 0.1 MM
phenylmethyl sulfonyl fluoride, 2 ug/ml aprotinin, and 2 ug/ml leupeptin) and centrifuged
for 5 minutes at 3000 x g at 40C. The pellet was then resuspended in 500 mL of Buffer 2
(20 mM HEPES, pH 7.9, 25% glycerol, 500 mM NaC1, 1.5 mM MgC12, 0.2 mM EDTA,
pH 8.0, 0.5 mM dithiothreitol, 0.2 mM phenylmethyl sulfonyl fluoride, 2 mg/mL
aprotinin, and 2 mg/mL leupeptin). Next, the sample was centrifuged for 5 minutes at
3000 x g at 40C. The supernatant was transferred to a 5000 nominal molecular weight
limit (NMWL), 4-ml Ultrafree Filter Unit (Millipore, Bedford, Ma) and centrifuged for
30 minutes at 4500 x g at 40C. This last step concentrated the sample, which was then
used for the measurement ofNF-KB activity.
Proteins were separated using 4-20% precast SDS-polyacrylamide gels (BMA,
Rockland) under denaturing conditions and electrotransferred onto nitrocellulose for 1 hr
at 0.28 amps. For TNF-R1, IKK3, IKKy, IKBc, p65, FADD and caspase-8, 30kg of
protein was added to each lane. After transfer, membranes were blocked with 5% nonfat
milk in PBS containing 0.05% Tween 20 (PBS-T/5% milk) overnight at 40C.
Membranes were then incubated with the following primary antibodies and
concentrations in PBS-T/5% milk for 1.5 hrs at room temperature: TNF-R1, monoclonal
antibody, 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA); IKK3, monoclonal
antibody, 1:100 (US Biological, Swampscott, MA); IKKy, monoclonal antibody, 1:100
(Santa Cruz Biotechnology, Santa Cruz, CA); IKBu, monoclonal antibody, 1:100
(Rockland, Gilbertsville, PA); p65, monoclonal antibody, 1:100 (Rockland, Gilbertsville,
PA); FADD, polyclonal antibody, 1:1000 (Stressgen, British Columbia, Canada), and
caspase-8, polyclonal antibody, 1:100 (Stressgen, British Columbia, Canada). After
washing (2 x 20 mins with PBS), membranes were incubated for 1.5 hrs at room
temperature with secondary antibody (anti-rabbit or anti-mouse IgG, horseradish linked
whole antibody; Amersham Life Science, United Kingdom) with an appropriate dilution
in PBS-T/5% milk. Blots were developed using ECL Western blotting detection reagents
(Amersham Pharmacia Biotech, United Kingdom) and protein bands analyzed using
Kodak ID Image analysis software (Eastman Kodak Company, Rochester, NY).
Arbitrary OD units were calculated by multiplying the area of each band by its optical
density. Groups were equally represented on each gel, and loading of equal amounts of
protein was controlled for by using Ponceau staining (Pierce Biochemicals, Rockford, IL)
of the nitrocellulose membrane.
Hematoxylin & Eosin stain. In an effort to characterize aging effects on the
skeletal muscles, muscle mass and representative muscle cross-sectional areas were
determined. Whole muscle samples, previously frozen in isopentane-cooled liquid
nitrogen and stored at -800C, were brought to the cryostat temperature (-200C) before
sectioning and placed in OCT mounting media (Miles, Elkhart, IN). Serial cross sections
(10 |tm thick) were cut from the mid-section of each muscle using a cryostat microtome
(Reichert-Jung) maintained at -200C and mounted on a microscope slide. Sections were
placed in 100% then 70% alcohol for 1 minute, respectively; hydrated in dH20 for 2
minutes then transferred to hematoxylin solution for 2 minutes. After washing in dH2O,
sections were placed in Scott's solution for 15 seconds, rinsed in dH20 and transferred to
70% alcohol. The sections were then stained in eosin solution for 2 minutes, dehydrated
and mounted. Sections were visualized using an Axiovert 200 light microscope (Carl
Zeiss Microimaging, Inc., Thornwood, NY) and computerized imaging processing
software (Scanalytics, Inc., Fairfax, VA). Two representative, random areas of interest
were chosen to determine muscle fiber cross-sectional area. Briefly, muscle fiber
membranes were traced and all areas within the membranes added to determine the cross-
section. Values were averaged for each sample and the results reported as cellular area
per mm .
Immunohistochemistry of skeletal muscle TNF-a. To expose TNF-a production
stemming from myocytes, consecutive sections obtained in the same manner (see
previous) were subjected to immunohistochemical analysis. In brief, sections were
incubated for 2hrs at 37C with the primary antibody for TNF-a (1:100; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). The sections were then washed in PBS (1.6 mM
NaH2PO4; 8.4 mM Na2HPO4; and 8.75g NaCl in 1L of dH20, pH 7.4) and incubated with
biotinylated anti-goat IgG (1:200, Vector Laboratories, Burlington, CA) for 30 minutes at
room temperature. After incubation with the secondary antibody, sections were washed
with PBS and incubated with ABC peroxidase reagent (Vector Elite ABC Kit, Vector
Laboratories, Burlington, CA) for 1 hr at room temperature. Following incubation, the
sections were washed 3 times in PBS and the enzyme substrate added (Vector NovaRed
substrate). Once the reaction proceeded for approximately 2-10 minutes, the section was
washed 3 times in dH2O.
Sections were viewed using an Axiovert 200 light microscope (Carl Zeiss
Microimaging, Inc., Thornwood, NY) and computerized imaging processing software
(Scanalytics, Inc., Fairfax, VA). For each sample, the number of TNF-a positive cells in
two representative regions of interest containing -100-200 fibers were counted manually,
and the mean used for statistical analysis. TNF-a labeled cells was reported as TNF-a
expression per number of fibers per mm2
Determination of TNF-a by ELISA. TNF-a was measured in the plasma using a
commercially available ELISA kit (R & D Systems, Minneapolis, MN) that employs the
quantitative sandwich enzyme immunoassay technique. Sample absorbances were read
at 450 nm off the standard curve and expressed in pg/mL.
NF-iB Activation. Skeletal muscle NF-KB activation was measured using an
ELISA kit (Active Motif, Carlsbad, CA). Briefly, sample nuclear extracts (96) were
treated according to manufacturers instructions, and loaded onto a microplate coated with
oligonucleotide containing the NF-KB consensus site. The primary antibodies used to
detect NF-KB recognized an epitope on p65 that is accessible only when NF-KB is
activated and bound to its target DNA. Following an incubation period, an
HRP-conjugated secondary antibody was added to provide a sensitive, colorimetric signal
from which absorbance at 450 nm was detected through use of a spectrophotometer.
Results were expressed as NF-KB activation optical density values per mg of protein at
DNA ladder assay. To enable detection of nucleosomal ladders in apoptotic cells
the DNA ladder assay was performed. Soleus and superficial vastus lateralis muscle
tissues (40-50 mg) were homogenized using a Teflon homogenizer in 1 mL DNAzol
(Molecular Research Center Inc., Cincinnati, OH). Proteinase K (Qiagen, Valencia, CA)
was added to the homogenates which, after a 3 hour incubation period, were centrifuged
(10 000 x g for 10 minutes at 40C) and the supernatants precipitated and washed with
100% and 75% ethanol, respectively. The isolated DNA was used in the PCR Kit for
DNA Ladder Assay (Maxim Biotech, Inc., San Francisco, CA) performed as directed by
the manufacturer. Lastly, samples were electrophoresed through 1.8% agarose gels
containing 0.5 [tg/mL ethidium bromide at 100 mA for 1 hour, and examined under UV
light for the presence of DNA ladders.
Antioxidant enzyme activity
Superoxide dismutase. Superoxide dismutase (SOD) activity was assayed
according to Oyanagui (1984) with slight modification (97) in muscle cytosolic fraction.
One unit (U) of SOD activity was defined as the concentration of enzyme that inhibited
nitrite formation from hydroxylamine in the presence of xanthine oxidase by 50%.
Cytosolic and nuclear protein concentrations were determined using the method
developed by Bradford (1976) (98). Samples and standard were pipetted in triplicate
onto a microplate and 200 [il of Coomassie Plus Protein Assay Reagent (Pierce) was
added. Optical density was determined using a microplate reader set at 595 nm.
A two-way analysis of variance was performed with age and fiber type, and calorie
restriction and fiber type as the independent variables using a statistical package from
Minitab Inc. (State College, PA). When appropriate, Tukey's post hoc analysis was
performed. A P-value of < 0.05 was considered significant.
Animal Body Weights
Compared to the 6-month-old ad libitum (6AL) rats (363.4 4.0 g; mean SEM),
the 26-month-old ad libitum (26AL) animals' body weights (413.6 5.4 g; mean SEM;
P < 0.001) increased -13%. Further, the 26AL animals' body weights were -35% greater
than those of the 26-month-old calorie restricted (26CR) group (307.1 2.3 g; mean +
SEM; P < 0.001) (Table 1).
Soleus muscle mass. The soleus muscle mass showed no difference in wet weight
between the 6AL (0.147 0.004 g; mean SEM) and 26AL (0.146 0.005 g; mean
SEM) groups with age (Table 1). Expression of soleus wet weight in conjunction with
body weight, however, indicated that the 6-month-old ad libitum animals had a
significantly greater soleus wet weight compared to the 26-month-old ad libitum group
(0.399 0.006 vs. 0.345 0.011 g; mean SEM; P = 0.02) (Fig 2). Alternatively, the
26CR animals displayed an -33% reduction (0.109 + 0.003 g; mean SEM; P < 0.001)
in soleus wet weight versus their ad libitum fed counterparts (Table 1); however, no
statistical differences were noted when body weight was brought into the equation (0.355
+ 0.011 vs 0.345 0.011 g; mean SEM) (Fig 2).
Table 1. Body mass and muscle mass of 6-month- and 26-month-old adlibitum and 26-
month-old calorie restricted male Fischer 344 rats.
6AL 26AL % change 26CR % change
(n = 8) (n = 8) 26AL vs. 6AL (n = 8) 26CR vs. 26AL
Bod mass 363.4 + 413.6+ T13% 307.1+ ,35%
4.0 5.4* 2.3
Soleus muscle 0.147 0.146 + 0.109 + ,33%
mass (g) 0.004 0.005** 0.003
vastus lateralis 0.419 0.364 15 0.332 + 9%
muscle mass 0.003 0.005* 0.006
Body mass and muscle mass of 6AL (6-month-old ad libitum), 26AL (26-month-old ad
libitum) and 26CR (26-month-old calorie restricted) male F344 rats. Data were
expressed as mean SEM. *P < 0.001 26AL vs 6AL and 26CR; *P < 0.001, 26AL vs.
Superficial vastus lateralis muscle mass. The predominantly type IIa and IIb
superficial vastus lateralis (SVL) (24, 25) demonstrated a significant reduction in wet
weight in the 26AL (0.364 0.005 g; mean SEM) compared to the younger animals
(0.419 0.003 g; mean SEM; P < 0.0001). In addition, calorie restriction also imparted
a reduced muscle wet weight (0.332 0.006 g; mean SEM; P = 0.0007) versus the
26AL group (Table 1). However, when expressed as a percentage of body weight, SVL
wet weight increased by 28% in the 26-month-old calorie restricted animals compared
with the 26-month-old ad libitum animals (1.089 0.022 g vs. 0.850 0.013 g; mean +
SEM; P < 0.0001). Further, when age was considered in the expression of SVL as a
percentage of body weight the 26AL group again exhibited a reduced wet weight
compared to the 6AL animals (0.850 + 0.013 vs. 1.126 0.021 g; mean + SEM; P <
0.0001) (Fig 2).
E 1.00- = 26AL
Figure 2. Soleus and SVL (superficial vastus lateralis) muscle wet weights (mg) of 6AL
(6-month-old ad libitum), 26AL (26-month-old ad libitum), and 26CR (26-
month-old calorie restricted) F344 rats expressed per gram of body weight.
Different from 6AL, P = 0.0205; *Different from 6AL, P < 0.0001; Different
from 26CR, P < 0.0001.
Muscle Cross Sectional Area
Histochemistry. Hematoxylin and eosin staining was conducted in an effort to
characterize aging on the soleus and superficial vastus lateralis muscle cross-sectional
areas. From each sample, two random areas of interest were chosen to determine muscle
fiber cellular area per mm2 as a representative measure of muscle cross sectional area
(Table 2). In soleus, the 6AL animals demonstrated a greater percentage of cellular area
per mm2 compared to the 26AL group (77.5 + 3.7 vs. 54.5 1.5 %; mean SEM; P =
0.002) (Fig 3A, B). We did not detect a statistical difference in percentage of cellular
area per mm2 between the 26AL and 26CR animals (54.5 + 1.5 vs. 61.9 6.0 %; mean +
SEM) (Fig 3B,C).
Table 2. Soleus and Superficial vastus lateralis cross sectional areas of 6-month- and 26-
month-old ad libitum and 26-month-old calorie restricted male F344 rats.
AL 2AL % difference R % difference
6AL 26AL 26CR
26AL vs. 6AL 26CR vs. 26AL
Soleus 77.5 54.5 61.9+ 13
12 142% 13%
(% Cell area/ mm ) 3.7 1.5 6.0
lateralis 77.2 + 68.1 + 13% 78.0 + "15%
lateralis 2 7 3 I13% 2.6 T15%
(% Cell area / mm) )
Representative cross sectional areas (expressed as % cell area / mm2) of 6AL (6-month-
old ad libitum), 26AL (26-month-old ad libitum) and 26CR (26-month-old calorie
restricted) male F344 rats obtained via H&E staining. Data were expressed as mean +
SEM. *P < 0.01, 26AL vs. 6AL; **P <0.05, 26AL vs. 6AL and 26CR.
In contrast, both age and diet effects were evident in the superficial vastus lateralis
(Fig 3D, E, F). Compared to the 6AL rats, the 26AL animals had -13% lower cell area
per mm2 (77.2 7.1 vs. 68.1 + 3.9 %; mean SEM; P = 0.0364) (Fig 3D, E). Also, when
compared to the 26CR animals, a similar scenario was found with the 26AL animals
demonstrating -15% reduction in cell area per mm2 (78.0 2.6 vs 68.1 3.9 %; mean +
SEM; P = 0.0406) (Fig 3E, F).
In agreement with previous findings (99) this data offered support to the idea that
a decline in muscle fiber size occurred in parallel with age. What's more, there appeared
to be a fiber type specific response to aging-associated muscle loss, highlighted by the
ability of the dietary intervention to impart a greater resistance in the SVL against muscle
fiber loss and cell area reduction compared to that which was displayed in the soleus.
Immunohistochemistry. Immunohistochemical analysis to expose TNF-ac
expression stemming from myocytes was performed in an attempt to discern whether
6AL 26AL 26CR
Figure 3. H&E staining in soleus (A-C) and SVL (superficial vastus lateralis) (D-F)
sections from 6AL (A,D), 26AL (B,E) and 26CR (C,F) Fischer-344 rats. Bar
muscle fiber type exerted an influence on the cytokines' expression. For each sample, the
number of TNF-a positive cells in two representative regions of interest containing -100-
200 fibers were counted manually, and the mean used for statistical analysis. In soleus,
we found no detectable differences between the 6AL and 26AL animals (0.03 0.005 vs.
0.043 0.003 TNF-a expression / fiber number / mm2; mean SEM) nor did we reveal
differences when comparing 26AL with 26CR (0.043 0.003 vs. 0.03 0.01 TNF-a
expression / fiber number / mm2; mean SEM) (Fig 4). There were, however, far
different results found in the superficial vastus lateralis muscle. The 26-month-old ad
libitum animals exhibited significantly greater TNF-a expression per fiber number per
mm2 compared to the 6-month-old ad libitum animals (0.069 0.02 vs. 0.039 0.003
TNF-a expression / fiber number / mm2; mean SEM; P < 0.05) and also compared to
the 26-month-old calorie restricted animals (0.069 0.02 vs. 0.033 0.008 TNF-a
expression / fiber number / mm2; mean SEM; P < 0.05) (Figs 4 and 5).
o E 0.100-
Figure 4. TNF-a expression from skeletal muscle myocytes. The number of TNF-a
positive cells in two representative regions containing -100-200 fibers were
counted manually, and the means used for statistical analysis No statistical
differences were detected between groups in soleus. In SVL, 26AL was
significantly different from 6AL and 26CR (*P < 0.05).
Elevated TNF-a levels have been reported in frail elderly individuals in myocytes
acquired from vastus lateralis muscle (14), a finding which the present data falls in
agreement with. However, to our knowledge few studies have reported the occurrence of
a fiber type-specific expression of this catabolic cytokine and a possible tendency for
type II over type I fibers to emanate greater expression.
Figure 5. TNF-a immunohistocheical staining in superficial vastus lateralis sections
from 6AL (A), 26AL (B) and 26CR (C) Fischer-344 male rats. Arrows
indicate muscle fibers positive for TNF-u. Bar 100 m.
,' ( / 1 ,y1 *
Figure 5. TNF-ca immunohistochemical staining in superficial vastus lateralis sections
from 6AL (A), 26AL (B) and 26CR (C) Fischer-344 male rats. Arrows
indicate muscle fibers positive for TNF-ca. Bar 100 |jm.
Antioxidant Enzyme Activity
Superoxide dismutase activity. As an index of superoxide production, activity
level of the antioxidant enzyme superoxide dismutase (SOD) was assayed in the cytosol
fractioned from the soleus and SVL of the 6-month and 26-month-old ad libitum and 26-
month-old calorie restricted animals (Table 3).
Table 3. Antioxidant enzyme activity in cytosol from 6-month- and 26-month-old ad
libitum and 26-month-old calorie restricted male F344 rats.
6AL 26AL 26CR
Soleus 31.6 0.9 38.4 + 1.9* 33.7 0.9
SVL 33.0 + 1.3 35.6 + 1.6* 30.1 0.5
Superoxide dismutase (SOD) activity is expressed as units / mg protein and was
measured according to Oyanagui (97). *Different from 6AL, P < 0.05; *Different from
26CR, P < 0.05
In soleus, SOD was found to be elevated in the 26AL animals compared to the 6AL
group (38.4 1.9 vs. 31.6 0.9 units / mg protein; mean SEM; P <0.05) but no
differences were observed between 26AL and 26CR groups. In the SVL, age effects
were not evident but there were differences accompanying diet with 26AL demonstrating
greater SOD activity compared to the 26CR animals (35.6 + 1.6 vs. 30.1 0.5 units / mg
protein; mean SEM; P <0.05).
Determination of Plasma TNF-a
Significant age and dietary effects on TNF-a levels. Greiwe et al. (14) showed
in human plasma and skeletal muscle that TNF-a levels elevated with age were
attenuated by resistance exercise. We investigated the efficacy of calorie restriction in
being able to exert similar effects. In agreement with others (100, 101), plasma TNF-a
levels were increased in 26-month- compared to 6-month-old ad libitum animals (21.8 +
0.7 vs. 7.2 1.4 pg / mL; mean + SEM; P =0.0001) (Fig 6).
6AL 26AL 26CR
Figure 6. Plasma TNF-a levels in 6AL (6-month-old ad libitum), 26AL (26-month-old
ad libitum) and 26CR (26-month-old calorie restricted) F344 rats. *Different
from 6AL, P = 0001; bDifferent from 26AL, P = 0.0001
Further, calorie restriction invoked a near 3-fold reduction in plasma TNF-a levels
as found when the 26AL animals were compared to the 26CR group (21.8 0.7 vs. 7.9 +
1.8 pg / mL; mean SEM; P = 0.0001) (Fig 6).
Western Blotting Detection of TNF-a / NF-cB Signaling Proteins
TNF-R1 protein content in muscle of 6AL, 26AL and 26CR rats. Despite the
presence of two receptors for TNF-c, multiple experimental approaches have revealed
that TNF-R1 initiates the majority of TNF-a's biological activities and has the ability to
direct both cell survival and cell death signals (56, 57). After homogenization of the
muscle samples, the homogenate was centrifuged at 1,000 x g for 10 minutes. The
supernatant was then centrifuged at 14,000 x g for 10 minutes and the cytosolic fraction
used for TNF-R1 protein determination (Fig 7).
u 20000- = 26AD
Figure 7. TNF-R1 in soleus and SVL (superficial vstus lateralis) muscle. Homogenates
were centrifuged at 1,000 x g for 10 minutes. The supernatant was then
centrifuged at 14,000 x g for 10 minutes and the cytosolic fraction assayed for
TNF-R1 content via Western blotting. There was no difference statistically in
TNF-R1 protein content between 6AL and 26AL, nor 26AL and 26CR in
We found no statistical differences in the protein content of TNF-R1 in the soleus
cytosol with age (14444 2530 vs. 12355 2850, 6AL vs. 26AL, respectively; mean +
SEM of arbitrary OD units / mg protein) nor when examined for effects of diet (12355
2850 vs. 19178 2773, 26AL vs. 26CR, respectively; mean SEM of arbitrary OD units
/ mg protein) (Fig 7). Furthermore, we did not detect differences in TNF-R1 expression
in the SVL muscle with age (10112 1987 vs. 11599 2484, 6AL vs. 26AL,
respectively; mean SEM of arbitrary OD units / mg protein) or dietary intervention
(11599 + 2484 vs. 15289 2148, 26AL vs. 26CR, respectively; mean SEM of arbitrary
OD units / mg protein).
IKK complex subunits, IKKp and IKKy, presence in soleus and superficial
vastus lateralis. In the IKK complex, IKKP and IKKy, kinase and regulatory subunits,
respectively, have been shown to be required for proinflammatory (e.g., TNF-a) induced
stimulation of NF-KB (61, 63-65). Accordingly, we examined these two subunits of the
IKK complex to determine how they would respond to the effects of age and calorie
restriction. In the soleus, we found that IKKP content was reduced with age (P = 0.0073
vs. 6AL) and was unaffected by calorie restriction (Fig 8). Conversely, calorie restriction
appeared to evoke a decrease in IKK3 content in the SVL compared to 26AL; however,
no age effect was noted in this muscle (Fig 8).
The regulatory noncatalytic component of the IKK complex, IKKy, has been
singled out as an essential requisite in order for NF-KB activation to be achieved by a
variety of stimuli (63, 64). We chose to investigate the response of this protein first, to
age and dietary intervention, and second, in muscles comprised of different fiber types.
Figure 8. Western blot analysis of IKKP content in soleus and SVL (superficial vastus
lateralis) muscle from 6AL (6-month-old ad libitum) (n=8), 26AL (26-month-
old ad libitum) (n=8) and 26CR (26-month-old calorie restricted) (n=8) male
F344 rats. Soleus IKK3 content decreased with age (246106 + 12408 vs.
184415 + 9170 arbitrary units / mg protein; mean + SEM; *P = 0.0073) with
no dietary effect detected. In the SVL, however, a dietary effect was found
and IKK3 appeared reduced in the 26CR group compared to the 26AL
animals (135844 + 26675 vs. 241832 + 6855 arbitrary units / mg protein;
mean + SEM; P = 0.0377). Age effects were not significant in the SVL.
In the SVL we did not detect any change in content of IKKy between the 6AL and
26AL animals (25987 5259 vs. 28644 3705 arbitrary units / mg protein, respectively;
mean SEM) nor did we see an influence of diet when comparing the 26AL to the 26CR
group (28644 3705 vs. 20168 + 3322 arbitrary units / mg protein, respectively; mean +
SEM) (Fig 9). Alternatively, when the effects of age and diet were evaluated in the
soleus significant differences were displayed. In terms of age, the 6AL animals had
substantially reduced protein content of IKKy compared to the 26AL group (43984 +
7150 vs. 92587 9794 arbitrary units / mg protein, respectively; mean SEM; P =
0.003). Calorie restriction imparted similar effects, with the 26CR rats demonstrating a
reduced IKKy content compared to the 26AL animals (29447 8410 vs. 92587 + 9794
arbitrary units / mg protein, respectively; mean + SEM; P < 0.0001) (Fig 9).
"I = 26AD
Figure 9. IKKy content in soleus and SVL muscle from 6AL, 26AL and 26CR male
F344 rats. There was no significant difference with age or diet in the SVL. In
soleus, age and diet evoked significant differences. 26AL was greater in
IKKy content compared to 6AL (*P = 0.003) and 26AL was also greater
compared to 26CR ( P < 0.0001). IKKy content is reported as arbitrary units /
The NF-KB inhibitor protein, IcBa, is significantly affected by age and diet in
soleus muscle. In quiescent cells, NF-KB is maintained in an inactive state through
binding to inhibitor of KB proteins, which cover the nuclear localization sequence of NF-
KB and prevent translocation of the transcription factor to the nucleus. We measured the
protein content of IKBa to determine how it would respond to age and calorie restriction
in different fiber types. Beginning with the soleus, we found that age and diet effects
existed. The 26-month-old ad libitum animals possessed a much greater content of IKBa
protein compared to the 6-month-old ad libitum animals (12069 1399 vs. 6758 1032
arbitrary units / mg protein, respectively; mean SEM; P = 0.0103) (Fig 10).
0 2 6AL
E 10000- 26CR
Figure 10. Western blot analysis of IKBa in soleus and SVL (superficial vastus lateralis)
muscle of 6-month-old- (6AL), 26-month-old ad libitum (26AL) and 26-
month-old calorie restriction (26CR) F344 rats. IKBa content is reported as
arbitrary units / mg protein. In soleus, significant differences were found
between 26AL and 6AL groups(12069 1399 vs. 6758 + 1032 arbitrary units
/ mg protein, respectively; mean SEM; *P = 0.0103). Also, calorie
restriction affected IKBa content in the same direction as age, with greater
levels found in 26AL compared to the 26CR animals (12069 1399 vs. 5866
+ 507 arbitrary units / mg protein, respectively; mean SEM; P = 0.0005).
In a similar fashion, differences were evidenced between the 26-month-old ad
libitum and 26-month-old calorie restricted groups (12069 + 1399 vs. 5866 507
arbitrary units / mg protein, respectively; mean SEM; P = 0.0005) (Fig 10). Upon
examination of IKBa in the superficial vastus lateralis, however, we did not detect
differences amongst groups for age (4599 868 vs. 6022 857 arbitrary units / mg
protein, 6AL vs. 26AL, respectively; mean SEM) nor diet (6022 857 vs. 4851 + 708
arbitrary units / mg protein, 26AL vs. 26CR, respectively; mean SEM).
Content of p65, a member of the family of dimers comprising NF-KB. The
predominant form of NF-KB is a heterodimer consisting of p50/p 105 and p65 (RelA) of
the Rel family of proteins. We elected to focus on the responses of p65 to aging and diet
as past studies have lamented on the frequent incidence of p65 as one of the elements of
the NF-KB dimer (73). In soleus, significant effects of age and diet were exhibited with
regard to p65 protein content (Fig 11). There were increases in p65 content in the 26AL
animals compared to the 6AL group (391925 + 45310 vs. 199460 19335 arbitrary units
/ mg protein, respectively; mean SEM; P = 0.0014). Also, 26AL demonstrated
significantly higher p65 content versus the 26CR animals (391925 45310 vs. 224699 +
20726 arbitrary units / mg protein, respectively; mean SEM; P
Figure 11. p65 protein content in soleus muscle of 6-month-old ad libitum (6AL), 26-
month-old ad libitum (26AL) and 26-month-old calorie restricted (26CR)
male F344 rats. Western blot analysis revealed a significant increase with age
for p65 content (199460 19335 vs. 391925 + 45310 arbitrary units / mg
protein, 6AL vs. 26AL, respectively; mean SEM; *P = 0.0014). Further,
calorie restriction reduced p65 content compared to the ad libitum fed group
(224699 + 20726 vs. 391925 45310 arbitrary units / mg protein, 26CR vs.
26AL, respectively; mean + SEM; P = 0.0423). p65 content is reported as
arbitrary units / mg protein.
To our surprise, we did not detect evidence of p65 content in the superficial vastus
lateralis muscle tissue using western blot analysis. The possibility exists that p65 may
not be the dominant protein subunit of NF-KB expressed in this muscle type. Rather,
other members of the Rel family, such as p50/pl05, RelB, c-Rel and p52/pl00 (73) may
experience a preferential expression in forming the NF-KB dimer. Accordingly, the
factors involved in shaping this intriguing prospect and determining the arrangement of
the NF-KB dimer in this tissue merit further investigation.
Nuclear Binding Activity of NF-KB
NF-KB activation is greater in the soleus compared to the superficial vastus
lateralis. Soleus and SVL muscle sample nuclear extracts were isolated (96) and NF-KB
activation was measured using an ELISA kit (Active Motif, Carlsbad, CA) (Fig 12). In
the soleus, we did not detect significant effects of age or diet on the activity levels of
NF-KB (Fig 12). Alternatively, we did reveal an effect of age in the SVL, which
identified greater NF-KB activity in the young compared to the old animals (1.662 +
0.134 vs. 1.327 0.137 OD values / mg protein; 6AL vs 26AL, respectively; mean +
SEM; P = 0.0332). Furthermore, when NF-KB activity was examined between muscle
types, soleus demonstrated -56% greater degree of activation compared to the superficial
vastus lateralis (2.151 0.051 vs. 1.378 0.072 OD values / mg protein; soleus vs. SVL,
respectively; mean SEM; P = 0.0001). This significantly different NF-KB activation
profile existent between the two muscles may be linked to upstream events at the level of
the receptor adaptor protein complex. The decision of which signal will be transmitted,
that is, NF-KB activation or apoptosis, is determined by the type of adaptor proteins
drawn to the receptor, TNF-R1. These current data lend support to the idea that there
may be a fiber type-specific characteristic directing downstream signaling pathways,
specifically, the soleus, a muscle comprised of type I fibers (39), electing to activate
NF-KB, compared to the SVL, composed predominantly of type IIa and IIb fiber types
(25, 39), opting for the apoptotic signaling paths.
Figure 12. NF-KB binding activity in soleus and SVL (superficial vastus lateralis)
nuclear extracts of 6-month-old ad libitum (6AL), 26-month-old ad libitum
(26AL) and 26-month-old calorie restricted (26CR) male F344 rats. In soleus,
we did not detect a significant age effect (2.129 0.07 vs. 2.073 0.077 OD
values / mg protein; 6AL vs 26AL, respectively; mean SEM) nor diet effect
(2.073 0.077 vs. 2.241 0.111 OD values / mg protein; 26AL vs. 26CR,
respectively; mean SEM). SVL NF-KB binding activity produced an age
effect with higher levels observed in the 6AL group (1.662 + 0.134 vs. 1.327
+0.137 OD values / mg protein; 6AL vs 26AL, respectively; mean SEM; P
= 0.0332), but no diet effect (1.327 0.137 vs. 1.220 0.137 values / mg
protein; 26AL vs 26CR, respectively; mean SEM). Also notable, however,
was the difference ( P = 0.0001) in NF-KB activity between muscles. NF-KB
binding activity is reported as OD values at 450nm per mg protein.
The TNF-a receptor-mediated pathway of apoptosis
Arising from our data describing the composition of TNF-a / NF-KB signaling in
skeletal muscle and the effects on this pathway of age and calorie restriction, we
proceeded to investigate the possibility of muscle fiber types playing a regulatory role in
directing the nature of the TNF-a signal conducted. Specifically, electing to recruit
adaptor proteins in the course of events that transmitted signals promoting inflammatory
events or apoptotic events. To achieve this end, we examined the occurrence of two of
the proteins intrinsic to the TNF-a receptor-mediated pathway of apoptosis, FADD
(Fas-associated death domain) and caspase-8 (57).
FADD content elevated with age
When TNF-a binds to TNF-R1, modifications to the TNF-R1 adaptor protein
complex promote the formation of a new complex which dissociates from TNF-R1 and
attracts FADD and other proapoptotic intermediates (102). Thus, we chose to assess how
age and calorie restriction in different muscle tissues would affect the expression of this
2 0 6AL
Figure 13. FADD protein content in soleus and SVL (superficial vastus lateralis) muscle
of 6AL, 26AL and 26CR male F344 rats. Western blot analysis revealed a
significant increase with age in the SVL for FADD content ( P = 0.0281).
Further, in the SVL calorie restriction reduced FADD content compared to the
ad libitum fed group (18429 2628 vs. 32876 + 2406 arbitrary units / mg
protein, 26CR vs. 26AL, respectively; mean SEM; P = 0.0477). We did
not observe significant differences in FADD protein levels in the soleus with
age or diet. FADD content is reported as arbitrary units / mg protein.
In the soleus, we did not detect a statistical difference in protein levels of FADD
with age (41924 4306 vs. 34993 + 2526 arbitrary units / mg protein, 6AL vs. 26AL,
respectively; mean SEM) nor with diet (34993 2526 vs. 27108 5493 arbitrary units
/ mg protein, 26AL vs. 26CR, respectively; mean SEM). When we examined FADD
content in the SVL (superficial vastus lateralis), however, we did observe a different
outcome. The 26AL group displayed a greater level of FADD content compared to the 6-
month-old adlibitum animals (32876 + 2406 vs. 21844 1048 arbitrary units / mg
protein, respectively; mean SEM; P = 0.0281) (Fig 13). Additionally, we found an
intervention effect on FADD content, with a reduced level of the protein being detected
in the 26CR animals compared to the 26AL group (Fig 13).
Caspase-8: zymogen and cleaved product
Another proponent of the TNF-ac stimulated receptor-mediated pathway of
apoptosis and affiliated with FADD is caspase-8. Caspase-8, the activated and cleaved
form of procaspase-8, belongs to the family of cysteine proteases, and functions to
activate other caspases with the potential to generate programmed cell death (103). We
chose to determine the effects of age and calorie restriction in the soleus and SVL on the
zymogen and cleaved product of caspase-8. When we analyzed procaspase-8 in the
soleus, we found no differences between the young and old ad libitum fed animals
(32261 + 7532 vs. 29451 3809 arbitrary units / mg protein, 6AL vs. 26AL,
respectively; mean SEM), nor between the old ad libitum fed and calorie restricted
groups (29451 3809 vs. 43367 + 6050 arbitrary units / mg protein, 26AL vs. 26CR,
respectively; mean SEM) (Fig 14). Likewise, our results for procaspase-8 content in
the SVL muscle indicated no differences with age (32547 + 4128 vs. 33884 4225
arbitrary units / mg protein, 6AL vs. 26AL, respectively; mean SEM) or with calorie
restriction (33884 4225 vs. 35636 5547 arbitrary units / mg protein, 26AL vs. 26CR,
respectively; mean + SEM) (Fig 14).
0 M 6AL
SE M 26CR
Figure 14. Procaspase-8 protein content in soleus and SVL (superficial vastus lateralis)
muscle of 6-month-old ad libitum (6AL), 26-month-old ad libitum (26AL)
and 26-month-old calorie restricted (26CR) male F344 rats. We did not detect
differences in protein content of the zymogen in either muscle with age or
dietary intervention using Western blot analysis. Procaspase-8 content is
reported as arbitrary units / mg protein.
Next we determined whether effects of age and diet would be evidenced in
caspase-8 content. In the soleus muscle, there were no differences between the 6AL and
the 26AL animals for caspase-8 protein content (14557 5879 vs. 10905 4300
arbitrary units / mg protein, 6AL vs. 26AL, respectively; mean SEM); similarly, we did
not observe differences when comparing the 26AL rats to the 26CR rats (10905 + 4300
vs. 15848 + 800 arbitrary units / mg protein, 26AL vs. 26CR, respectively; mean SEM)
(Fig 15). In the superficial vastus lateralis, we found a significant effect with age (7605 +
1296 vs. 22918 4486 arbitrary units / mg protein, 6AL vs. 26AL, respectively; mean +
SEM; *P = 0.0478) in caspase-8 content.
0 E 20000-
Figure 15. Caspase-8 protein content in soleus and SVL (superficial vastus lateralis)
muscle of 6-month-old ad libitum (6AL), 26-month-old ad libitum (26AL)
and 26-month-old calorie restricted (26CR) male F344 rats. Western blot
analysis revealed a significant increase with age in the SVL for Caspase-8
content (7605 1296 vs. 22918 4486 arbitrary units / mg protein, 6AL vs.
26AL, respectively; mean + SEM; *P = 0.0478). Further, in the SVL calorie
restriction reduced caspase-8 content compared to the ad libitum fed group
(8497 + 1522 vs. 22918 + 4486 arbitrary units / mg protein, 26CR vs. 26AL,
respectively; mean + SEM; P = 0.0142). We did not observe significant
differences in caspase-8 protein levels in the soleus with age or diet. Caspase-
8 content is reported as arbitrary units / mg protein.
Further, the dietary intervention reduced caspase-8 levels compared to the age-
matched ad libitum fed counterparts (8497 1522 vs. 22918 4486 arbitrary units / mg
protein, 26CR vs. 26AL, respectively; mean SEM; P = 0.0142) (Fig 15).
DNA was isolated from the soleus and superficial vastus lateralis muscles and
assessed on agarose gels containing 0.5 [lg/mL ethidium bromide for the presence of
DNA ladders, a characteristic of apoptosis. The presence of DNA ladders were revealed
in the superficial vastus lateralis samples from the 26-month-old ad libitum fed animals;
however, DNA ladders were absent in both the 6-month-old ad libitum and 26-month-old
calorie restricted groups (Fig 16A). When we examined the soleus for DNA
fragmentation a different profile was found. In contrast to the superficial vastus lateralis,
the only trace of DNA laddering was observed in the 6-month-old ad libitum fed animals
(Fig 16B). Neither the 26-month-old ad libitum group nor the 26-month-old calorie
restricted group displayed signs of DNA fragmentation in the soleus.
1 2 3 4 5 6 7 8 9 10 11 12
Figure 16. DNA fragmentation in superficial vastus lateralis (panel A) and soleus (panel
B). DNA ladders were observed in the superficial vastus lateralis muscles of
the 26-month-old ad libitum animals (lanes 5 to 7) but were not observed in
the 6-month-old ad libitum animals (lanes 2 to 4) nor the 26-month-old calorie
restricted animals (lanes 8 to 10). In soleus muscles, traces of DNA ladders
were observed in the 6-month-old ad libitum animals (lanes 2 to 4; panel B)
but were not detected in the 26-month-old ad libitum or the 26-month-old
calorie restricted animals (lanes 5 to 7, and lanes 8 to 10, respectively; panel
B). Lane 1 comprised the molecular weight marker; lane 11 the negative
control and lane 12 the positive control for both panels A and B. One
representative image is shown from two experiments that yielded similar
Aging and Sarcopenia
Sarcopenia (describing the decline in skeletal muscle mass with age) and an
elevated inflammatory milieu are among the physiological changes deemed synonymous
with the aging process, and have been cited as significant indicators of mortality in older
populations (104). Given the social and economic impact of sarcopenia and subclinical
inflammation with age our research has focused on elucidating the mechanisms driving
such occurrences. The direct healthcare costs attributable to sarcopenia in the United
States alone in 2000 were estimated as $18.5 billion, representing approximately 1.5% of
the total healthcare expenditures for that year (105). Sources responsible for sarcopenia
are believed to be multivariate in nature, and include a reduction in hormones promoting
growth (106, 107), increased fat mass (108), a decline in central motor system alpha
motor neurons (109), elevated apoptosis (110) and increases in catabolic cytokines (104).
We sought to investigate sarcopenia from the perspective of involvement of the
proinflammatory and catabolic cytokine, tumor necrosis factor-a (TNF-c) as little
research has explored a role for this pleiotropic cytokine in the pathology of the
TNF-a, Aging and Skeletal Muscle
We measured plasma TNF-a to establish the presence of an age effect with regard
to systemic levels of the cytokine, and in agreement with findings from previous work
(104) observed an age-associated increase in plasma TNF-a which the intervention of
calorie restriction attenuated. The impact of aging on skeletal muscle mass was next
explored. In an attempt to discern whether TNF-a played an active role in muscle mass
reduction with age we evaluated the expression of TNF-a from the soleus and superficial
vastus lateralis (SVL) muscles immunohistochemically. These particular muscles were
selected as they are composed of vastly different fiber types, with soleus containing
primarily type I fibers and SVL type IIa and lib, representing slow and fast fiber types,
respectively (23, 25). Furthermore, the soleus and SVL have been reported to display
differing degrees of age-associated atrophy (26). A reduction in the SVL wet weight
only was evidenced with age, concurring with previous reports of a greater susceptibility
of fast type muscles to yield to age-linked muscle mass loss or atrophy, compared to slow
fiber type muscle (26, 27). A recent study by McKiernan et al., (111) substantiated the
greater resistance to age-linked atrophy demonstrated by the soleus muscle (composed of
type I fibers) than was evident in the vastus lateralis and rectus femoris muscles
(predominantly of type II fibers). These researchers in a paralleled study (112) hinted
toward the involvement of mitochondrial DNA deletions and associated electron
transport system enzymatic abnormalities as providing the trigger for loss of skeletal
muscle fibers with age, and for this sequela to be more pronounced in muscles comprised
predominantly of type II fibers (99). They reasoned that the lower mitochondrial content
in the type II fibers presented a more vulnerable target with respect to the frequency of
mitochondrial DNA mutations occurring, thereby increasing the likelihood of these fibers
ultimately succumbing to atrophy.
Age Decline in Muscle Cell Area and Increased TNF-a Expression
Skeletal muscle atrophy with age is thought to be manifest by either a reduction in
fiber mass, fiber number or some combination thereof (5, 25, 27). Our findings from
hematoxylin and eosin stains of muscle fiber sections fell in agreement with these reports.
Both soleus and superficial vastus lateralis muscles displayed reductions in the areas
measured for myocytes per square millimeter in the aged animals than was found in the
young animals. Notable also, was the ability of calorie restriction to elicit a reversal of
this age-associated decline in cell area in the superficial vastus lateralis. Concomitant
with the decrease in SVL mass and cellular area with age, was an increase in TNF-a
expression. The age-associated elevation in TNF-a stemming from myocytes has been
acknowledged by earlier work (14). The study by Greiwe and coworkers (14), deployed
an exercise intervention which successfully resulted in a reduction of TNF-a expression
in skeletal muscle of elderly humans.
In the current study, the dietary intervention of calorie restriction was used to
suppress age-associated increases in the cytokine. We found lowered TNF-a expression
in the calorie restricted group compared to their age-matched, ad libitum fed counterparts.
Furthermore, when we performed the same analysis in soleus we found no detectable
differences in myocyte-expressed TNF-a levels, presenting the possibility of a fiber type
dependent regulation of TNF-a expression. Alternatively, the presence of other cell
types that may reside in skeletal muscle, for example adipocytes and macrophages, both
of which also have the propensity to produce TNF-u, may also be part contributors to the
higher levels of TNF-a observed in the SVL with age. Moreover, the lowered superficial
vastus lateralis muscle TNF-a expression accompanying calorie restriction may be
attributable to either the reduced fat mass which this intervention imparts (113) or may be
the result of a decline in inflammatory genes expressed, as has been reported in DNA
microarray studies profiling gene expression in skeletal muscle subjected to calorie
Fiber Type Variations in NF-KB Signaling
The actions of TNF-a are transmitted via two receptors and an intricate assortment
of signaling proteins. The roles of most of these components have been ascertained;
however, certain points of contention remain, particularly with respect to the manner in
which adaptor proteins are recruited to the TNF-a receptors. With these considerations
in mind, we wanted to determine in skeletal muscle the response to aging and calorie
restriction of the signaling intermediaries integral to TNF-a stimulation of NF-KB. With
the majority of TNF-a's biological actions initiated through TNF-R1 (57) we began our
examination of the signaling pathway at this juncture. We did not observe differences in
protein content of TNF-R1 with age or calorie restriction in either muscle tissue, an
outcome that may have been influenced by the cellular fraction we chose to examine,
namely the cytosolic fraction. Although this receptor does persist in a soluble form, a
population also prevails that is membrane bound and this population may have responded
differently to age and calorie restriction.
Kim and associates (15) in their study of rat kidney from young and old animals,
showed signaling molecules of the NF-KB family to be amplified with age (15); we
wanted to establish whether skeletal muscle acted in a similar fashion. A focal point in
the pathway of NF-KB activation by TNF-a is the IKK complex. The catalytic subunit of
the complex, IKKP, was affected by age in the soleus but not in the superficial vastus
lateralis muscle. The lowered IKK3 protein content observed in the aged animals falls in
contrast to the response of this protein reported in the aged kidney (15). When we
proceeded to examine the regulatory unit of the IKK complex, IKKy or NEMO, a stark
increase in this protein was found with age in the soleus but no age effects were found in
the superficial vastus lateralis. What's more, in the calorie restricted animals, the levels
of IKKy were significantly reduced compared to those demonstrated in the age-matched
ad libitum fed counterparts. There is much debate surrounding the extent of each of the
subunits' contribution in promoting the downstream events leading to NF-KB activation;
however, several lines of evidence contend that NF-KB activation is contingent on the
presence of IKKy (63, 64) to a much greater degree than that of the other subunits. Thus,
if this relationship holds true for skeletal muscle, then the current data would support the
notion that in spite of the reduced protein level of IKK3, the elevated IKKy level ensured
an increased stimulation of NF-KB with age. Further work is necessitated in order to
corroborate this idea.
The target of the IKK complex and the protein inhibiting NF-KB translocation from
cytosol to nucleus, IKBc, was next subjected to analysis. Others have reported a decline
in this inhibitor of KB with age (15) but in the current study we found the opposite to be
true. Reflective of the IKKy responses observed earlier, we found all of the significant
differences in IKBa content to occur in the soleus and not the superficial vastus lateralis
muscle. The young ad libitum fed animals and the old calorie restricted animals both
exhibited substantially lower levels of IKBa compared to the old ad libitum fed animals.
This illustration of increased IKBa content with age may act as an indirect indicator of
elevated NF-KB activity. Specifically, contained in the NF-KB program of genes targeted
for transcription (71) are members of the Rel / NF-KB / IKB family. When NF-KB is
subject to activation it prompts the new synthesis of IKB, and limits its own activation
(74, 75). Given that the current data demonstrated an increased content of IKBa with age
supports the proposition of an age-associated elevation in NF-KB activity necessitating an
elevated IKBa transcription to quash NF-KB's activation for an ill-desired extended
period of time.
Fiber Type Variations in NF-KB Subunits and Binding Activities
Following a very similar pattern to those uncovered for the upstream signaling
proteins, was the response of p65 to age and diet. An elevation with age, which calorie
restriction was able to attenuate, was exuded in the soleus for this subunit of NF-KB.
Others have reported an analogous relationship in non-muscle tissue for p65 expression
obtained from aged and aged calorie restricted rats (15). Unexpectedly, however, we
could not find evidence of p65 protein in the superficial vastus lateralis muscle, nor
references in the literature that have documented this anomaly. Given that there are five
members of the Rel family that are relied upon to form the hetero- or homodimer unit that
embodies NF-KB (73) presents the possibility that in the SVL, the other family member
subunits may experience a preferential expression over and above p65 in forming the NF-
KB dimer. In any event, further research is warranted to explore whether or not this is the
case and the underlying elements that may regulate this prospect.
Binding of NF-KB to the DNA nuclear localization site represents the green light
for transcriptional activities to be initiated. To gauge the effects of age and calorie
restriction on this event, we measured NF-KB binding activity in nuclear extracts
obtained from the soleus and superficial vastus lateralis muscles. We did not detect age
or diet effects on the degree of NF-KB activity in the soleus, and the only significant
difference, symbolized by increased NF-KB binding, observed in the superficial vastus
lateralis fell in the young group, who displayed greater NF-KB activation compared to the
old animals. An intriguing finding did emerge, however, with respect to the NF-KB
binding activities exhibited by the different muscles. The superficial vastus lateralis
exuded a notably lower level (-56%) of transcription factor nuclear binding compared to
the soleus. This profile of NF-KB binding may be representative of an intrinsic fiber
type-specific capability, or in other words, a fiber type driven preference for signaling to
NF-KB. Support for this proposition is derived from the pattern that emerges in soleus of
the inflammatory factors' responses with age. The amplified presence of the
inflammatory advocates, i.e., IKKy, IKBa, p65 and NF-KB binding, consistently
appearing in the soleus more so than the superficial vastus lateralis, puts forth a strong
case for fiber type holding some authority in the decision to promote inflammatory
signaling, a decision which appears to be coupled with the aging process. Furthermore,
the ability of calorie restriction to attenuate most of the age-associated elevations in the
components of inflammation studied, may be representative of the effectiveness of this
intervention against chronic, sub clinical levels of inflammation which accompanies
aging (104) and which has also been suggested by others (13).
It must also be recognized, however, that when signaling to NF-KB predominates
over the other TNF-a signaling paths, (i.e., proapoptotic activation) that alongside
inflammatory signaling, NF-KB promotes gene transcription responsible for cell survival
and repression of apoptosis (54, 76). Therefore, the pattern of signal conductance in the
soleus of old animals may be indicative of signaling efforts in this tissue to promote
growth and survival (71). If indeed fiber type does exert a regulatory influence over the
cell signal conveyed, then the balance of cell survival or cell death signaling may differ in
skeletal muscle with different fiber type profiles. In order to investigate this conjecture, a
closer examination of TNF-a signaling apoptotic pathways was undertaken.
Fiber Type Differences in Apoptotic Signaling
An increase in apoptosis has been described as a contributor to age-associated
atrophy (110); thus, to address the possibility of TNF-a transmitting signals to recruit
apoptotic proteins versus those linked to NF-KB, we measured the content of two proteins
in the programmed cell death cascade, FADD and caspase-8, and sought out evidence of
DNA fragmentation, one of the final morphological events in the conserved sequence of
In the old ad libitum fed animals, we observed significant increases in the content
of both FADD and caspase-8 in the superficial vastus lateralis compared to the young and
the old calorie restricted animals, with no differences with age or diet detected in the
soleus for either protein. Furthermore, DNA fragmentation (as denoted by DNA
laddering) persisted in the superficial vastus lateralis muscles of the 26-month-old ad
libitum fed animals but was absent in the 6-month-old ad libitum and the 26-month-old
calorie restricted animals. In the soleus, traces of DNA fragmentation emerged in the
young animals, but were not observed in either of the old groups. This may have been a
reflection of normal apoptotic activity to maintain cellular homeostasis in the young
Scrutiny of the data comparing apoptotic signaling in the type I soleus and the type
II superficial vastus lateralis muscle suggests a more pronounced stimulation of cell death
signaling in the type II muscle which would concur with the greater loss of muscle mass
demonstrated with age in this particular muscle (26, 27). Previous research has reported
a tendency for fast (type II) muscles to be more prone to develop apoptosis than slow
(type I) muscles (116, 117). These researchers explored the skeletal muscle myopathy
linked with muscle bulk loss in a model of congestive heart failure. In the tibialis
anterior muscle, composed primarily of type II fibers, the magnitude of apoptosis
detected in the tissue mirrored the increase in circulating TNF-a and was accompanied
by muscle atrophy (117). At variance, however, was the degree of apoptosis and muscle
atrophy detected in the soleus. Despite, the levels of TNF-a being equivalent to those
reported in the sister study (117) the investigators did not observe any degree of muscle
atrophy in the soleus, and noted the near three fold reduction in myocyte apoptosis
compared to that determined in the type II fiber muscle (116). Granted, additional
investigation is required to confirm that with aging similar signaling events occur.
Nonetheless, on the basis of the current findings, type II muscles possessing a
predisposition to develop apoptosis to a greater extent than type I muscles with age,
remains a distinct possibility.
Final Conclusions: TNF-a Promoting Skeletal Muscle Cell Survival or Cell Death?
Differences in fiber type responses to age-associated atrophy have been attributed
to a collection of intrinsic and systemic factors (26, 27, 104, 106-110). The prospect of a
variation in TNF-a signaling representing one such fiber type difference in the regulation
of age-linked atrophy, however, is a novel proposition. Age was associated with greater
signaling to NF-KB in the soleus than was observed in the superficial vastus lateralis. In
contrast, a greater presence of TNF-a (the upstream signaling cytokine of NF-KB) was
expressed in the superficial vastus lateralis compared to the soleus, presenting a
somewhat paradoxical picture. However, reexamination of the cell signaling capabilities
of TNF-a taken in conjunction with the apoptotic signaling pattern exhibited, may offer a
potential explanation for these inconsistencies.
TNF-a bound to TNF-R1 may not only elicit intracellular signaling to NF-KB, but
may also signal activation of cellular apoptosis via recruitment of cysteine proteases (57).
The current data presents the idea that as we age, the manner of TNF-a signaling in
skeletal muscle (promoting cell survival or cell death) may to some degree be fiber type
dependent (Fig 17). This postulate would provide an alternative explanation addressing
why age coincides with differential atrophy in muscles with different fiber types. What's
more, further questions are raised surrounding the specific muscle attributes that may
play a role in shaping the cells' decision to opt for TNF-a signaling to NF-KB versus
apoptosis. Recent speculation (102) has been directed toward the fidelity of the
stimulation of NF-KB and subsequent transcriptional activity of NF-KB as determining
the cells' fate. Specifically, cells accommodating defective NF-KB signals (resulting in
low quantities of antiapoptotic proteins) undergo TNF-a-induced apoptotic elimination.
This scenario may provide one explanation for the different signaling profiles observed in
the soleus and superficial vastus lateralis. The absence of measurable p65 protein in the
superficial vastus lateralis may embody one potential flaw (compromising the integrity of
NF-KB signaling fidelity), and in so doing, prompting the tissue to resort to apoptotic
signaling pathways. However, given that four alternative subunits of NF-KB exist,
implies a sense of redundancy and safeguard against any one subunit deciding the success
of the NF-KB signal. Nonetheless, further detailing the role of p65 (particularly in
skeletal muscles exhibiting a greater predisposition to age-linked atrophy) may unfold the
requisite of this NF-KB subunit to the success of the NF-KB signaling pathway.
Modifications / FI clAP
p65 protein 4 d
265S Proteasome FLIF clAP
T Phllip 2004
Figure 17. Proposed model of TNF-a signaling with age in soleus and superficial vastus
lateralis (SVL) skeletal muscles
Achieving a 10% reduction in sarcopenia prevalence in the United States would
result in healthcare savings of $1.1 billion per year (105). Accordingly, further
examination of muscle specific attributes from the perspective of TNF-a signal
transmission (in an attempt to make known why sarcopenia affects different muscles
disproportionately) presents an avenue worthy of additional investigation. Realizing this
l Cell mahi
goal may potentially improve or generate new therapeutic interventions geared toward
the pathogenesis of sarcopenia, and drastically curb the physical and financial burdens
imposed by sarcopenia.
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Tracey Phillips was born in Edinburgh, Scotland, growing up in the city suburb of
Clermiston. After attending Edinburgh University for one year, she embarked upon a
bachelor's degree at Moray House Institute of Education which she completed at Phillips
University, in Enid, Oklahoma, in May 1998. She then moved to Charleston, Illinois to
undertake a master's degree in cardiac rehabilitation and exercise physiology. In 1999,
Tracey began work on her Doctor of Philosophy degree in exercise physiology at the
University of Florida. Following graduation, she will begin a degree in medicine at