(Received for publication, July 16, 1995; and in revised form, September 7, 1995)
From the
Fatty acids and thiazolidinediones act as potent activators of
the adipose differentiation program in established preadipose cell
lines. In this report, the effects of these agents on the
differentiation pathway of myoblasts have been investigated. Exposure
of C2C12N myoblasts (a subclone of the C2C12 cell line) to
thiazolidinediones or fatty acids prevents the expression of myogenin,
-actin, and creatine kinase, thus abolishing the formation of
multinucleated myotubes. These treatments lead in parallel to the
expression of a typical adipose differentiation program including
acquisition of adipocyte morphology and activation of adipose-related
genes. A similar transition toward the adipose differentiation pathway
also occurs in mouse muscle satellite cells maintained in primary
culture. Thiazolidinediones exert their adipogenic effects only in
non-terminally differentiated myoblasts; myotubes are insensitive to
the compounds. Continuous exposure to inducers after growth arrest is
not required to maintain the adipose phenotype, but proliferation of
adipose-like C2C12N cells leads to a complete reversion toward
undifferentiated cells able to undergo either myogenic or adipogenic
differentiation depending on the composition of culture medium. These
results indicate that adipogenic inducers, such as thiazolidinediones
or fatty acids, specifically convert the differentiation pathway of
myoblasts into that of adipoblasts.
Formation of muscle, bone, and adipose tissue are multistep
processes that include the determination of a common progenitor
mesodermal cell toward a specific differentiation pathway followed by
the expression of various terminal differentiation phenotypes. The
multipotentiality of progenitor mesodermal cells has been illustrated in vitro by the spontaneous and concomitant differentiation of
embryonal carcinoma cells (1) or of clonal cell populations
derived from fetal rat calvaria (2) into myotubes, adipocytes,
and chondrocytes. Multiple differentiated phenotypes can also be
chemically induced by 5-azacytidine treatment of fibroblasts, such as
C3H10T1/2 or Swiss 3T3 cells(3) . Several lines of evidence
indicate that exogenous regulatory factors play a crucial role in the
determination of common progenitor cell into specific differentiation
lineages. Glucocorticoids exert positive effects on the differentiation
of the RCJ 3.1 clonal line from fetal rat calvaria into myotubes,
adipocytes, and chondrocytes(2) , whereas transforming growth
factor- exerts negative effects on all these differentiation
processes(4) . Other factors, such as bone morphogenetic
protein-2(5) , triggers specific determination of pluripotent
fibroblasts (6) or L6 and C2C12 myoblasts (7, 8) toward the osteoblast lineage. To our
knowledge, factors that are able to promote transition from the
myoblast lineage to that of the adipoblast have not been yet
discovered. Muscle and adipose differentiation involve complex
processes that lead to the induction of several differentiation-linked
genes specifically expressed either in muscle cells, i.e. MyoD, myogenin,
-actin, or muscle creatine kinase (MCK), (
)or in adipose cells, i.e. adipocyte lipid binding
protein (ALBP) or hormone-sensitive lipase(9, 10) .
Other genes, such as glycerol-3-phosphate dehydrogenase (GPDH),
lipoprotein lipase, insulin-responsive glucose transporter-4 (Glut-4),
or fatty acid transporter, are expressed in both tissues but at higher
levels in adipose tissue. Factors controlling muscle and adipose
differentiation processes are clearly different. Differentiation of
myoblasts in cell culture is initiated by peptide growth factor
withdrawal (11, 12) while adipose differentiation is
controlled by addition of various hormones and nutrients(10) .
We have shown that long chain fatty acids act in preadipose cells as
adipogenic agents(13, 14, 15, 16) .
These effects of fatty acids are mediated by activation of a nuclear
receptor called fatty acid-activated receptor (FAAR) expressed in a
variety of tissues including adipose tissue and muscle(17) .
FAAR is activated by fatty acids (17) and
thiazolidinediones(18) , a new class of antidiabetic agents,
which have also been described as exerting potent adipogenic effects in
preadipose cell lines(19, 20) . The mode of action of
thiazolidinediones as antidiabetic agents is not yet understood, but it
has been shown that their administration to diabetic animals improves
insulin sensitivity of muscle and adipose
tissue(21, 22) .
In this study, we investigated the effects of fatty acids and thiazolidinediones, on the differentiation pathway of C2C12N myogenic cells and satellite cells from newborn mouse muscle. We report that these compounds prevent myotube formation and induce the expression of a new phenotype resembling that of adipose cells.
Figure 1: Effects of BRL 49653 on C2C12N morphological differentiation. Cells were maintained to day 5 post-confluence in standard medium without additions (A) or in the presence of 5 µM BRL 49653 added at confluence (B) or 2 days before confluence (C). Bars, 0.1 mm.
Figure 2:
Effects of various thiazolidinediones and
fatty acids on muscle and adipose-like differentiation of C2C12N cells.
MCK and GPDH activities were determined in 5 day post-confluent cells
maintained in standard medium (a) or exposed from day -2
to day +5 relative to confluence to 5 µM BRL 49653 (b), 10 µM CS 045 (c), 10 µM pioglitazone (d), 100 µM palmitate (e), 100 µM -linolenate (f), or 100
µM 5,8,11,14-eicosatetraynoic acid (g). Values
obtained with cells maintained in standard medium are taken as 1 and
represent the mean ± S.D. from three separate
experiments.
Northern
blot analyses were performed to investigate the effects of BRL 49563 on
the expression of RNA markers characteristic of either muscle or
adipose differentiation in C2C12N cells (Fig. 3). Cells
maintained in standard medium were clearly differentiated into myotubes
since high levels of muscle-specific form of -actin mRNA and
myogenin mRNA were expressed 5 days after confluence. By contrast,
these cells did not express detectable levels of ALBP, GPDH, and
hormone-sensitive lipase mRNAs, and they showed only weak signals for
Glut-4, lipoprotein lipase, and fatty acid transporter mRNAs. Exposure
to BRL 49653 strongly reduced the expression of myogenin and
-actin mRNAs and led to the emergence of adipose markers including
ALBP, GPDH, and hormone-sensitive lipase mRNAs. Significant increases
in Glut-4, lipoprotein lipase, and fatty acid transporter mRNA
expression were also observed. The level of expression of these mRNAs
at this stage were strikingly similar to those found in fully
differentiated adipose cells from Ob1771 (31) or 3T3-442A (32) cell lines (not shown).
Figure 3: Effects of BRL 49653 on the expression of myogenic or adipose differentiation markers in C2C12N cells. Cells were maintained in standard medium until day 5 post-confluence and exposed (lane 2) or not (lane 1) from confluence to 5 µM BRL 49653. 20 µg of total RNA was analyzed by Northern blot as described under ``Experimental Procedures.'' Similar results have been obtained in three separate experiments.
We next examined the effects of
the compound on the differentiation process of satellite cells from
thigh muscles of 1-day-old mice. After plating until day 5
post-confluence, satellite cells were maintained in standard medium
supplemented or not with 5 µM BRL 49653. At that time,
cells maintained in standard medium differentiated into myotubes (Fig. 4A), whereas cells exposed to BRL 49653 presented
an adipose-like morphology (Fig. 4B). Northern blot
analyses confirmed these morphological observations, since control
cells expressed high levels of myogenin and -actin mRNAs and did
not express ALBP and fatty acid transporter mRNAs, whereas BRL
49653-treated cells strongly expressed the adipose markers ALBP and
fatty acid transporter and only weakly expressed the muscle markers (Fig. 4C). Similar results were obtained for satellite
cells exposed to 10 µM CS 045 or 100 µM
linolenic acid instead of BRL 49653 (not shown). Taken together, these
observations strongly suggest that thiazolidinediones and fatty acids
inhibit myogenic differentiation and induce the expression of a typical
adipose differentiation program in C2C12N cells and in primary cultures
of muscle cells.
Figure 4: Effects of BRL 49653 on satellite cell differentiation. A and B, satellite cells were isolated from muscle of newborn mice as described under ``Experimental Procedures'' and maintained in standard medium in the absence (A) or presence (B) of 5 µM BRL 49653 from seeding to 5 days post-confluence. Bars, 0.1 mm. C, RNA (20 µg/lane) from 5 days post-confluent untreated cells (lane 1) or cells exposed to 5 µM BRL 49653 (lane 2) was analyzed as described in the legend to Fig. 3. Similar results have been obtained in three separate experiments.
Figure 5:
Dose-response effects of BRL 49653 and CS
045 on myogenic and adipose marker expression in C2C12N cells. Cells
cultured in standard medium were exposed from day -2 to day
+5 to increasing concentrations of BRL 49653 (filled
symbols) or CS 045 (open symbols). A, RNA was
analyzed as described under ``Experimental Procedures.''
Results are expressed by taking the maximal value obtained for each
probe as 100. Symbols are: ,
, myogenin mRNA;
,
, ALBP mRNA. B, MCK (
,
) and GPDH (
,
) enzymatic activities were determined in the same cells as in A. Results are presented as in Fig. 2and represent the
mean ± S.D. from three separate
experiments.
Figure 6: Time dependence of BRL 49653 effects on C2C12N cells. Cells cultured in standard medium were exposed for the indicated period of time to 5 µM BRL 49653. MCK and GPDH activities were determined at day 5 post-confluence. Results are presented as described in the legend to Fig. 2and are the mean ± S.D. from three separate experiments.
To investigate whether or not the adipose phenotype is inherited, C2C12N cells exposed to 5 µM BRL 49653 from day -2 to day +3 relative to confluence were replated in standard medium at a 20-fold dilution in order to promote cell proliferation. These cells were already differentiated into adipocytes since expressing a high level of GPDH activity (Fig. 7). After attachment, cells began to proliferate actively to reach confluence after 5 days. At that time, cells presented the same morphology as cells which had never been exposed to the compound and expressed low GPDH activity (35 milliunits/mg of protein). In addition, these dedifferentiated cells were found to have recovered the ability to undergo either new myogenic differentiation when maintained after confluence in standard medium, illustrated by the induction of MCK and the lack of expression of GPDH, or new adipose differentiation when exposed to BRL 49653, illustrated by the induction of GPDH and the lack of expression of MCK (Fig. 7). These observations demonstrate that induction of the adipose phenotype by BRL 49653 does not require the continuous exposure of nonproliferative confluent cells. Rather, cell proliferation leads to complete reversion of this differentiated phenotype.
Figure 7:
Cell proliferation reverses the
adipose-differentiated phenotype of C2C12N cells. Cells exposed from
day -2 to day +3 to 5 µM BRL 49653 were
replated at a 20-fold dilution and maintained in standard medium until
confluence (day 0). After that cells were maintained in the absence (open symbols) or the presence (filled symbols) of 5
µM BRL 49653. MCK (,
) and GPDH (
,
) activities were determined at the indicated time. Results are
presented in milliunits/mg of protein and are the mean ± S.D.
from three separate experiments.
Figure 8:
Time course of muscle and adipose marker
expression in C2C12N cells exposed or not to BRL 49653. Cells were
maintained in standard medium and exposed (filled symbols) or
not (open symbols) from confluence to 5 µM BRL
49653. RNA was prepared at the indicated time and analyzed as described
in the legend to Fig. 5A. Results are presented as in Fig. 5A and are representative of three independent
experiments. A: ,
;
-actin mRNA;
,
, ALBP mRNA. B:
,
, FAAR mRNA;
,
, PPAR
mRNA;
,
, C/EBP
mRNA.
The present study demonstrated that thiazolidinediones and
fatty acids prevent the myogenic differentiation of myoblasts from a
clonal cell line or from primary muscle cell cultures, and also promote
their differentiation into adipose-like cells. Exposure to
thiazolidinediones or to fatty acids drives the developmental fate of
C2C12N myoblasts in an adipogenic direction. At the gene level, these
agents prevent the expression of muscle specific genes such as MCK,
-actin, and myogenin and lead to the expression of a typical
adipose differentiation program. It is noteworthy that in C2C12N
adipose-like cells the levels of expression of all the adipose-related
genes investigated in this study are quite similar to those found in
fully differentiated cells from adipose cell lines. For the most potent
adipogenic agent, BRL 49653, this dual regulation takes place at low
concentrations with a half-maximal effect of about 100 nM,
indicating that C2C12N myoblasts are more sensitive to BRL 49653 than
preadipose Ob1771 cells in which a stimulatory effect of the compound
on ALBP gene expression is observed over a range of concentration at
least one order of magnitude higher (18) .
Thiazolidinediones and fatty acids are also able to promote a
similar change in the cell fate of satellite cells from newborn mouse
muscle (36, 37) . When kept in standard medium from
the time of seeding to day 5 post-confluence, about 50% of these cells
differentiate into myotubes and express the muscle markers -actin
and myogenin, whereas chronic exposure to BRL 49653 leads to a nearly
homogeneous monolayer of lipid-containing cells which express high
levels of the adipose markers ALBP and fatty acid transporter, and only
moderate levels of myogenin and
-actin mRNAs.
Several lines of evidence support the conclusion that thiazolidinediones exert their effects on non-terminally differentiated C2C12N cells. First, exposure of fully differentiated myotubes to the drug fails to reverse myotube formation and to induce adipose differentiation. Second, maximal adipogenic action of BRL 49653 is observed in cells treated before or just after confluence, i.e. before expression of the specific myotube markers. This is also evident morphologically as a complete differentiation into adipose-like cells is observed in cells exposed before confluence to the drug, whereas some myotubes still appear in cultures treated following confluence (Fig. 1, C versus B). It can also be concluded from our results that chronic treatment by the inducer is not required for adipose differentiation of C2C12N cells. Furthermore, the adipoblast commitment is not an inherited trait since the proliferation of adipose C2C12N cells in standard medium reverses the differentiated phenotype and leads to the appearance of cells capable of undergoing either myogenic or adipose differentiation depending upon the absence or presence of BRL 49653 in the culture medium. These findings demonstrate that thiazolidinediones and fatty acids promote the transition from myogenic lineage to that of adipogenic lineage and that this conversion event occurs at the end of the growing phase of committed myoblasts. Similar features emerge from a recent report describing the conversion of the differentiation pathway of C2C12 myoblasts into osteoblasts upon bone morphogenetic protein-2 treatment(8) . Taken together, these observations provide an illustration of the plasticity of the myoblast cell commitment and of the crucial role exerted by external regulatory factors, i.e. fatty acids and thiazolidinediones or bone morphogenetic protein-2, on the fate of these cells toward adipogenic or osteoblastic cell lineages, respectively.
The identification of
the regulatory mechanisms implicated in the action of these external
stimuli could provide interesting information for understanding
mesodermal cell commitment. Clearly, the molecular mechanisms mediating
the adipogenic action of thiazolidinediones and fatty acids in
myoblasts remain to be determined. However, it is tempting to postulate
that FAAR could be involved in this signaling pathway since (i) it has
previously been demonstrated that this nuclear receptor for fatty acids (17) is also activated by thiazolidinediones in preadipose
cells(18) ; and (ii) FAAR mRNA is expressed in C2C12N myoblasts
during the period of time at which thiazolidinediones and fatty acids
exert their adipogenic effects, as shown above. By contrast, the lack
of expression of PPAR and C/EBP
at this cell stage argues
against a regulatory role for these nuclear proteins in the
myoblast/adipoblast cell lineage transition. Rather, it is more likely
that C/EBP
and PPAR
participate in the regulation of gene
expression during the terminal stages of adipose differentiation given
their late expression in C2C12N cells exposed to BRL 49653.
In conclusion, thiazolidinediones and fatty acids are able to withdraw committed myoblasts from the myogenic differentiation lineage and to promote the expression of a typical adipose conversion program in these cells. These findings offer a valuable cellular model to study the cellular and molecular events of mesodermal cell commitment. In addition, even if it is premature to conclude that transition from the myoblast to the adipoblast cell lineage could occur in vivo, it would be of interest to investigate this possibility in some pathological states characterized by lipid accumulation in muscle cells. For instance, such lipid deposition in muscle cells is occurring in the dystrophic mouse (38) and in mitochondrial myopathy(39, 40) . In these cases, this phenomenon could be related to the increase of fatty acid disposal due to both an increase of fatty acid synthesis and decrease of mitochondrial fatty acid oxidation. Lipid accumulation has also been described in cardiac cells from diabetic rats (41) which are characterized by a high blood fatty acid concentration.