Protein profiling of 3T3-L1 adipocyte differentiation and (tumor necrosis factor ?-mediated) starvation

Maastricht Proteomics Center, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Human Biology, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
Cellular and Molecular Life Sciences CMLS (Impact Factor: 5.81). 03/2005; 62(4):492-503. DOI: 10.1007/s00018-004-4498-9
Source: PubMed
ABSTRACT
The increased incidence of obesity and related disorders in Western societies requires a thorough understanding of the adipogenic process. Data at the protein level of this process are scarce. Therefore we performed a proteome analysis of differentiating and starving 3T3-L1 cells using two-dimensional gel electrophoresis combined with mass spectrometry. Effects of different starvation conditions were examined by subjecting 3T3-L1 adipocytes to caloric restriction, either in the absence or the presence of the lipolysis inducer tumor necrosis factor-alpha. Ninety-three differentially expressed proteins were found during differentiation and starvation of 3T3-L1 cells, 50 of which were identified. GenMAPP/MAPP-finder software revealed a non-reciprocal regulation of the glycolytic pathway during 3T3-L1 differentiation followed by starvation. Furthermore, proteins involved in growth regulation, cytoskeletal rearrangements and protein modification, 16 of which have not been described before in 3T3-L1 cells, were identified. In conclusion, our data provide valuable information for further understanding of the adipogenic process.

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Research Article
Protein profiling of 3T3-L1 adipocyte differentiation
and (tumor necrosis factor
aa
-mediated) starvation
J. Renes
a,
*, F. Bouwman
a
, J.-P. Noben
b
,C.Evelo
c
,J.Robben
b
and E. Mariman
a
a
Maastricht Proteomics Center, Nutrition and Toxicology Research Institute Maastricht (NUTRIM),
Department of Human Biology, Maastricht University, P.O. Box 616, 6200 MD Maastricht (The Netherlands),
Fax: +31 (0)43 3670976, e-mail: j.renes@hb.unimaas.nl
b
Biomedical Research Institute, Limburgs Universitair Centrum and School of Life Sciences,
Transnational University Limburg, 3590 Diepenbeek (Belgium)
c
BiGCaT Bioinformatics, Technical University Eindhoven and Maastricht University, P.O. Box 616, 6200 MD
Maastricht (The Netherlands)
Received 9 November 2004; received after revision 21 December 2004; accepted 28 December 2004
Abstract. The increased incidence of obesity and related
disorders in Western societies requires a thorough under-
standing of the adipogenic process. Data at the protein
level of this process are scarce. Therefore we performed
a proteome analysis of differentiating and starving 3T3-
L1 cells using two-dimensional gel electrophoresis com-
bined with mass spectrometry. Effects of different starva-
tion conditions were examined by subjecting 3T3-L1
adipocytes to caloric restriction, either in the absence or
the presence of the lipolysis inducer tumor necrosis fac-
tor-
a
. Ninety-three differentially expressed proteins were
CMLS, Cell. Mol. Life Sci. 62 (2005) 492–503
1420-682X/05/040492-12
DOI 10.1007/s00018-004-4498-9
© Birkhäuser Verlag, Basel, 2005
CMLS
Cellular and Molecular Life Sciences
found during differentiation and starvation of 3T3-L1
cells, 50 of which were identified. GenMAPP/MAPP-
finder software revealed a non-reciprocal regulation of
the glycolytic pathway during 3T3-L1 differentiation fol-
lowed by starvation. Furthermore, proteins involved in
growth regulation, cytoskeletal rearrangements and pro-
tein modification, 16 of which have not been described
before in 3T3-L1 cells, were identified. In conclusion,
our data provide valuable information for further under-
standing of the adipogenic process.
Key words. 3T3-L1; proteomics; differentiation; caloric restriction; TNF-
a.
In Western societies, obesity is taking on epidemic pro-
portions, which will lead to an increased population risk
for obesity-related complications such as type II diabetes
and cardiovascular diseases [1]. Treatment and, more im-
portantly, prevention of obesity are necessary to reduce
the risk for these disorders. Hence, targets for future in-
tervention are required, which necessitates a thorough un-
derstanding of the development of obesity.
Obesity is the result of a chronic imbalance between en-
ergy intake and energy expenditure that leads to an in-
* Corresponding author.
crease in fat cell size and number [2, 3]. Several studies
with transciptomics data from in vitro and in vivo exper-
iments on obesity-related model systems have already
provided insight into gene regulation during adipogenesis
[4–7]. This facilitates further detailed studies to dissect
molecular pathways involved in obesity.
Although the power of the DNA array is highly appreci-
ated, the predictive value of mRNA expression is limited
with respect to cellular physiology. Expression levels of
mRNA often do not parallel the levels of protein expres-
sion from a particular gene [8, 9] and protein turnover and
post-translational modifications, essential for cellular be-
havior, are not covered by the information obtained from
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CMLS, Cell. Mol. Life Sci. Vol. 62, 2005 Research Article 493
DNA arrays [10]. Consequently, a broader understanding
of the adipogenic process requires independent examina-
tion of protein expression and protein function comple-
menting the mRNA expression analyses.
We used a combined two-dimensional (2D) electrophore-
sis/mass spectrometry approach to further understand the
molecular mechanisms involved in fat storage and fat de-
pletion in mouse 3T3-L1 cells which serve as a well-
known model system for adipogenesis. Thus far, only a
limited number of reports have described profiling of cel-
lular proteins with a focus on 3T3-L1 differentiation
[11–14], with Welsh et al. [13] and Choi et al. [14] using
similar techniques as ours. Moreover, in addition to 3T3-
L1 differentiation, we also investigated differences in the
proteome during starvation of 3T3-L1 adipocytes, be-
cause understanding of the conversion of adipocytes to a
fat-depleted status may further contribute to knowledge
about the response of adipose cells to different nutritional
conditions. The response of mature 3T3-L1 adipocytes to
starvation was examined by caloric restriction in either
the absence or the presence of the lipolysis inducer tumor
necrosis factor-
a
(TNF-
a
) [15, 16]. The aim of this study
was to obtain a more comprehensive view of fat cell dif-
ferentiation and starvation. This may possibly result in
potential targets for improved future intervention strate-
gies with respect to obesity and obesity-related disorders.
Materials and methods
Chemicals were purchased from Sigma (Zwijndrecht,
The Netherlands) unless stated otherwise.
Cell culture and cellular Oil Red O accumulation
Mouse 3T3-L1 fibroblasts were purchased from the
American Type Culture Collection and were differenti-
ated into adipocytes as described elsewhere [17], only
with 18 days of differentiation. Differentiation was mon-
itored by the visual appearance of fat droplets in the cells.
Subsequently, adipocytes were subjected to a starvation
period of 4 days by culturing them in DMEM (Invitrogen,
Breda, The Netherlands) without glucose and insulin con-
taining 4% fetal calf serum either in the absence or the
presence of 1 nM mouse TNF-
a
.
At appropriate time points, cells were fixed with 3.7%
formamide in DMEM/F12 (Invitrogen) for 10 min at
room temperature. Cells were incubated with a filtered
Oil Red O (ORO) solution (1% in isopropanol) for 30 min
at room temperature. Cells were washed with 70%
ethanol and dissolved in dimethylsulfoxide (DMSO) to
determine the intracellular ORO content by spectropho-
tometry. The amount of intracellular ORO staining was
corrected for the quantity of genomic DNA, since the
number of living 3T3-L1 cells correlates with the amount
of intact genomic DNA [18]. Images of the cells were
taken with a Nikon TE 200 eclipse phase contrast micro-
scope equipped with digital image acquisition.
Protein sample preparation and 2D electrophoresis
Protein sample preparation was performed as described
previously [17]. Protein concentrations were determined
by a Bradford-based protein assay (Bio-Rad, Veenendaal,
The Netherlands). Protein concentrations were verified
by densitometry with a GS-800 Calibrated Desitometer
(Bio-Rad) of a silver-stained [19] SDS-PAGE gel that
was used to control the protein sample contents. After
correction according to the densitometry results, equal
amounts of protein samples were subjected to 2D elec-
trophoresis.
Separation of the protein samples by 2D electrophoresis
was performed as described elsewhere [17, 20]. For re-
producible results, 12 gels were prepared, run and stained
simultaneously. Gels were stained with silver according
to Shevchenko et al. [19] with minor modifications using
our in house-developed automated gel-staining machine.
Gel images were taken by densitometrical scanning (GS-
800 Calibrated Densitometer; Bio-Rad) and gel images
were further processed to determine differentially ex-
pressed proteins by image analysis software (PD-Quest
7.2, Bio-Rad) as described by Wang et al. [20]. To obtain
more protein identities, preparative gels of the same sam-
ples were generated with fivefold more sample loads.
These gels were stained with Coomassie Brilliant Blue
(CBB) and were further processed in a manner similar to
the silver-stained gels. The CBB gels were matched with
the silver-stained gels and spots earlier pinpointed as dif-
ferentially expressed were excised from the CBB-stained
gels and subjected to mass spectrometry.
Protein identification
Differentially expressed proteins were excised from the
gels by an automated spot cutter (Bio-Rad) according to
the manufacturer’s instructions. Generation of tryptic di-
gests from the proteins by in-gel digestion, Maldi-TOF
analysis and subsequent database searching were per-
formed as described previously [17].
Samples that could not be identified by Maldi-TOF were
subjected to liquid chromatography tandem mass spec-
trometry (LC-MS/MS) [21]. Protein identification was
performed by database searching as described elsewhere
[20, 21].
Data processing
Differentially expressed proteins were categorized into
seven clusters according to their expression pattern dur-
ing the experimental conditions. Data from all clusters
were analyzed using the GenMAPP-Mappfinder tandem
of gene expression mapping software (version 2.0)
(http://www.genmapp.org) [22, 23] in order to find rele-
vant biological pathways. For this purpose, proteins were
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494 J. Renes et al. Protein profiling of 3T3-L1 cells
identified with their Swiss-Prot primary accession num-
ber and categorized in a single column using a positive
numerical identifier for the experimental condition. The
Mappfinder criterion was set to match all positive values
(and thus all proteins present for any experimental condi-
tion). The dataset and criterion file were evaluated using
Mappfinder [23] with both mapps derived from the Gene
Ontology (http://www.geneontology.org) and mapps
specifically build for GenMAPP (the so called local
mapps), using the map set developed for mouse. A ranked
list of mapps with higher numbers of changed proteins
was created and mapps showing three or more changed
proteins were considered relevant.
Results
Intracellular ORO accumulation
Intracellular fat contents during differentiation and star-
vation of 3T3-L1 cells were measured by ORO staining.
Figure 1A shows that in 3T3-L1 pre-adipocytes, ORO
staining of triglycerides was not observed (A). Differen-
tiation of 3T3-L1 cells for 18 days resulted in an accu-
mulation of triglycerides as shown by the red-colored
cells (B). Starvation of these cells by caloric restriction
without TNF-
a
reduced the amount of accumulated
triglycerides (C); however, in the presence of TNF-
a
this
reduction was stronger (D). These results were confirmed
by measurement of the total amount of intracellular ORO,
corrected for the number of living cells (fig. 1B). The
content of intracellular ORO in differentiated 3T3-L1
adipocytes was strongly increased compared to pre-
adipocytes and was set to 100%. Caloric restriction of
3T3-L1 adipocytes in the absence of TNF-
a
reduced the
intracellular ORO contents by 46%, while in the presence
of TNF-
a
this was reduced by 77%.
Protein profiling from 3T3-L1 cells
Changes in protein expression during differentiation and
starvation of 3T3-L1 cells were monitored by 2D elec-
trophoresis. With the image analysis procedure, 93 spots
were found matching the criteria for differentially ex-
pressed proteins. Maldi-TOF analysis of silver-stained
protein spots revealed the identity of 33 proteins (35%).
Spots that could not be identified by the Maldi-TOF pro-
cedure were further analyzed by LC-MS/MS. In total,
our mass spectrometry analysis resulted in the identity of
50 spots (54%) representing 32 different genes. The lo-
cation of these spots in a 2D pattern is depicted in figure
2. Numbers of the protein spots on the gel images corre-
spond with the proteins listed in table 1. The enlarged gel
sections in figure 2 are from 2D gels derived from pro-
tein samples of our four experimental conditions and
show the expression patterns of particular protein spots
during the experiment. Boxed areas in the large gel im-
age indicate the location of these sections in the respec-
tive 2D gels. The gel sections are chosen so that exam-
ples of proteins from every cluster in table 1 are dis-
played. To our best knowledge, 16 proteins listed in table
1 have not been reported before as being expressed in
3T3-L1 cells.
The identified proteins were clustered into seven groups
according to their expression patterns during 3T3-L1 dif-
ferentiation and starvation (table 1). Similar identified
spots in one cluster such as
a
-enolase (table 1, cluster 3,
spot no. 7 and 8), nucleotide diphosphate kinase (NPDK)
B (cluster 3, spot no. 13 and 14) and annexin II (cluster 6,
spot no. 33, 34 and 35) are possibly isoforms or post-
translationaly modified forms of the same protein. Un-
fortunately, with the method we used, we were not able to
distinguish between these possibilities and the functional
significance with respect to 3T3-L1 cells remains elu-
Figure 1. (A) Fat accumulation in 3T3-L1 cells during differentia-
tion and starvation. Detection of triglycerides occurred with ORO
in 3T3-L1 pre-adipocytes (A), 3T3-L1 adipocytes (B), 3T3-L1
adipocytes subjected to caloric restriction without TNF-
a
(C) and
3T3-L1 adipocytes subjected to caloric restriction in the presence of
TNF-
a
(D). (B) Fat accumulation in 3T3-L1 cells during differenti-
ation and starvation corrected for the number of living cells. The
amount of fat storage in 3T3-L1 adipocytes was set to 100%.
(A)
(B)
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CMLS, Cell. Mol. Life Sci. Vol. 62, 2005 Research Article 495
sive. Alternatively, due to technical conditions, some pro-
teins may also be truncated during 2D electrophoresis
which results in different spots derived from the same
protein, e.g. glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) in cluster 1 and protein disulfide isomerase A3
in cluster 5 and 6.
Expression patterns of identified proteins were analyzed
using GenMAPP/Mappfinder in order to find relevant bi-
ological pathways involved in differentiation and starva-
tion of 3T3-L1 (pre)-adipocytes. We found the gly-
colytic/gluconeogenesis pathway that met our criteria of
at least three changed proteins (fig. 3).
Proteins involved in 3T3-L1 differentiation
Clearly, several proteins from the glycolysis/gluco-
neogenic pathway and associated reactions show a differ-
ential expression pattern during 3T3-L1 differentiation
(fig. 3A, table 1, clusters 1 and 3). Spots identified as the
glycolytic enzymes GAPDH and
a
-enolase showed an in-
creased expression during differentiation. Surprisingly,
these enzymes were also found to be down-regulated dur-
ing 3T3-L1 differentiation (fig 3A). Figure 4 shows the
expression patterns of identified protein spots represent-
ing these two proteins during 3T3-L1 differentiation and
starvation. Obviously, the appearance of
a
-enolase (spot
no. 1) and GAPDH (spot no. 3) paralleled the disappear-
ance of
a
-enolase (spot no. 7 and 8) and GAPDH (spot
no. 9) during differentiation. In addition, during starva-
tion, the expression pattern returned to the pre-adipocyte
status. This suggests that one form of these proteins is
converted into another form during 3T3-L1 differentia-
tion and vice versa during starvation.
Expression of the final enzyme of the glycolysis pathway,
pyruvate kinase was found to be down-regulated during
3T3-L1 differentiation, while expression of the mito-
chondrial malate dehydrogenase (MDH) was induced
(fig. 3A). MDH converts malate into oxaloacetate which
is used as a carrier for acetyl-CoA across the mitochon-
drial membrane. Acetyl CoA, once released in the cy-
tosol, is the first substrate in fatty acid synthesis [24]. An-
other glycolysis-associated protein, phosphoglycerate de-
hydrogenase (spot 10a) was down-regulated during
3T3-L1 differentiation (fig. 3A).
Other metabolic enzymes that were down-regulated dur-
ing 3T3-L1 differentiation were ornitine aminotrans-
ferase (OAT), NDPK A/B (table 1, cluster 3) and py-
rophosphatase (cluster 4). OAT is involved in amino acid
metabolism, while NDPK A and B play important roles in
synthesis of non-adenylic nucleotides. The function of
pyrophosphatase in 3T3-L1 cells remains elusive.
Figure 2. Identified proteins marked on a representative 2D gel image from pre-adipocytes. Enlarged gel sections are from 2D gels derived
from protein samples of 3T3-L1 pre-adipocytes (A), 3T3-L1 adipocytes (B), 3T3-L1 adipocytes subjected to caloric restriction without
TNF-
a
(C) and 3T3-L1 adipocytes subjected to caloric restriction in the presence of TNF-
a
(D). The location of these sections in the re-
spective 2D gels is indicated by boxed areas in the large gel image. The gel sections show expression patterns of proteins which are exam-
ples of all clusters in table 1. Numbers on the gel images correspond to the protein numbers in table 1.
Page 4
496 J. Renes et al. Protein profiling of 3T3-L1 cells
Table 1. Proteins identified during 3T3-L1 differentiation and starvation.
Proteins are grouped into seven clusters according to their expression profiles during the experiment. Bars represent relative expression ra-
tios of the proteins under the following conditions: 3T3-L1 pre-adipocytes (A), 3T3-L1 adipocytes (B), 3T3-L1 adipocytes subjected to
caloric restriction without TNF-
a
(C) and 3T3-L1 adipocytes subjected to caloric restriction in the presence of TNF-
a
(D). Accession num-
bers refer either to the Swiss-Prot database (p and q numbers) or to the NCBI protein database (NP numbers). Proteins indicated by aster-
isks have not been described before in 3T3-L1 cells.
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CMLS, Cell. Mol. Life Sci. Vol. 62, 2005 Research Article 497
Figure 3. Regulated proteins in the glycolysis/gluconeogenesis pathway during 3T3-L1 differentiation (A) and during 3T3-L1 starvation
(B). Proteins found to be up- as well as down-regulated during 3T3-L1 differentiation or starvation (GAPDH,
a
-enolase and pyruvate ki-
nase M2) are double colored.
A
Page 6
498 J. Renes et al. Protein profiling of 3T3-L1 cells
Figure 3 (continued)
B
Page 7
CMLS, Cell. Mol. Life Sci. Vol. 62, 2005 Research Article 499
In addition to metabolic enzymes, we observed a differ-
ential expression of proteins with growth-regulatory
properties. These were peptidyl-prolyl cis-trans iso-
merase C and galectin-1 (clusters 1 and 2) which were in-
duced during differentiation, and galectin-3, cystatin B
(cluster 3) and calreticulin (cluster 4) which were down-
regulated. We also observed regulation of proteins with
functions in cytoskeletal rearrangements. These were de-
strin/actin-depolymerizing factor (ADF) (cluster 3),
b
-
tubulin and cofilin (cluster 4) which showed down-regu-
lation during 3T3-L1 differentiation. Another protein
down-regulated during differentiation was lamin C and
C2 (spot 10b). This protein is present in a mixed spot with
phosphoglycerate dehydrogenase (spot 10a). Lamin C, a
component of the nuclear lamina, has been detected be-
fore by us in 3T3-L1 cells [17] and is also found in human
adipose tissue [25]. Mutations in the gene encoding this
protein cause familiar lipodystrophy [26].
Proteins involved in 3T3-L1 starvation
Proteins in cluster 1 and 3 (table 1) with altered expres-
sion during differentiation (compare bars A and B)
showed a reciprocal regulation when differentiated 3T3-
L1 cells were subjected to starvation (compare bars B vs
bars C and D). This suggests a specific functional associ-
ation of these proteins with the transition of 3T3-L1 pre-
adipocytes to adipocytes. In addition, both starvation pro-
tocols resulted in a return of these proteins to their pre-
adipocyte status.
Figure 3B shows proteins with different expression pat-
terns in the glycolytic/gluconeogenic pathway during
starvation. Compared to 3T3-L1 differentiation (fig. 3A),
beside common regulated proteins, some proteins are
specifically influenced by starvation. These are tri-
osephosphate isomerase, phosphoglycerate kinase and
lactate dehydrogenase.
Next to metabolic enzymes, other proteins were specifi-
cally regulated by starvation. Cluster 5 shows up-regula-
tion of the chaperone protein disulfide isomerase A3 and
heat shock protein 60. These proteins are linked to cellu-
lar stress, a condition that may be induced by caloric re-
striction of 3T3-L1 cells. Cluster 6 shows proteins that
are related to the cytoskeletal network such as annexins,
tubulins, actin, myosin and cofilin. In addition, we found
the redox regulator peroxiredoxin 2 and ubiquilin, a pro-
tein involved in protein degradation. These proteins were
all down-regulated during starvation.
TNF-
aa
regulates a different set of proteins during
caloric restriction
We investigated the effect of a known lipolysis inducer
(TNF-
a
) on the proteome of differentiated 3T3-L1 cells
on a background of caloric restriction (table 1, bars B vs
bars D). We also compared the effect of both starvation
protocols (caloric restriction in the absence or in the pres-
ence of TNF-
a
) on 3T3-L1 cells (table 1, bars C vs bars
D). Beside common regulated proteins during both star-
vation protocols (table 1, clusters 1, 3, 5 and 6), a set of
differentially expressed proteins was observed between
caloric restriction and caloric restriction combined with
TNF-
a
(see table 1, clusters 2, 4 and 7).
One protein where expression was specifically down-reg-
ulated by TNF-
a
was galectin-1. During starvation, ex-
pression of galectin-1 was not influenced by caloric re-
striction while its expression was reduced by TNF-
a
(table
1, cluster 2). On the other hand, pyrophosphatase,
b
-tubu-
lin, calreticulin and cofilin were specifically up-regulated
by TNF-
a
(table 1, cluster 4). A striking difference in pro-
tein expression was observed with respect to metabolic
enzymes (see also fig. 3B). For example, expression of
MDH (table 1, cluster 2) was not changed during caloric
restriction but was down-regulated by TNF-
a
.
Cluster 7 shows several metabolic proteins that were
down-regulated when differentiated 3T3-L1 cells were
subjected to caloric restriction but did not show a change
in expression when these cells were treated with TNF-
a
.
These are the glycolytic enzymes
a
-enolase (spot no. 42),
GAPH (spot no. 43), phosphoglycerate kinase 1, tri-
osephosphate isomerase and the nucleotide synthesizer
NDPK B (spot no. 46). Other proteins specifically down-
regulated by caloric restriction without TNF-
a
are the en-
doplasmic reticulum proteins ERp29 and calreticulin and
the cytoskeleton-related protein annexin II. TNF-
a
seems
to prevent down-regulation of these proteins when differ-
entiated 3T3-L1 cells are subjected to the combination of
caloric restriction and TNF-
a
.
Figure 4. Reciprocal regulation of
a
-enolase and GAPDH during
3T3-L1 differentiation and starvation.
Page 8
500 J. Renes et al. Protein profiling of 3T3-L1 cells
Discussion
The complex etiology of obesity requires a thorough un-
derstanding of the molecular mechanisms of the adi-
pogenic process. To gain a broader understanding of the
molecular events during adipogenesis and to overcome
the limits of transcriptomics with respect to cellular be-
havior, we examined changes in the proteome of 3T3-L1
cells. Concerning differentiation of 3T3-L1 cells, our
data show similar expression patterns for several proteins
which confirm previous results [12–14]. However, we
also identified proteins that are specifically involved in
starvation of 3T3-L1 cells. Moreover, we found proteins
whose expression has not been reported before in 3T3-L1
cells. Therefore, our data add valuable information for a
better understanding of the molecular mechanism in-
volved in fat storage and fat depletion in 3T3-L1
adipocytes.
Four categories of proteins were identified during 3T3-
L1 differentiation and starvation: metabolic enzymes,
proteins with growth regulatory properties, proteins with
a function in cytoskeletal rearrangements and protein
modifiers. With respect to metabolic enzymes, the in-
creased expression of GAPDH,
a
-enolase and MDH dur-
ing differentiation is in agreement with enhanced gly-
colytic activity and fatty acid synthesis [24]. The expres-
sion pattern of these proteins resembles the mRNA
expression profiles from the same genes during adipoge-
nesis in vitro and in vivo [6]. The down-regulation of
phosphoglycerate dehydrogenase may reduce the exit of
3-phosphoglycerate from the glycolytic pathway and con-
sequently stimulate the conversion of glucose into acetyl-
CoA. The pyruvate dehydrogenase complex (PDC) is the
link between glycolysis and fatty acid synthesis by con-
version of pyruvate into acetyl-CoA. One of the control
mechanisms for regulation of the PDC is the energy sta-
tus in the cell. GTP, in particular, is able to inhibit PDC
activity [24] and GTP-binding proteins are known to reg-
ulate PDC activity [27]. The main function of NDPK A
and B is synthesis of non-adenylic nucleotides [28].
Therefore, down-regulation of NDPK A and B during dif-
ferentiation of 3T3-L1 cells results in a decreased level of
nucleotides that may counteract the inhibition of PDC
and promote fatty acid synthesis. In addition, NDPK A
and B are also able to regulate cell growth, which will be
discussed below. The surprising disappearance of the spot
representing the major glycolytic enzyme pyruvate ki-
nase during differentiation might be due to dephosphory-
lation, in analogy with its isoform pyruvate kinase L. This
enzyme is activated by insulin via a dephosphorylation
event [29, and references therein].
(De-)phosphorylation may be one explanation for the ob-
served shift in the position of certain spots in our 2D gels.
However, the mass difference between the spots identi-
fied as GAPDH and
a
-enolase showing a reciprocal ex-
pression pattern during differentiation and subsequent
caloric restriction (fig. 4) is too large to be explained by
(de-)phosphorylation. For both enzymes, several iso-
forms have been identified with suggested different cel-
lular locations and functions such as apoptosis, mem-
brane dynamics, excretion, receptor function and growth
regulation [reviewed in refs. 30, 31]. Which isoforms are
involved in each process and whether these isoforms are
active in 3T3-L1 cells is currently not known. Alterna-
tively, degradation of these proteins cannot be excluded
but because of the reciprocal expression patterns, this is
unlikely to be due to technical conditions. Instead, this
may imply the activity of specific proteases during 3T3-
L1 differentiation and starvation.
Based on our protein expression data, we conclude that
the glycolytic pathway is not completely reciprocally reg-
ulated when differentiated 3T3-L1 cells are subjected to
caloric restriction. Beside common regulated proteins
during both conditions, such as GAPDH and
a
-enolase,
we observed three glycolytic proteins, triosephosphate
isomerase, phosphoglycerate kinase 1 and lactate dehy-
drogenase, whose expression was stable during 3T3-L1
differentiation but was altered during caloric restriction.
In addition, the up-regulated expression of MDH during
differentiation was not reversed during caloric restriction
which indicates a residual activity in fatty acid synthesis.
These results clearly demonstrate that the regulation of
the glycolysis pathway during caloric restriction of 3T3-
L1 cells differs from the regulation during differentiation.
For other identified proteins, a non-reciprocal regulation
was also observed when differentiated 3T3-L1 cells were
subjected to caloric restriction. This shows that although
caloric restriction results in fat release, it induces only a
limited pre-adipocyte-like protein expression pattern.
Previously, at the mRNA level, TNF-
a
was shown to in-
duce a conversion to a pre-adipocyte genotype when
added to differentiated 3T3-L1 cells [32]. We found a
similar effect at the protein level. Compared to solely
caloric restriction, a combination of caloric restriction
and TNF-
a
showed that more proteins change to a pre-
adipocyte-like expression pattern. Moreover, TNF-
a
counteracted effects of caloric restriction on differenti-
ated 3T3-L1 (see table 1, clusters 2, 4 and 7).
We observed a specific TNF-
a
-mediated up-regulation of
calreticulin (table 1, clusters 4 and 7). This protein is able
to repress translation of CCAAT/enhancer-binding pro-
tein (C/EBP)
a
and C/EBP
b
[33]. Repression of C/EBP
has also been observed when 3T3-L1 adipocytes were
subjected to TNF-
a
[15, 34]. We expect that calreticulin
is involved in this process. Overexpression of either
CEBP
a
or CEBP
b
in 3T3-L1 cells is sufficient to induce
their differentiation into mature fat cells. In conjunction
with peroxisome proliferator-activated receptor
g
(PPAR
g
), both proteins are involved in the transcriptional
cascade that plays an important role in the differentiation
Page 9
CMLS, Cell. Mol. Life Sci. Vol. 62, 2005 Research Article 501
of 3T3-L1 cells [reviewed in ref. 35]. In our experiments,
TNF-
a
regulates a set of proteins that seem to induce a
pre-adipocyte phenotype that results in a further decrease
in intracellular fat content. This is in agreement with the
observed inhibition of PPAR
g
by TNF-
a
in mature 3T3-
L1 adipocytes [36, 37]. Furthermore, stimulation of
PPAR
g
inhibits the action of TNF-
a
on 3T3-L1
adipocytes [38]. Thus, in contrast to solely caloric re-
striction, we expect that a forced down-regulation of
C/EBP proteins and PPAR
g
by TNF-
a
might play a role
in a stronger depletion of fat content in 3T3-L1 cells (see
fig. 1).
During differentiation, 3T3-L1 cells loose proliferative
potential and acquire resistance against apoptotic stimuli
which is accompanied by induced expression of a neu-
ronal apoptosis inhibitory protein [39]. We found seven
differentially expressed proteins which possess growth-
regulatory properties: galectin 1 and 3, NDPK A and B,
calreticulin, peptidyl-prolyl cis-trans isomerase C and
cystatin B. Dependent on the cell type, galectin 1 and 3
are anti-apoptotic [40]. Galectin 1 arrests T cells in the S
and G2/M phase of the cell cycle, while low expression of
galectin 1 induces cell proliferation [41]. Transfection
with antisense galectin 3 cDNA inhibited the prolifera-
tion of MDA-MB435 breast cancer cells [42]. NDPK
gene expression is positively correlated with proliferating
tumor cells, while down-regulation of NDPK proteins by
RNA antisense techniques suppresses tumor cell growth
as reviewed by Kimura et al. [43]. Mouse embryonic fi-
broblasts deficient in calreticulin are resistant to apopto-
sis, probably via Ca
2+
-mediated signaling [44], and over-
expression of calreticulin is associated with increased
malignancy of breast cancer cells [45, 46]. Peptidyl-pro-
lyl cis-trans isomerase C is a member of a large conserved
family of peptidyl-prolyl cis-trans isomerases which in-
cludes FK506-binding proteins (FKBP), cyclophilins and
parvulins [47]. Recently, cyclophilin D and FKBP38
were shown to be anti-apoptotic [48, 49]. Finally, cystatin
B is suggested to be involved in progression of tumor cell
growth [50, 51]. When differentiated 3T3-L1 cells are
subsequently treated with our starvation protocols, the
expression of these proteins returns to their pre-adipocyte
status. Based on their expression profiles during our ex-
periments, the seven proteins indicated here are likely ac-
tively involved in cell growth arrest during 3T3-L1 dif-
ferentiation and in an anti-apoptotic phenotype of differ-
entiated 3T3-L1 cells. Upon starvation, the expression
patterns of these proteins are switched to a profile that is
associated with induced cell proliferation and increased
sensitivity to apoptosis, particularly when TNF-
a
is used.
Indeed, TNF-
a
is known to activate pre-adipocyte genes
in 3T3-L1 adipocytes [32] and to induce apoptosis in
3T3-L1 pre-adipocytes [52].
During 3T3-L1 differentiation, a dramatic remodeling of
the cytoskeleton occurs. While the tubulin network is ex-
panded by the action of insulin [53], the actin fiber net-
work is depolymerized and transformed into a cortical
network lining the inner face of the plasma membrane
[54, 55]. We found three proteins, annexin II, cofilin and
destrin/ADF, which are actively involved in actin dynam-
ics [56, 57]. Recently, increased expression of coactosin
was found during 3T3-L1 differentiation. Overexpression
of this protein induced a depolymerization of actin [13].
This indicates that several different proteins are involved
in remodeling of the cytoskeleton during 3T3-L1 differ-
entiation. Little is known, however, about the behavior of
the cytoskeleton during fat depletion in 3T3-L1 cells.
Brasaemle et al. [58] showed that cytoskeleton-disrupting
agents did not inhibit isoproterenol-induced lipolysis in
3T3-L1 cells. Thus a remodeling of the cytoskeleton does
not hinder lipolysis. The decreased expression of annexin
II, actin, tubulin and myosin IX, an actin-binding protein
[59], that we observed during starvation of differentiated
3T3-L1 cells is in agreement with this.
Our data show that the glycolysis/gluconeogenic path-
ways are differentially regulated during 3T3-L1 differen-
tiation and subsequent starvation. Differentiated 3T3-L1
cells express a protein profile that is associated with cell
growth arrest, resistance to apoptosis and a remodeling of
the cytoskeleton. Most of the proteins involved in these
processes show a reversed expression pattern upon 3T3-
L1 starvation, especially with TNF-
a
. In conclusion, our
results demonstrate that an independent survey of protein
expression provides valuable information for the broader
understanding of adipogenesis. New proteins were dis-
covered with expected important roles in 3T3-L1 differ-
entiation and starvation. These provide potential new tar-
gets for future intervention studies with respect to obe-
sity.
Acknowledgements. Dr. P. Verhaert (University of Leuven, Bel-
gium) is acknowledged for assistance with protein identification by
mass spectrometry and Dr. W. Voncken (Maastricht University) for
assistance with microscopy. This study was supported by the Maas-
tricht Proteomics Center, the Brede Onderzoek Strategie and the re-
search institute NUTRIM of the Maastricht University.
1 Kopelman P. G. (2000) Obesity as a medical problem. Nature
404: 635–643
2 Spiegelman B. M. and Flier J. S. (2001) Obesity and the regu-
lation of energy balance. Cell 104: 531–543
3 Prins J. B. and O’Rahilly S. (1997) Regulation of adipose cell
number in man. Clin. Sci. 92: 3–11
4 Guo X. and Liao K. (2000) Analysis of gene expression profile
during 3T3-L1 preadipocyte differentiation. Gene 251: 45–53
5 Burton G. R., Guan Y., Nagarajan R. and McGehee R. E. Jr
(2002) Microarray analysis of gene expression during early
adipocyte differentiation. Gene 293: 21–31
6 Soukas A., Socci N. D., Saatkamp B. D., Novelli S. and Fried-
man J. M. (2001) Distinct transcriptional profiles of adipogen-
esis in vivo and in vitro. J. Biol. Chem. 276: 34167–34174
7 Nadler S. T., Stoehr J. P., Schueler K. L., Tanimoto G., Yandell
B. S. and Attie A. D. (2000) The expression of adipogenic genes
Page 10
502 J. Renes et al. Protein profiling of 3T3-L1 cells
is decreased in obesity and diabetes mellitus. Proc. Natl. Acad.
Sci. USA 97: 11371–11376
8 Gygi S. P., Rochon Y., Franza B. R. and Aebersold R. (1999)
Correlation between protein and mRNA abundance in yeast.
Mol. Cell. Biol. 19: 1720–1730
9 Futcher B., Latter G. I., Monardo P., McLaughlin C. S. and
Garrels J. I. (1999) A sampling of the yeast proteome. Mol.
Cell. Biol. 19: 7357–7368
10 Richards J., Le Naour F., Hanash S. and Beretta L. (2002) Inte-
grated genomic and proteomic analysis of signaling pathways
in dendritic cell differentiation and maturation. Ann. N. Y.
Acad. Sci. 975: 91–100
11 Sidhu R. S. (1979) Two-dimensional electrophoretic analyses
of proteins synthesized during differentiation of 3T3-L1
preadipocytes. J. Biol. Chem. 254: 11111–11118
12 Wilson-Fritch L., Burkart A., Bell G., Mendelson K., Leszyk J.,
Nicoloro S. et al. (2003) Mitochondrial biogenesis and remod-
eling during adipogenesis and in response to the insulin sensi-
tizer rosiglitazone. Mol. Cell. Biol. 23: 1085–1094
13 Welsh G. I., Griffiths M. R., Webster K. J., Page M. J. and
Tavare J. M. (2004) Proteome analysis of adipogenesis. Pro-
teomics 4: 1042–1051
14 Choi K. L., Wang Y., Tse C. A., Lam K. S., Cooper G. J. and Xu
A. (2004) Proteomic analysis of adipocyte differentiation: evi-
dence that alpha2 macroglobulin is involved in the adipose con-
version of 3T3 L1 preadipocytes. Proteomics 4: 1840–1848
15 Ron D., Brasier A. R., McGehee R. E. Jr and Habener J. F.
(1992) Tumor necrosis factor-induced reversal of adipocytic
phenotype of 3T3-L1 cells is preceded by a loss of nuclear
CCAAT/enhancer binding protein (C/EBP). J. Clin. Invest. 89:
223–233
16 Petruschke T. and Hauner H. (1993) Tumor necrosis factor-al-
pha prevents the differentiation of human adipocyte precursor
cells and causes delipidation of newly developed fat cells. J.
Clin. Endocrinol. Metab. 76: 742–747
17 Bouwman F., Renes J. and Mariman E. (2004) A combination
of protein profiling and isotopomer analysis using matrix-as-
sisted laser desorption/ionization-time of flight mass spectrom-
etry reveals an active metabolism of the extracellular matrix of
3T3-L1 adipocytes. Proteomics 4: 3855–3863
18 Zorenc A., Bouwman F. and Bakker A. (2002) Induction of
lipodystrophy in 3T3-L1 cells. Int. J. Obes. Rel. Metab. Dis. 26:
654
19 Shevchenko A., Wilm M., Vorm O. and Mann M. (1996) Mass
spectrometric sequencing of proteins silver-stained polyacry-
lamide gels. Anal. Chem. 68: 850–858
20 Wang P., Mariman E., Keijer J., Bouwman F., Noben J. P.,
Robben J. et al. (2004) Profiling of the secreted proteins during
3T3-L1 adipocyte differentiation leads to the identification of
novel adipokines. Cell. Mol. Life Sci. 61: 2405–2417
21 Dumont D., Noben J. P., Raus J., Stinissen P. and Robben J.
(2004) Proteomic analysis of cerebrospinal fluid from multiple
sclerosis patients. Proteomics 4: 2117–2124
22 Dahlquist K. D., Salomonis N., Vranizan K., Lawlor S. C. and
Conklin B. R. (2002) GenMAPP, a new tool for viewing and an-
alyzing microarray data on biological pathways. Nat. Genet.
31: 19–20
23 Doniger S. W., Salomonis N., Dahlquist K. D., Vranizan K.,
Lawlor S. C. and Conklin B. R. (2003) MAPPFinder: using
Gene Ontology and GenMAPP to create a global gene-expres-
sion profile from microarray data. Genome Biol 4: R7
24 Stryer L. (1988) Biochemistry, Freeman, New York
25 Lelliott C. J., Logie L., Sewter C. P., Berger D., Jani P., Blows F.
et al. (2002) Lamin expression in human adipose cells in rela-
tion to anatomical site and differentiation state. J. Clin. En-
docrinol. Metab. 87: 728–734
26 Speckman R. A., Garg A., Du F., Bennett L., Veile R., Arioglu
E. et al. (2000) Mutational and haplotype analyses of families
with familial partial lipodystrophy (Dunnigan variety) reveal
recurrent missense mutations in the globular C-terminal do-
main of lamin A/C. Am. J. Hum. Genet. 66: 1192–1198
27 Curto M., Piccinini M., Rabbone I., Mioletti S., Mostert M.,
Bruno R. et al. (1997) G proteins and regulation of pyruvate de-
hydrogenase activity by insulin in human circulating lympho-
cytes. Int. J. Biochem. Cell Biol. 29: 1207–1217
28 Lacombe M. L., Milon L., Munier A., Mehus J. G. and Lambeth
D. O. (2000) The human Nm23/nucleoside diphosphate ki-
nases. J. Bioenerg. Biomembr. 32: 247–258
29 Carrillo J. J., Ibares B., Esteban-Gamboa A. and Feliu J. E.
(2001) Involvement of both phosphatidylinositol 3-kinase and
p44/p42 mitogen-activated protein kinase pathways in the
short-term regulation of pyruvate kinase L by insulin. En-
docrinology 142: 1057–1064
30 Sirover M. A. (1999) New insights into an old protein: the func-
tional diversity of mammalian glyceraldehyde–3-phosphate de-
hydrogenase. Biochim. Biophys. Acta 1432: 159–184
31 Pancholi V. (2001) Multifunctional alpha-enolase: its role in
diseases. Cell. Mol. Life Sci. 58: 902–920
32 Ruan H., Hacohen N., Golub T. R., Van Parijs L. and Lodish H.
F. (2002) Tumor necrosis factor-alpha suppresses adipocyte-
specific genes and activates expression of preadipocyte genes
in 3T3-L1 adipocytes: nuclear factor-kappaB activation by
TNF-alpha is obligatory. Diabetes 51:
1319–1336
33 Timchenko L. T., Iakova P., Welm A. L., Cai Z. J. and Tim-
chenko N. A. (2002) Calreticulin interacts with C/EBPalpha
and C/EBPbeta mRNAs and represses translation of C/EBP
proteins. Mol. Cell. Biol. 22: 7242–7257
34 Stephens J. M. and Pekala P. H. (1991) Transcriptional repres-
sion of the GLUT4 and C/EBP genes in 3T3-L1 adipocytes by
tumor necrosis factor-alpha. J. Biol. Chem. 266: 21839–21845
35 Rosen E. D. and Spiegelman B. M. (2000) Molecular regulation
of adipogenesis. Annu. Rev. Cell Dev. Biol. 16: 145–171
36 Zhang B., Berger J., Hu E., Szalkowski D., White-Carrington
S., Spiegelman B. M. et al. (1996) Negative regulation of per-
oxisome proliferator-activated receptor-gamma gene expres-
sion contributes to the antiadipogenic effects of tumor necrosis
factor-alpha. Mol. Endocrinol. 10: 1457–1466
37 Xing H., Northrop J. P., Grove J. R., Kilpatrick K. E., Su J. L.
and Ringold G. M. (1997) TNF alpha-mediated inhibition and
reversal of adipocyte differentiation is accompanied by sup-
pressed expression of PPARgamma without effects on Pref-1
expression. Endocrinology 138: 2776–2783
38 Souza S. C., Yamamoto M. T., Franciosa M. D., Lien P. and
Greenberg A. S. (1998) BRL 49653 blocks the lipolytic actions
of tumor necrosis factor-alpha: a potential new insulin-sensitiz-
ing mechanism for thiazolidinediones. Diabetes 47: 691–695
39 Magun R., Gagnon A., Yaraghi Z. and Sorisky A. (1998) Ex-
pression and regulation of neuronal apoptosis inhibitory pro-
tein during adipocyte differentiation. Diabetes 47: 1948–1952
40 Hsu D. K. and Liu F. T. (2004) Regulation of cellular home-
ostasis by galectins. Glycoconj. J. 19: 507–515
41 Yang R. Y. and Liu F. T. (2003) Galectins in cell growth and
apoptosis. Cell. Mol. Life Sci. 60: 267–276
42 Honjo Y., Nangia-Makker P., Inohara H. and Raz A. (2001)
Down-regulation of galectin-3 suppresses tumorigenicity of
human breast carcinoma cells. Clin. Cancer Res. 7: 661–668
43 Kimura N., Shimada N., Fukuda M., Ishijima Y., Miyazaki H.,
Ishii A. et al. (2000) Regulation of cellular functions by nucle-
oside diphosphate kinases in mammals. J. Bioenerg. Bio-
membr. 32: 309–315
44 Nakamura K., Bossy-Wetzel E., Burns K., Fadel M. P., Lozyk
M., Goping I. S. et al. (2000) Changes in endoplasmic reticu-
lum luminal environment affect cell sensitivity to apoptosis. J.
Cell Biol. 150: 731–740
45 Franzen B., Linder S., Alaiya A. A., Eriksson E., Uruy K., Hi-
rano T. et al. (1996) Analysis of polypeptide expression in be-
nign and malignant human breast lesions: down-regulation of
cytokeratins. Br. J. Cancer 74: 1632–1638
Page 11
CMLS, Cell. Mol. Life Sci. Vol. 62, 2005 Research Article 503
46 Franzen B., Linder S., Alaiya A. A., Eriksson E., Fujioka K.,
Bergman A. C. et al. (1997) Analysis of polypeptide expression
in benign and malignant human breast lesions. Electrophoresis
18: 582–587
47 Galat A. (2003) Peptidylprolyl cis/trans isomerases (im-
munophilins): biological diversity-targets-functions. Curr. Top.
Med. Chem. 3: 1315–1347
48 Schubert A. and Grimm S. (2004) Cyclophilin D, a component
of the permeability transition-pore, is an apoptosis repressor.
Cancer Res. 64: 85–93
49 Shirane M. and Nakayama K. I. (2003) Inherent calcineurin in-
hibitor FKBP38 targets Bcl-2 to mitochondria and inhibits
apoptosis. Nat. Cell Biol. 5: 28–37
50 Kos J. and Lah T. T. (1998) Cysteine proteinases and their en-
dogenous inhibitors: target proteins for prognosis, diagnosis
and therapy in cancer. Oncol. Rep. 5: 1349–1361
51 Calkins C. C., Sameni M., Koblinski J., Sloane B. F. and Moin
K. (1998) Differential localization of cysteine protease in-
hibitors and a target cysteine protease, cathepsin B, by im-
muno-confocal microscopy. J. Histochem. Cytochem. 46:
745–751
52 Niesler C. U., Urso B., Prins J. B. and Siddle K. (2000) IGF-I
inhibits apoptosis induced by serum withdrawal, but potentiates
TNF-alpha-induced apoptosis, in 3T3-L1 preadipocytes. J. En-
docrinol. 167: 165–174
53 Olson A. L., Eyster C. A., Duggins Q. S. and Knight J. B. (2003)
Insulin promotes formation of polymerized microtubules by a
phosphatidylinositol 3-kinase-independent, actin-dependent
pathway in 3T3-L1 adipocytes. Endocrinology 144: 5030–
5039
54 Kawaguchi N., Sundberg C., Kveiborg M., Moghadaszadeh B.,
Asmar M., Dietrich N. et al. (2003) ADAM12 induces actin cy-
toskeleton and extracellular matrix reorganization during early
adipocyte differentiation by regulating beta1 integrin function.
J. Cell Sci. 116: 3893–3904
55 Kanzaki M. and Pessin J. E. (2001) Insulin-stimulated GLUT4
translocation in adipocytes is dependent upon cortical actin re-
modeling. J. Biol. Chem. 276: 42436–42444
56 Gerke V. and Moss S. E. (2002) Annexins: from structure to
function. Physiol. Rev. 82: 331–371
57 Remedios C. G. dos, Chhabra D., Kekic M., Dedova I. V.,
Tsubakihara M., Berry D. A. et al. (2003) Actin binding pro-
teins: regulation of cytoskeletal microfilaments. Physiol. Rev.
83: 433–473
58 Brasaemle D. L., Levin D. M., Adler-Wailes D. C. and Londos
C. (2000) The lipolytic stimulation of 3T3-L1 adipocytes pro-
motes the translocation of hormone-sensitive lipase to the sur-
faces of lipid storage droplets. Biochim. Biophys. Acta 1483:
251–262
59 Bahler M. (2000) Are class III and class IX myosins motorized
signalling molecules? Biochim. Biophys. Acta 1496: 52–59
60 Alexander M., Curtis G., Avruch J. and Goodman H. M. (1985)
Insulin regulation of protein biosynthesis in differentiated 3T3
adipocytes: regulation of glyceraldehyde-3-phosphate dehy-
drogenase. J. Biol. Chem. 260: 11978–11985
61 Lee Y. H., Harada S., Smith R. M., Friedman R. and Jarett L.
(1996) The expression of and insulin binding to cellular thyroid
hormone binding protein, but not insulin degrading enzyme, is
increased during 3T3-L1 adipocytes differentiation. Biochem.
Biophys. Res. Commun. 222: 839–843
62 Laszlo L., Doherty F. J., Osborn N. U. and Mayer R. J. (1990)
Ubiquitinated protein conjugates are specifically enriched in
the lysosomal system of fibroblasts. FEBS Lett. 261: 365–
368
63 Guilherme A., Emoto M., Buxton J. M., Bose S., Sabini R.,
Theurkauf W. E. et al. (2000) Perinuclear localization and in-
sulin responsiveness of GLUT4 requires cytoskeletal integrity
in 3T3-L1 adipocytes. J. Biol. Chem. 275: 38151–38159
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  • Source
    • "Thus, progranulin may be a valuable therapeutic target for treating obesity and obesity-related metabolic diseases. There have been several reports based on 2-DE analysis during the adipogenic differentiation of 3T3-L1 cells52535455. In 2009, the adipocyte proteome during differentiation was reported by in vivo labeling using a five-plex SILAC-based strategy [56]. "
    [Show abstract] [Hide abstract] ABSTRACT: Obesity is a chronic disease that is associated with significantly increased levels of risk of a number of metabolic disorders. Despite these enhanced health risks, the worldwide prevalence of obesity has increased dramatically over the past few decades. Obesity is caused by the accumulation of an abnormal amount of body fat in adipose tissue, which is composed mostly of adipocytes. Thus, a deeper understanding of the regulation mechanism of adipose tissue and/or adipocytes can provide a clue for overcoming obesity-related metabolic diseases. In this review, we describe recent advances in the study of adipose tissue and/or adipocytes, focusing on proteomic approaches. In addition, we suggest future research directions for proteomic studies which may lead to novel treatments of obesity and obesity-related diseases.
    Full-text · Article · Mar 2015 · International Journal of Molecular Sciences
  • Source
    • "Yet, protein analysis studies using samples obtained at 18 days of differentiation [Fig. 2e, f (Renes et al. 2005)] and studies performed by others (Tilgner et al. 2009) showed an evident decrease in protein levels of lamins A/C and emerin upon adipose conversion. Emerin is thought to be a key player in adipogenesis because of its role in nuclear-cytoplasmic shuttling of b-catenin (Markiewicz et al. 2006 ). "
    [Show abstract] [Hide abstract] ABSTRACT: A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the ‘linker of nucleus and cytoskeleton’ (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy. Electronic supplementary material The online version of this article (doi:10.1007/s00418-011-0792-4) contains supplementary material, which is available to authorized users.
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  • Source
    • "But, proteomic analysis of the adipocyte was performed using cell lines such as 3T3-L1 preadipocytes. Many studies involving the effect of hormones (Fasshauera et al., 2002), apopototic factors (Renes et al., 2005) and oligosaccharides (Rahman et al., 2008) on differentiating 3T3-L1 preadipocytes are in literature. Our proteomic analyses displayed about 16 differently expressed proteins, and they belong to similar categories observed in proteomic analysis of primary cultures of human adipose-derived stem cells (James et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Anatomically separate fat depots differ in size, function, and contribution to pathological states such as the metabolic syndrome. We isolated pre-adipocytes from different adipose depots, omental, subcutaneous and intramuscular, of beef cattle, and cultured in vitro to determine the basis for the variations and attribute these variations to the inherent properties of adipocyte progenitors. The proliferating cells from all depots before the confluence were harvested and the proteome was analyzed by a functional proteomic approach, involving 2-DE and MALDI-TOF/TOF. More than 252 protein spots were identified, selected and analyzed by Image Master (ver 7.0) and MALDI-TOF/TOF. Further, our analysis showed that there were specific differences in proteome expression patterns among proliferating precursor cells from the three depots. Sixteen proteins were found to be differentially expressed and these were identified as proteins involved in cellular processes, heat shock/chaperones, redox proteins, cytoskeletal proteins and metabolic enzymes. The results also enabled us to understand the basic roles of these proteins in different inherent properties exhibited by adipose tissue depots.
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