Research Article

Are Peroxisome Proliferator-Activated Receptors Involved in Skeletal

Muscle Wasting during Experimental Cancer Cachexia?
Role of B2-Adrenergic Agonists
Gemma Fuster, Sı́lvia Busquets, Elisabet Ametller, Mireia Olivan, Vanessa Almendro,
Cibely Cristine Fontes de Oliveira, Maite Figueras, Francisco J. López-Soriano,
and Josep M. Argilés
Cancer Research Group, Departament de Bioquı́mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

Abstract view, especially if there are different mediators involved. Taking

Implantation of the Yoshida AH-130 ascites hepatoma to rats
resulted in a decrease in muscle weight 7 days after the
inoculation of the tumor. These changes were associated with
increases in the mRNA content for both peroxisome prolifer-
ator-activated receptor (PPAR) ; and PPARD in skeletal muscle.
The increase in gene expression for these transcription factors
was related to increases in the expression of several genes
involved in fatty acid transport, activation, and oxidation.
Tumor burden also resulted in increases in PPAR; coactivator-
1A gene expression and pyruvate dehydrogenase kinase 4. All
these changes in lipid metabolism genes suggest that a
metabolic shift occurs in skeletal muscle of tumor-bearing rats
toward a more oxidative phenotype. Formoterol treatment to
tumor-bearing rats resulted in an amelioration of all the
changes observed as a result of tumor burden. Administration
of this B2-adrenergic agonist also resulted in a decrease in
mRNA content of muscle PPARA, PPARD, and PPAR;, as well as
in mRNA levels of many of the genes involved in both lipid and
mitochondrial metabolism. All these results suggest an
involvement of the different PPARs as transcription factors
related with muscle wasting and also indicate that a possible
mode of action of the anticachectic compound formoterol may
involve a normalization of the levels of these transcription
factors. [Cancer Res 2007;67(13):6512–9]
Introduction
Muscle wasting is a common feature in many pathologic states,
including infection and cancer (1). Muscle wasting, the main trend
of cachexia, is responsible for the death of at least 30% of cancer
patients (2). Although we know the main events related with
muscle wasting [activation of myofibrillar protein degradation,
induction of apoptosis, and activation of uncoupling proteins
(UCP); refs. 3, 4], we have contradictory evidence about the possible
mediators involved. Indeed, whereas involvement of different
cytokines, mainly tumor necrosis factor-a (TNFa) and interleu-
kin-6 (IL-6), has been postulated, other studies describe a more
direct role for tumor-derived factors, such as proteolysis-inducing
factor (PIF) and lipid-mobilizing factor (5, 6). The intracellular
signaling pathway may have a key role, from a therapeutic point of
Requests for reprints: Josep M. Argilés, Cancer Research Group, Departament de
Bioquı́mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona,
Diagonal 645, 08028 Barcelona, Spain. Phone: 34-934021002; Fax: 34-934021559;
E-mail: jargiles@ub.edu.
I2007 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-0231
this into consideration, a lack of knowledge about signaling
pathways and transcription factors involved in muscle wasting
exists. Some work has postulated a role for nuclear factor-nB
(NF-nB) in muscle wasting associated with cytokines (7) and
tumor-derived factors (8). Other transcription factors, such as
activator protein-1 (AP-1) and CCAAT/enhancer binding protein
(C/EBP), have also been involved in sepsis-induced muscle
cachexia (9). Results from our laboratory indicate that the
transcription factor AP-1 could also be involved during cancer
cachexia (10, 11). Not much attention has been focused on the role
of peroxisome proliferator-activated receptors (PPAR) in skeletal
muscle. These transcription factors are associated with changes in
lipid metabolism as well as UCP expression (12) and apoptosis (13).
PPARs are transcription factors belonging to the superfamily of
nuclear receptors. Three isoforms (a, y, and g) have been described
(14). They act on DNA response elements as heterodimers with the
nuclear retinoic acid receptor. Their natural activating ligands are
fatty acids and lipid-derived substrates. PPARa is present in liver,
heart, and, to a lesser extent, skeletal muscle; when activated, it
promotes fatty acid oxidation, ketone body synthesis, and glucose
sparing. PPARg is expressed in adipose tissue, lower intestine,
skeletal muscle, and immune cells; activation of PPARg induces the
differentiation of preadipocytes into adipocytes and stimulates
triglyceride storage. The PPARs are thus major regulators of lipid
and glucose metabolism, allowing adaptation to the prevailing
nutritional environment (14). PPARy has a broad expression
pattern in adult and is expressed very early during embryogenesis
(15). These past few years, it has been shown that treatment with
PPARy agonists normalizes blood lipids and also reduces insulin
resistance and adiposity in rodents and primates. Utilization of
both cellular and animal models revealed that this nuclear receptor
plays a central role in the control of fatty acid burning in adipose
tissue and skeletal muscle. Furthermore, PPARy seemed to be
important for adaptive response of skeletal muscle to environ-
mental changes, such as physical exercise (15).
h2-adrenergic agonists are potent muscle growth promoters in
many animal species (16, 17), resulting in skeletal muscle
hypertrophy (18–20) and reducing body fat content (21, 22).
Interestingly, results from our laboratory clearly indicate that
formoterol is a very efficient agent preventing muscle weight loss in
tumor-bearing rats (23). In vivo treatment can effectively reverse
muscle wasting loss decreasing protein degradation and increasing
the rate of protein synthesis in skeletal muscle, therefore favoring
protein accretion (23).
Bearing this in mind, the aim of the present investigation was to
ascertain if tumor burden induces any changes in PPARs gene
transcription in skeletal muscle and if these changes are associated

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 PPARs and Cancer Cachexia

Table 1. Food intake, body weight, and muscle weight in tumor-bearing rats

Experimental groups

C, n (mean F SE) C + F, n (mean F SE) TB, n (mean F SE) TB + F, n (mean F SE) P, ANOVA

IBW-FBW 5 (49 F 3a) 5 (50 F 2a) 6 (11 F 5b) 7 (17 F 5b) <0.001
Food intake 5 (133 F 1a) 5 (131 F 2a) 6 (106 F 1b) 7 (108 F 1b) <0.001
Muscle weights
GSN 5 (718 F 10.5a) 4 (774 F 8b) 5 (505 F 17c) 7 (611 F 18d) <0.001
Soleus 5 (52 F 2.2a) 4 (52 F 1.6a) 5 (41 F 2.7b) 6 (44 F 1.4b) 0.001
EDL 5 (59 F 2a) 5 (60 F 1.6a) 5 (42 F 1.8b) 6 (51 F 2c) <0.001
Tibialis 5 (236 F 2.5a) 5 (266 F 4.5b) 5 (171 F 4.8c) 7 (200 F 9d) <0.001

NOTE: For further details, see Materials and Materials. n is the number of animals. Food intake (grams) refers to the ingestion for each rat during the
period of the experiment before sacrifice (7 d). Initial body weight-final body weight without tumor is expressed as grams. Tissue weights are expressed
as mg/100 g of initial body weight. Formoterol was given for 7 d s.c. (0.3 mg/kg body weight). Statistical significance of the results by one-way ANOVA
and statistically significant difference by post-hoc Duncan test. Different superscripts indicate differences between groups.
Abbreviations: IBW, initial body weight; FBW, final body weight without tumor; GSN, gastrocnemius; C, control; F, formoterol-treated animals; TB,
tumor-bearing animals.

with alterations in gene transcription of different types of proteins
involved in lipid metabolism. A second objective in the present
investigation was to see if these changes could be reversed by
administration of the h2-agonist formoterol, a molecule that shows
a clear anticachectic action in skeletal muscle during cancer (23).
Materials and Methods
Animals. Male Wistar rats (Interfauna) of 5 weeks of age were used. The
animals were maintained at 22 F 2jC with a regular light-dark cycle (light
on, 08:00–20:00) and had free access to food and water. The food intake was
measured daily. All animal manipulations were made in accordance with
the European Community guidelines for the use of laboratory animals.
Tumor inoculation and treatment. Rats were divided into two groups,
namely controls and tumor hosts. The latter received an i.p. inoculum of
108 AH-130 Yoshida ascites hepatoma cells obtained from tumors grow-
ing in the exponential phase (24). Both groups were further divided into
treated and untreated, the former being given daily i.p. dose of formoterol
(0.3 mg/kg body weight), dissolved in physiologic solution, and the latter a
corresponding volume of solvent. On day 7 after tumor transplantation, the
animals were weighed and anesthetized with an i.p. injection of ketamine/
xylazine mixture (3:1; Imalgene and Rompun, respectively). The tumor was
harvested from the peritoneal cavity and its volume and cellularity were
evaluated. Tissues were rapidly excised, weighed, and frozen in liquid nitrogen.
Biochemicals. They were all reagent grade and obtained either from
Roche S.A. or from Sigma Chemical Co.
RNA isolation. Total RNA from soleus and extensor digitorum longus
(EDL) muscle was extracted by TriPure kit (Roche), a commercial modifica-
tion of the acid guanidinium isothiocyanate/phenol/chloroform method (25).
Real-time PCR. First-strand cDNA was synthesized from total RNA
with oligonucleotide dT15 primers and random primers p(dN)6 by using a
cDNA synthesis kit (Transcriptor Reverse Transcriptase, Roche). Analysis of
mRNA levels for PPARa, PPARy, PPARg, muscle carnitine palmitoyltrans-
ferase-I (MCPTI), and CPTII were done with primers designed to detect gene
products as described previously (26). Oligonucleotide sequences for fatty
acid translocase (FAT) were 5¶-CTCTGACATTTGCAGGTC-3¶ and 5¶-CAC-
AGGCTTTCCTTCTTTGC-3¶ (gi: 98674378), for fatty acid transport protein
(FATP) were 5¶-GCATGGATGATCGGCTATTT-3¶ and 5¶-GATGTTCCCTGC-
TGAGTGGT-3¶ (gi: 18811712), for PPARg coactivator-1a (PGC1a) were
5¶-AAGGTCCCCAGGCAGTAGAT-3¶ and 5¶-TTCAGACTCCCGCTTCTCAT-3¶
(gi: 13786187), for pyruvate dehydrogenase kinase 4 (PDK4) were 5¶-CCTT-
TGCTGGTTTTGGTTA-3¶ and 5¶-CACCAGTCATCAGCCTCAGA-3¶ (gi:
16758425), and for acyl-CoA synthetase 4 (ACS4) were 5¶-CACCATTGCCAT-
TTTCTGTG-3¶ and 5¶-GCCTTCAGTTTGCTTTCCAG-3¶ (gi: 16758425). Ampli-
fication conditions consisted in 5 s of denaturation at 94jC, 9 s of annealing
at 60jC, and 9 s of extension at 72jC for each step for 45 cycles for FAT,
PGC1a, PDK4, and ACS4 and for FATP consisted in 5 s of denaturation at 94jC,
9 s of annealing at 60jC, 9 s of extension at 72jC, and 3 s at 87jC to detect
amplification product avoiding unspecific products after each cycle, for
45 cycles. Myosin heavy-chain I (MHCI) and MHCIIA primers were designed
by Mortensen et al. (27). To avoid the detection of possible contamination
by genomic DNA, primers were designed in different exons. The real-time PCR
was done using a commercial kit (LightCycler FastStart DNA Master PLUS
SYBR Green I, Roche). The relative amount of all mRNA was calculated using
comparative C T method. 18S mRNA was used as the invariant control for all
studies.
Protein extraction and Western blotting. For PDK4 analysis, EDL and
soleus muscles were homogenized in 10 mmol/L HEPES (pH 7.5),
containing 10 mmol/L MgCl2, 5 mmol/L KCl, 0.1 mmol/L EDTA, 0.1%
Triton X-100, 1 mmol/L DTT, and 5 AL of a protease inhibitor cocktail
(Sigma)/mL of buffer. They were then centrifuged at 7,000 rpm for 5 min at
4jC, and the supernatants were collected. For MCPTI, PGC1a, and
mitochondrial complex II analysis, an enriched mitochondrial/nuclear
fraction was obtained: the pellets being resuspended in 500 AL of buffer
[10 mmol/L HEPES (pH 7.9) containing 0.5 mol/L NaCl, 1.5 mmol/L MgCl2,
0.2 mmol/L EDTA, 30% glycerol, 1 mmol/L DTT, 5 AL of a protease inhibitor
cocktail/mL of buffer] and left for 30 min on ice. They were later
centrifuged at 7,000 rpm for 5 min at 4jC, and the supernatants were
collected. About FAT analysis, an enriched plasma membrane was obtained
from the total protein extracts; these were obtained by ultracentrifugation
at 47,000 rpm for 1 h at 4jC. Protein concentrations were determined
according to the method of bicinchoninic acid (Pierce). Equal amounts of
protein (50 or 100 Ag) were heat denatured in sample loading buffer
[50 mmol/L Tris-HCl (pH 6.8), 100 mmol/L DTT, 2% SDS, 0.1% bromphenol
blue, 10% glycerol], resolved by SDS-PAGE (10% polyacrylamide, 0.1% SDS),
and transferred to Immobilon membranes (Immobilon polyvinylidene
difluoride, Millipore). The filters were blocked with 5% PBS-nonfat dry milk
and then incubated with polyclonal antibodies: anti-PDK4, anti-PGC1a,
anti-MCPTI, anti-FAT (Santa Cruz Biotechnology), and monoclonal anti-
body (mAb) anti-OxPhos complex II (Molecular Probes). Polyclonal anti-
bodies anti-Na+/K+ ATPase a-subunit (Developmental Studies Hybridoma
Bank), anti–glyceraldehyde-3-phosphate dehydrogenase, and mAb anti-
porin (Calbiochem) were used as invariant controls for the different studies.
Donkey anti-mouse peroxidase-conjugated IgG (Jackson ImmunoResearch,
Laboratories, Inc.), rabbit anti-goat peroxidase-conjugated IgG (Acris
Antibodies GmbH), and goat anti-rabbit horseradish peroxidase conjugate

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Cancer Research

(Bio-Rad) were used as secondary antibodies. The membrane-bound
immune complexes were detected by an enhanced chemiluminescence
system (EZ-ECL).
Statistical analysis. Statistical analysis of the data was done by means of
one-way ANOVA.
Results and Discussion
Muscle wasting during different pathologic conditions exerts a
negative effect on survival and quality of life in the patient.
Therefore, understanding the molecular mechanisms and the
different mediators involved provides for the design of efficient
therapeutic approaches (28). From this point of view, the therapy
against wasting during cachexia has concentrated on either
increasing food intake or normalizing the persistent metabolic
alterations that take place in the patient. It is difficult to apply a
therapeutic approach based on the neutralization of the
potential mediators involved in muscle wasting (i.e., TNFa,
IL-6, IFN-g, and PIF) because many of them are involved at the
same time in promoting the metabolic alterations and the
anorexia present in the cancer patients (29). Bearing this in
mind, it is obvious that a good understanding of the molecular
mechanisms involved in the signaling of these mediators may be
very positive in the design of the therapeutic strategy. This is
especially relevant because different mediators may be sharing
the same signaling pathways (30, 31). However, very few
investigations have focused on the signaling of these mediators
in skeletal muscle, especially about transcription factors. Penner
et al. (32) described, in a model of sepsis, the involvement of
NF-nB and AP-1 in muscle wasting. Other reports, using expe-
rimental cancer models, have also suggested that NF-nB is
involved in the signaling of muscle wasting (30, 31). In our own
laboratory, we have shown recently that there is an increased
activation of AP-1 in skeletal muscle of tumor-bearing rats,
therefore suggesting that this factor is indeed involved in the
muscle events that take place during cancer cachexia (10).
Indeed, the i.m. administration of adenoviruses carrying TAM-67
[a negative-dominant of c-jun (AP-1)] resulted in an improve-
ment of the muscle weight during tumor growth (11).

Table 2. Gene expression of different proteins related to lipid metabolism

Experimental groups

C, n (mean F SE) C + F, n (mean F SE) TB, n (mean F SE) TB + F, n (mean F SE) P, ANOVA

(A) Skeletal muscle mRNA content of the different PPARs

PPARa
EDL 4 (100 F 13a) 5 (49 F 10b) 5 (102 F 26a) 5 (23 F 18b) <0.05
Soleus 4 (100 F 24ab) 4 (111 F 12ab) 5 (137 F 12a) 4 (76 F 22b) 0.1
PPARg
EDL 4 (100 F 8ab) 5 (45 F 5ac) 4 (148 F 31b) 5 (30 F 12c) <0.05
Soleus 5 (100 F 28 )
a
6 (119 F 6a) 6 (308 F 31b) 6 (77 F 19a) <0.05
PPARy
EDL 5 (100 F 12a) 5 (93 F 9a) 4 (241 F 20b) 5 (85 F 22a) <0.001
Soleus 4 (100 F 13a) 5 (109 F 12a) 5 (476 F 46b) 6 (140 F 29ab) 0.09

(B) Skeletal muscle mRNA content of the different genes related to lipid metabolism
Fatty acid transport and activation
FAT
EDL 4 (100 F 18a) 4 (22 F 11a) 5 (315 F 34b) 5 (26 F 10a) <0.05
Soleus 4 (100 F 6ab) 5 (57 F 28a) 4 (186 F 12c) 4 (135 F 5b) <0.001
FATP
EDL 4 (100 F 9a) 4 (51 F 10b) 4 (151 F 17c) 4 (31 F 14b) <0.001
Soleus 4 (100 F 22 )
a
5 (68 F 15 )a
5 (181 F 14b) 4 (105 F 11a) <0.05
ACS4
EDL 5 (100 F 18a) 5 (68 F 6ab) 4 (100 F 14a 5 (58 F 9b) <0.05
Soleus 4 (100 F 5a) 5 (160 F 9b) 4 (138 F 7bc) 5 (123 F 9ac) <0.05
Oxidation
MCPTI
EDL 4 (100 F 11a) 4 (38 F 5b) 3 (253 F 38c) 5 (35 F 5b) <0.001
Soleus 5 (100 F 7a) 6 (110 F 5a) 4 (216 F 17b) 4 (145 F 10a) <0.001
CPTII
EDL 3 (100 F 16a) 4 (40 F 9b) 3 (188 F 19c) 5 (20 F 16b) <0.001
Soleus 4 (100 F 12 )
a
6 (91 F 6 )
a
4 (156 F 11b) 4 (112 F 20a) <0.05

NOTE: For further details, see Materials and Methods. n is the number of animals. The results are expressed as a percentage of controls. Statistical
significance of the results by one-way ANOVA and statistically significant difference by post-hoc Duncan test. Different superscripts indicate differences
between groups.
Abbreviations: C, control; F, formoterol-treated animals; TB, tumor-bearing animals; FAT, fatty acid translocase; PPAR, peroxisome proliferator-activated
receptor; FATP, fatty acid transport protein; ACS4, acyl-CoA synthetase 4; MCPTI, muscle carnitine palmitoyltransferase I; CPTII, carnitine
palmitoyltransferase II.

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 PPARs and Cancer Cachexia

Table 3. Skeletal muscle protein content of different proteins related to lipid metabolism

Experimental groups

C, n (mean F SE) C + F, n (mean F SE) TB, n (mean F SE) TB + F, n (mean F SE) P, ANOVA

FAT
EDL 4 (100 F 14ab) 6 (99 F 17ab) 3 (198 F 97a) 3 (66 F 12b) ns
Soleus 5 (100 F 5a) 7 (108 F 17ab) 5 (160 F 25b) 3 (99 F 18a) 0.09
MCPTI
EDL 5 (100 F 7ab) 6 (38 F 14a) 5 (256 F 93b) 3 (97 F 12ab) <0.05
Soleus 4 (100 F 16a) 7 (94 F 22a) 5 (319 F 50b) 4 (185 F 35a) <0.001
PDK4
EDL 4 (100 F 16a) 4 (77 F 6a) 3 (175 F 21b) 4 (99 F 29a) <0.001
Soleus 5 (100 F 20) 6 (109 F 13) 3 (170 F 31) 4 (135 F 55) ns
PGC1a
EDL 5 (100 F 11a) 6 (92 F 13a) 4 (341 F 83b) 4 (131 F 20a) <0.05
Soleus 4 (100 F 9a) 7 (64 F 9a) 4 (196 F 26b) 3 (95 F 21a) <0.001

NOTE: For further details, see Materials and Methods. n is the number of animals. The results are expressed as a percentage of controls. Statistical
significance of the results by one-way ANOVA and statistically significant difference by post-hoc Duncan test. Different superscripts indicate differences
between groups.
Abbreviations: C, control; F, formoterol-treated animals; TB, tumor-bearing animals; FAT, fatty acid translocase; MCPTI, muscle carnitine
palmitoyltransferase I; PDK4, pyruvate dehydrogenase kinase-4; PGC1a, PPAR coactivator-1a; ns, nonsignificant differences.

In spite of the involvement of PPARs in several metabolic path-
ways that are altered during cancer cachexia, no information is
available on the mRNA levels of these transcription factors in skeletal
muscle during this catabolic situation. This was the aim of the
present investigation: to elucidate any changes of PPARs in skeletal
muscle during tumor growth and also to relate them to the changes
of mRNA content of different genes involved in lipid metabolism.
Table 1 shows the effect of tumor growth on body weight, food
intake, and muscle weights in animals bearing the Yoshida AH-130
ascites hepatoma. The growth of this tumor causes in the host a
rapid and progressive loss of body weight and tissue waste,
particularly in skeletal muscle (33). Acceleration of tissue protein
breakdown accounts for most of the waste in the AH-130 bearers
(24, 34). The effects of the tumor on weight loss were very

Figure 1. A, skeletal muscle protein

content of mitochondrial complex II. Porin
was used as invariant control. B, soleus
mRNA content of the different MHCs. 18S
was used as invariant control. For further
details, see Materials and Methods.
Columns, mean of four animals in each
group; bars, SE. The results are expressed
as a percentage of controls. C, control;
F, formoterol-treated animals; TB,
tumor-bearing animals. Statistical
significance of the results by one-way
ANOVA and statistically significant
difference by post-hoc Duncan test. a, b,
and c, differences between groups.

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Cancer Research
significant, with a decrease of 28% in the tumor-bearing group.
This effect on body weight was accompanied by a marked anorexia:
the tumor-bearing rats ate 20% less than the control ones (Table 1).
It can also be seen that the implantation of the tumor resulted in a
general decrease of muscle weights (30% gastrocnemius, 29% EDL,
28% tibialis, and 21% soleus) 7 days after the inoculation of the
tumor. These changes were associated with increases in the mRNA
content for PPARg (48% in EDL and 208% in soleus) and PPARy
(141% in EDL and 376% in soleus) but no changes in PPARa mRNA
content (Table 2A). It is interesting to note here that PPARy is
especially important for adaptable response in skeletal muscle and
precisely the largest changes occur at the level of this transcription
factor (15). The increase in gene expression for these two
transcription factors was related with increases in the expression
of genes involved in fatty acid transport activation and oxidation.
Thus, FAT and FATP (both proteins associated with fatty acid
cellular transport) were increased 215% and 51% in EDL and 86%
and 81% in soleus, respectively (Table 2B). In addition, FAT protein
content was also increased in soleus (60%) of tumor-bearing
animals (Table 3). Similarly ACS4 (the protein that activates the
intracellular fatty acids for their subsequent use in oxidation
pathways) was increased 38% in soleus (Table 2B). Finally, MCPTI
and CPTII mRNA content (both involved in the transport of fatty
acid across the mitochondrial membrane) were also increased as a
result of tumor burden by 153% and 116% for MCPTI in EDL and
soleus, respectively, and 88% and 56% for CPTII in EDL and soleus,
respectively. MCPTI protein content was also increased in EDL
(156%) and in soleus (219%) muscles of tumor-bearing animals
(Table 3). All these results agree with previous reports that have
already suggested that lipid metabolism is increased in skeletal
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Figure 2. A, mRNA content of PDK4 and
PGC1a in EDL muscle of tumor-bearing
animals. 18S was used as invariant control.
B, mRNA content of PDK4 and PGC1a in
soleus muscle of tumor-bearing animals.
18S was used as invariant control. For
further details, see Materials and Methods.
Columns, mean values of four animals in
each group; bars, SE. The results are
expressed as a percentage of controls.
Statistical significance of the results by
one-way ANOVA and statistically
significant difference by post-hoc Duncan
test. a and b, differences between groups.
muscle of tumor-bearing rats (35). In addition, all these changes in
lipid metabolism genes clearly suggest that a metabolic shift occurs
in skeletal muscle of tumor-bearing rats toward a more oxidative
phenotype. Previous studies have shown that PPARy is involved in
muscle fiber composition; an increase in this transcription factor
determines a more oxidative fiber phenotype (36). Bearing this in
mind, the results found in this study clearly agree with this
previously reported data. In fact, as can be seen in Fig. 1A, tumor
burden resulted in a clear increase (49%) in oxidative muscle fibers,
as measured by the expression of the MHCI gene (27). Conversely,
the presence of the tumor did not have any effects on the mRNA
content of MHCIIA, a clear glycolytic marker (Fig. 1A; ref. 27). The
same tendency toward a more oxidative phenotype in the muscles
of tumor-bearing rats was confirmed when analyzing the
mitochondrial complex II protein content as observed in Fig. 1B
(37). PGC1a is a transcriptional coregulator that coordinates the
formation/maintenance of slow twitch fibers in skeletal myocytes,
and this seems to be directly controlled by PPARy (37). In addition,
PPARs seem to independently regulate specific PDK isoforms
transcript levels, which are likely to impart important metabolic
mediation of fuel utilization by the muscle. Experiments to date
suggest that PDK4 is the major isoenzyme responsible for changes
in pyruvate dehydrogenase complex activity in response to
various different metabolic conditions (38). Tumor burden also
resulted in increases in the PGC1a gene expression (83% and
102% in EDL and soleus, respectively) and PDK4 (373% and 172%
in EDL and soleus, respectively; Fig. 2). In addition PDK4 and
PGC1a protein content was also increased both in EDL (75% and
241%, respectively) and in soleus (70% and 96%, respectively)
muscles of tumor-bearing animals (Table 3). These data agree
6516 www.aacrjournals.org

with the work by Puigserver et al. (39) that stated that cytokine-
induced activation of PGC1a in culture muscle cells or muscle
in vivo causes increased respiration and expression of genes
linked to mitochondrial uncoupling and energy expenditure. It is
therefore possible to suggest that the cytokine changes related
with cancer cachexia are altering the mRNA content of PGC1a
and this may affect the transcriptional activity of both PPARg and
PPARy. In connection with this, it is interesting to remark that
the activation of UCP2 and UCP3 is also observed in tumor-
bearing rats (40). This could be very well linked with PPARs
activation, possibly through PGC1a coactivation.
Formoterol, a h2-adrenergic agonist, is a very efficient agent
preventing muscle weight loss in tumor-bearing rats (23). Bearing
this in mind, we decided to see if the effects of formoterol on
muscle wasting during experimental tumor growth were connected
with changes in PPARs and the genes involved in lipid metabolism
studied above. The results obtained seem to indicate that
formoterol treatment can normalize the changes induced by the
tumor (Fig. 2; Tables 1–3). Several studies have shown that h2-
adrenergic agonists act on skeletal muscle by changing the fiber
composition toward a more glycolytic pattern (41). Taking this into
account, the different effects of formoterol on different types of
muscles were studied; therefore, these studies were done in a
predominantly oxidative muscle (soleus) and in a predominantly
glycolytic muscle (EDL). The results found here are in line with
previously described observations (41). As seen in Fig. 1A,
formoterol treatment resulted in a significant increase in MHCIIA
(a glycolytic marker) in mRNA soleus content, in both control and
tumor-bearing animals. In addition, formoterol also significantly
decreased mitochondrial complex II content in tumor-bearing rats
(Fig. 1B), this observation supporting further the change in fiber
composition.
Figure 3. Hypothetical involvement of
the different transcription factors during
muscle wasting in cancer cachexia. ARb2,
h2-adrenoceptor; IL-6R, IL-6 receptor;
PIFR, PIF receptor; TNFR1, TNF receptor
1; TNFR2, TNF receptor 2.
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PPARs and Cancer Cachexia
It is interesting to note that formoterol also had an effect on
nontumor-bearing animals (Table 3A and B), therefore suggesting
that the action [possibly linked with variations of the intracellular
concentration of cyclic AMP (cAMP) in cells] may be linked with
changes in PPARs (42, 43). In fact, a previous report using the h2-
agonist phenylephrine already suggests this possibility (44). On the
other hand, it is well known that h2-agonists also affect lipid
metabolism on adipose tissue, decreasing lipid synthesis and
favoring lipid oxidation (45), but this is the first report that clearly
shows a possible action of h2-agonists on skeletal muscle lipid
metabolism. The effects of the h2-agonists via h2-adrenergic
receptors increase cAMP and interfere with PPARs gene expression
(Fig. 3). However, another possibility is that formoterol actually
interferes with the signaling of TNFa on skeletal muscle cells.
Indeed, a report already suggests that the h2-agonist clenbuterol
decreases the circulating levels and activity of TNFa (46). This
makes particular sense for the tumor model involved in this study
because the Yoshida AH-130 ascites hepatoma seems to be highly
dependent on the increase of TNFa circulating levels (47). In
addition, in this study, formoterol treatment also reduced the
TNFa mRNA content in gastrocnemius muscle [tumor, 185 F 15a
arbitrary units (4); tumor treated with formoterol, 115 F 9b
arbitrary units (3); P < 0.05]. (Statistical significance of the results
by one-way ANOVA and statistically significant difference by post-
hoc Duncan test; different superscripts indicate differences
between groups).
The data presented here, in a way, complicate the understanding
of the different transcription factors in muscle wasting because
some reports have suggested that activation of different PPARs may
be useful in preventing metabolic alterations linked with
inflammation by cytokines (48). However, other reports suggest
the opposite, emphasizing that PPARs may have a role in mediating
Cancer Res 2007; 67: (13). July 1, 2007
Cancer Research

cytokine action in skeletal muscle (39). In addition, several studies
(49) indicate that PPARs can partially interfere in NF-nB signaling
in skeletal muscle, therefore controlling to some extent the action
exerted by cytokines through these transcription factors (Fig. 3).
Bearing all this in mind, one has to look at the plethora of
transcription factors involved in muscle wasting during cancer
cachexia because possibly some of them play a more important
role in some cases, depending on the species, the tumor model, and
the tumor stage. It is perfectly conceivable to understand that by
agonizing PPARg and PPARy, one can produce a reduction in
muscle weight partly through the interference in the NF-nB
signaling system. It can also be suggested that the elevation of the
different PPARs during muscle wasting in cancer may be a counter
metabolism triggered by the host to control an excessive
degradation of muscle protein and favoring lipid utilization for
muscle.
In conclusion, the results presented here contribute to a better
understanding of the role of transcription factors in the muscle
tissue during cancer cachexia. It becomes clear that future research
into this field is necessary and may provide important tools to
design effective drugs for the treatment of wasting in skeletal
muscle during pathologic conditions.
Acknowledgments
Received 1/18/2007; revised 3/22/2007; accepted 4/12/2007.
Grant support: Ministerio de Educación y Ciencia Dirección General de
Investigación Cientı́fica y Técnica BFI2002-02186 (G. Fuster); Ministerio de Sanidad
y Consumo Instituto de Salud Carlos III grant 03/0100 (E. Ametller); La Marató TV3
grant 04/1010 (M. Olivan); Programme Alhan, the European Union Programme of High
Level Scholarships for Latin America, scholarship E05D059293BR (C.C. Fontes de
Oliveira); Fondo de Investigaciones Sanitarias de la Seguridad Social of the Ministerio
de Sanidad y Consumo grant 06/0907; and Ministerio de Ciencia y Tecnologı́a grant
SAF 4744-2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Industriale Chimica s.r.l. for providing micronized formoterol fumarate;
Dr. Carles Barceló-Vidal for his statistical support.

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Are Peroxisome Proliferator-Activated Receptors Involved in
Skeletal Muscle Wasting during Experimental Cancer
Cachexia? Role of β2-Adrenergic Agonists
Gemma Fuster, Sílvia Busquets, Elisabet Ametller, et al.

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