1 Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582; and 2 Division of Clinical Immunology, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0919, Japan
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ABSTRACT |
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Adipose differentiation-related protein
(ADRP) is a lipid droplet-associated protein that is expressed early
during adipose differentiation. The present study was undertaken to
reveal the role of ADRP in adipose differentiation. In murine
fibroblasts infected with green fluorescent protein (GFP)-ADRP fusion
protein expression adenovirus vector, confocal microscopic analysis
showed the number and size of lipid droplets apparently increased
comparing with those of control cells. Overexpressed GFP-ADRP were
mainly located at the surface of lipid droplets and appeared to be
"ring-shaped." Triacylglycerol content was also significantly
(P < 0.001) increased in GFP-ADRP-overexpressed cells
compared with control cells. ADRP-induced lipid accumulation did not
depend on adipocyte-specific gene induction, such as peroxisome
proliferator-activated receptor-, lipoprotein lipase, or other
lipogenic genes, including acyl-CoA synthetase, fatty acid-binding
protein, and fatty acid transporter. In conclusion, ADRP stimulated
lipid accumulation and lipid droplet formation without induction of
other adipocyte-specific genes or other lipogenic genes in murine
fibroblasts. The detailed molecular mechanisms of ADRP on lipid
accumulation remain to be elucidated.
adipose differentiation-related protein; triacylglycerol; adipocyte
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INTRODUCTION |
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OBESITY, AN EXCESSIVE accumulation of adipose tissue, is associated with the development of serious medical conditions, including type 2 diabetes mellitus, coronary artery disease, and hyperlipidemia via an insulin resistance state (13, 14, 21). Therefore, an understanding about the detailed mechanisms of adipogenesis is essential for the development of rational treatments for these disorders.
Adipogenesis is a complex process controlled by the interplay of
intracellular factors and signals from the environment. During this
differentiation, a large number of genes have to be regulated in a
selective, coordinated manner, and dramatic changes occur in both cell
morphology and gene expression (17, 20). Peroxisome proliferation-activated receptor- (PPAR
) and CCAAT/enhancer binding protein-
(C/EBP
) are prominent adipogenic transcription factors, and they are induced in the early stage of adipose
differentiation (5, 19, 24). PPAR
and C/EBP
alone or
in cooperation with each other induce the transcription of many
adipocyte-specific genes encoding proteins and enzymes involved in
creating and maintaining the adipose phenotype, such as lipogenesis,
lipolysis, glucose metabolism, and endocrine functions
(9).
Adipose differentiation-related protein (ADRP) was first identified by Jiang et al. (11, 12); they demonstrated that ADRP mRNA is expressed most strongly in adipose tissue and is inducted very early during adipose differentiation in murine 1246 cells. In 3T3-L1 cells, ADRP protein levels increased by day 1 of differentiation and then decreased by day 4 (3). ADRP protein localizes at the surface of lipid droplets in cultured 3T3-L1 preadipocytes and early differentiated adipocytes (3). Although these findings suggest that ADRP may play a certain role in the early stage of adipose differentiation, little is known about its function in this stage.
In the present study, we examined the function of ADRP in murine fibroblast cell lines (3T3-L1, NIH-3T3, and Swiss-3T3) by GFP-ADRP fusion protein overexpression. These may provide the direct evidence about the roles of ADRP during adipose differentiation.
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RESEARCH DESIGN AND METHODS |
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Plasmids. A plasmid containing the mouse ADRP was constructed as follows. Based on a sequence from GenBank (M93275), the open reading frame of mouse ADRP was amplified from C57BL/6J mouse adipose tissue cDNA. Primers for ADRP were 5'-CCCAAGCTTGTTAGGCGTCTCTTTTCTCCA-3' and 5'-TGCTCTAGACTGGTGACAAGGAGGGGTTTA-3', including Hind III and Xba I restriction sites, respectively. The ADRP gene was restricted by Hind III and Xba I and then inserted in the multicloning site of pBluescript SK (TOYOBO, Osaka, Japan).
Construction of green fluorescent protein-ADRP fusion protein expression adenovirus vector. Recombinant adenovirus vector derived from the human type 5 adenovirus was used for this study (Takara, Osaka, Japan). ADRP cDNA was restricted by Hind III and Sac II and inserted in the multicloning site of pEGFP-C2 (Clontech, Palo Alto, CA). pEGFP-ADRP was linearized with Nhe I and Xba I, blunt-ended, and then inserted in the Swa I site of the recombinant cosmid vector pAxCAwt (Takara). The recombinant green fluorescent protein (GFP)-ADRP adenovirus (Ad.GFP-ADRP) was obtained as described in a previous report (18).
Cell culture and adenovirus treatment.
NIH-3T3 cells (American Type Culture Collection, Manassas, VA) and
Swiss-3T3 cells (Health Science Research Resources Bank, Osaka, Japan)
were maintained in Dulbecco's modified Eagle medium (DMEM) containing
10% calf serum. The cells were infected by adenovirus vector with a
multiplicity of infection of ~30 plaque-forming units/cell. For most
of the assays, recombinant adenovirus expressing -galactosidase (Ad.LacZ) was used as a control.
3T3-L1 preadipocytes (kindly provided by Dr. J. M. Olefsky,
California University, San Diego, CA) were grown in DMEM containing
10% FCS. Differentiation of 3T3-L1 cells into adipocytes was
accomplished by incubating confluent monolayers of cells in DMEM with
10% FCS and 1µM dexamethasone (Sigma), 0.5 mM isobutyl
methylxanthine (IBMX; Sigma), and 10µg/ml insulin (Sigma) for 72 h. After 72 h, the medium was withdrawn and changed to DMEM with
10% FCS and 5µg/ml insulin. The medium was changed every 2 days.
Immunofluorescence microscopy. For determination of GFP-ADRP fluorescence, cells were cultured on 35-mm coverslip-bottomed dishes (Magtek, Ashland, MA) and infected adenovirus vector as described above. Fluorescence imaging of GFP-ADRP was assessed by confocal laser scanning microscopy (Leica TCS-SP system; Leica Microsystems, Heidelberg, Germany). The cells were imaged for GFP by excitation with the 488-nm line from an argon laser, and the emission was viewed through a 496- to 505-nm band-pass filter. To correlate the localization of GFP-ADRP with intracellular structure, cells were viewed with phase-contrast images and fluorescence images.
Oil red O staining. Cells were cultured in 10-cm dishes or 35-mm coverslip-bottomed dishes with adenovirus vector as described above. After 14 days, the cells cultured in 10-cm dishes were washed three times with PBS and then fixed by soaking in 10% formalin. After being washed two times with PBS, cells were stained for 30 min at 37°C in fleshly diluted Oil red O (Chroma, Mueuster, Germany) solution (six parts Oil red O stock and four parts H2O; Oil red O stock solution is 0.5% Oil red O in isopropanol). The stain was then removed, and the cells were washed two times with water. Nuclei were then stained with hematoxylin (Nichirei, Tokyo, Japan), and the stained cells were examined under a light microscope (Nikon, Tokyo, Japan). As for dual staining with Oil red O and GFP-ADRP, 5 days after adenovirus infection, the cells cultured in 35-mm coverslip-bottomed dishes were washed three times with PBS and then fixed by soaking in 4% paraformaldehyde for 10 min. After being washed two times with PBS, cells were stained for 2 min at room temperature in fleshly diluted Oil red O solution. Fluorescence imaging of GFP-ADRP and Oil red O was assessed by confocal laser scanning microscopy. Imaging of Oil red O was provided by excitation with the 563- and 633-nm line, and the emission was viewed through a 650- to 700-nm band-pass filter.
Lipid analysis. For determination of intracellular triacylglycerol content, cells were cultured on 10-cm dishes. After adenovirus vector infection and incubation in 10% FCS or 10% delipidated calf serum (Sigma, St. Louis, MO) for 7 days, cells were washed by PBS, and total lipids were extracted with 2:1 chloroform-ethanol and centrifuged for 5 min at 12,000 g. The chloroform layer, containing triacylglycerol, was extracted and placed under vacuum to evaporate chloroform and methanol. Triacylglycerol content was measured using an enzymatic method (Mizuho Medy, Saga, Japan) and was expressed relative to total cellular protein content measured by the bicinchoninic acid method (Pierce, Rockford, IL).
Northern blot analysis.
A plasmid-encoding mouse PPAR was kindly provided by Dr.
Kazuhiko Umesono (Kyoto University, Kyoto, Japan). cDNA probes for murine lipoprotein lipase (LPL), adipose fatty acid-binding protein (aP2/FABP), acyl-CoA synthase (ACS), and CD36/fatty acid transporter (FAT) were prepared by RT-PCR by use of differentiated 3T3-L1 adipocyte
cDNA. cDNA probe for human perilipin was prepared by RT-PCR by use of
human mesenteric adipose tissues, which were obtained from a resected
organ from a 58-yr-old female patient who had undergone surgery for
ovarian cancer (with informed consent for use of tissues).
Immunostaining of perilipin. 3T3-L1 cells were cultured on 35-mm coverslip-bottomed dishes and infected with Ad.GFP-ADRP. After incubation for 15 days, cells were fixed in 10% formalin for 10 min at room temperature, washed with PBS, permeabilized by 0.2% Triton X-100 (Katayama Chemistry, Osaka, Japan) in PBS for 2 min, and washed with PBS. Anti-perilipin polyclonal antibody (Research Diagnostics, Flanders, NJ) was diluted 1:400 in PBS with 1% dried bovine milk and incubated for 1 h at room temperature. Alexa Fluor 594, fluorescein-conjugated secondary antibody (Molecular Probes, Eugene, OR), was diluted 1:200 in PBS with 1% dried bovine milk and incubated for 30 min at room temperature. The cells were imaged for Alexa Fluor 594 by excitation with the 633-nm line from an argon laser, and the emission was viewed through a 650- to 700-nm band-pass filter.
Statistical analysis. Statistical analysis was done by ANOVA followed by Student's t-test.
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RESULTS |
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Effect of overexpression of ADRP in murine fibroblast cell lines.
Two murine fibroblast cell lines, NIH-3T3 cells and Swiss-3T3 cells,
which don't differentiate into adipocytes, were infected with control
adenovirus (Ad.LacZ) or Ad.GFP-ADRP with a multiplicity of
infection of ~30 plaque-forming units/cell and then incubated. Cells
were viewed with a confocal laser-scanning microscope. GFP images and
phase-contrast images allowed us to correlate the localization of
adenovirus-induced GFP-ADRP protein with an intracellular structure. In
NIH-3T3 cells, overexpressed GFP-ADRP protein localized around tiny
lipid droplets and in the cytosol at day 2 (Fig.
1, A and D). As
shown in Fig. 1, B and C, E, and
F, the number and size of lipid droplets apparently
increased in GFP-ADRP-overexpressed cells. The fluorescence of GFP-ADRP
increased around the lipid droplets and decreased in the cytosol at
day 7. In Swiss-3T3 cells, overexpressed GFP-ADRP stimulated
lipid accumulation more remarkably than in NIH-3T3 cells (Fig.
2). A lot of tiny lipid droplets
surrounded by GFP-ADRP appeared at day 2 (Fig. 2,
A and D), and the droplets became larger at
day 7 (Fig. 2, B and E) and at
day 14 (Fig. 2, C and F). The
fluorescence of GFP-ADRP appeared to be "ring-shaped." Analysis by
Oil red O staining confirmed that overexpressing ADRP stimulated lipid
accumulation in both NIH-3T3 cells and Swiss-3T3 cells (Fig.
3). Dual-staining images clearly showed
that the lipid droplets stained with Oil red O were surrounded by
GFP-ADRP (Fig. 4, A-D). In
addition, the cells contained increased levels of lipid droplets in
parallel with the overexpression level of GFP-ADRP (Fig. 4,
I-L, yellow arrow). In contrast, the GFP-undetectable cells
(Fig. 4, I-L, blue arrow) had few amounts of lipid droplets at the similar levels as Ad.LacZ-infected control cells (Fig. 4,
E-H). Intracellular triacylglycerol content in
GFP-ADRP-overexpressed Swiss-3T3 cells was significantly increased by
225% (P < 0.001) compared with that of
Ad.LacZ-infected cells when the cells were cultured in 10% normal
serum (Fig. 5). Even if the cells were cultured in delipidated serum, the intracellular triacylglycerol content in GFP-ADRP-overexpressed cells was still significantly (P < 0.005) increased compared with that in
Ad.LacZ-infected cells to a similar degree as in normal serum (Fig. 5).
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Effect of ADRP overexpression on adipocyte-specific gene
expression.
To investigate the mechanism of ADRP-induced lipid accumulation,
we analyzed PPAR, which is an early marker and regulator of adipose
differentiation, and other adipocyte-specific gene expression in
ADRP-overexpressed Swiss-3T3 cells. Swiss-3T3 cells infected with
Ad.GFP-ADRP or Ad.LacZ were incubated with induction medium containing
insulin, IBMX, and dexamethasone, as described in RESEARCH DESIGN
AND METHODS. Figure 6 shows that
ADRP overexpression did not induce PPAR
, LPL, or perilipin mRNA
expression at days 4 or 8.
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Correlation of ADRP and other adipocyte-specific gene expression
during 3T3-L1 cell differentiation.
To correlate the temporal pattern of expression of ADRP mRNA and other
adipocyte-specific gene expression relative to each other, we analyzed
gene expression by Northern blot hybridization during the conversion of
3T3-L1 preadipocytes to adipocytes (Fig. 8). ADRP mRNA was present at low levels
in 3T3-L1 cells before the initiation of differentiation and increased
at day 3 of differentiation. The induction of PPAR was
activated at day 2 before that of ADRP. Expressions of other
adipocyte-specific genes, LPL and aP2, were induced at day
2. Perilipin mRNA was absent until day 2, and its induction was activated at day 3.
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GFP-ADRP localizes small lipid droplets in differentiating 3T3-L1
cells.
In 3T3-L1 cells, overexpressed GFP-ADRP localized at the surface of
lipid droplets and stimulated lipid accumulation similar to Swiss-3T3
and NIH-3T3 cells. At day 15 after infection, some of the
cells had started differentiation and had several larger droplets (Fig.
9). Ad.LacZ-infected cells also had large
droplets in the same degree as Ad.GFP-ADRP-infected cells (data not
shown). Immunostaining using anti-perilipin antibody showed that the
larger droplets were surrounded by perilipin instead of GFP-ADRP (Fig. 9, C and G). No staining was seen using nonimmune
serum, confirming that perilipin staining is specific. In contrast,
tiny or middle-sized droplets were still surrounded by GFP-ADRP (Fig.
9, B and F). In certain cells, GFP-ADRP and
perilipin colocalized (Fig. 9, J-L).
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DISCUSSION |
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ADRP is a lipid droplet-associated protein, and its expression increases in the early stage of adipose differentiation (3, 11). Previous reports demonstrated that ADRP was expressed in a wide range of lipid-accumulated cells and the ADRP was a marker of lipid accumulation (10). Although the localization and expression pattern of ADRP has been well examined, little is known about its function in murine preadipocyte cells or other lipid-accumulated cells. Whether increased ADRP promotes lipid accumulation or if the increased ADRP merely reflects the increased storage of lipid remained to be elucidated. Therefore, we analyzed the function of ADRP in murine fibroblasts by GFP-ADRP fusion protein overexpression. In GFP- ADRP-overexpressed cells, the number and size of lipid droplets apparently increased, and overexpressed GFP-ADRP protein localized around lipid droplets. Analysis by Oil red O staining and measurement of intracellular triacylglycerol content confirmed that overexpressing ADRP stimulated lipid accumulation in both NIH-3T3 cells and Swiss-3T3 cells. Furthermore, dual-staining images clearly showed that the cells contained increased levels of lipid droplets, in parallel with the overexpression level of GFP-ADRP. Taken together, these results suggested that ADRP stimulated lipid accumulation and lipid droplet formation in murine fibroblasts.
To determine the mechanism of ADRP-induced lipid accumulation, the
effect of ADRP overexpression on adipocyte-specific gene expression,
such as PPAR, LPL, or perilipin, was examined. ADRP overexpression
could not induce adipocyte-specific gene expression, suggesting that
ADRP-induced lipid accumulation is not the result of activation of the
adipose differentiation program. This notion is consistent with the
evidence that ADRP is expressed in a wide range of cells and tissues
(3, 10). Next, the expression of three lipogenic genes
(ACS, aP2/FABP, and CD36/FAT) was also analyzed in ADRP-overexpressed
cells. ADRP overexpression did not induce these lipogenic genes,
showing that ADRP-induced lipid accumulation was independent of other
lipogenic genes, such as ACS, aP2/FABP, and CD36/FAT.
One possible mechanism is that the free fatty acid-transporting function of ADRP may be associated with lipid accumulation. Several investigators have mentioned ADRP as a free fatty acid transporter (1, 6, 22). ADRP specifically enhances uptake of long-chain fatty acids by increasing the initial rate of uptake in COS-7 cells (6). A recent report demonstrated that recombinant ADRP protein bound 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate at high affinity, and its binding was completed by natural fatty acid (22). ADRP binding fatty acid with high affinity may contribute to the rapid appearance of fatty acids at the surface of lipid droplets, and subsequently the fatty acids may be used for triacylglycerol synthesis (22). One report showed that ADRP protein translocated to the cell periphery in murine 1246 cells during the differentiating process (6), suggesting that ADRP function as a protein involved in carrier-mediated fatty acid influx from the extracellular environment into the cells. In contrast, in the present study, overexpressed GFP-ADRP protein localized around tiny lipid droplets and in cytosol at day 2, and then it increased around the lipid droplets, not in the cell periphery, and decreased in the cytosol at day 7. These results suggested that ADRP might transport endogenous free fatty acid from the cytosol to the surface of lipid droplets. This notion was supported by the present results showing that ADRP overexpression induced an increase in intracellular triacylglycerol content even if the cells were cultured in delipidated serum to a similar degree as in nondelipidated serum. Thus, in murine fibroblasts, ADRP may stimulate free fatty acid accumulation by shuttling of long-chain free fatty acids, mainly from the cytosol to the surface of lipid droplets to supply the source of triacylglycerol synthesis.
Similar to ADRP, perilipin localizes at the surface of lipid droplets (2, 8), and perilipin also stimulates lipid accumulation (4, 23). A previous report showed that the transition in the surface protein composition of lipid droplets from ADRP to perilipin occurs from 3 to 5 days after the initiation of differentiation (3). In our observation of 3T3-L1 cell differentiation, overexpressed GFP-ADRP appeared to localize at the surface of only small lipid droplets, whereas in turn endogenous perilipin located around large-sized lipid droplets. In the early stage of lipid accumulation, ADRP may play an important role.
The molecular mechanisms for regulation of ADRP expression have not
been well understood. ADRP mRNA expression is stimulated by either
indomethacin (25) or long-chain free fatty acids
(7), and both of them are found to bind directly to
PPAR and to act as a PPAR
agonist (15, 16, 24), so
it is possible to assume that their effect on ADRP expression could be
secondary to the prior activation of PPAR
. This notion was in
agreement with the present finding that ADRP mRNA expression was
induced ~24 h later than that of PPAR
mRNA. Promoter
activity analysis of ADRP would give us further information about the
regulation of ADRP induction. At the ADRP-to-perilipin switch, the ADRP
protein level was reported to be decreased, whereas the ADRP mRNA level
still increased, suggesting that ADRP protein levels would also be
regulated by a posttranscriptional mechanism (3).
In conclusion, ADRP overexpression stimulated lipid accumulation and lipid droplet formation without induction of other adipocyte-specific genes or other lipogenic genes in murine fibroblast cells. The detailed molecular mechanisms of ADRP on lipid accumulation remain to be elucidated.
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ACKNOWLEDGEMENTS |
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This work was supported in part by Grant-in-Aid for Scientific Research no. 11671126 from the Ministry of Education, Science, and Culture, Japan.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Inoguchi, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan (E-mail: toyoshi{at}intmed3.med.kyushu-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 4, 2002;10.1152/ajpendo.00040.2002
Received 31 January 2002; accepted in final form 23 May 2002.
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