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J. Biol. Chem., Vol. 277, Issue 10, 8267-8272, March 8, 2002
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From the
Received for publication, August 29, 2001, and in revised form, October 28, 2001
Perilipin (Peri) A is a phosphoprotein
located at the surface of intracellular lipid droplets in adipocytes.
Activation of cyclic AMP-dependent protein kinase (PKA)
results in the phosphorylation of Peri A and hormone-sensitive lipase
(HSL), the predominant lipase in adipocytes, with concurrent
stimulation of adipocyte lipolysis. To investigate the relative
contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we
utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably
overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport
protein 1 (FATP1). When incubated with exogenous fatty acids,
ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as
compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of
Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited
lipolysis, whereas expression of HSL fused to green fluorescent protein
(GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin
treatment induced Peri A hyperphosphorylation and abrogated the
inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri
A Adipocytes are the major reservoir of energy stored in the form of
triacylglycerol (TG).1 TG is
stored within intracellular lipid droplets, from which it can be
rapidly mobilized. It is thought that the hydrolysis of stored TG in
adipocytes is regulated primarily by hormones such as catecholamines,
which activate cyclic AMP-dependent protein kinase (PKA).
PKA phosphorylates and activates hormone-sensitive lipase (HSL), the
major hormonally regulated lipase in adipocytes (1, 2). HSL hydrolyzes
TG at the surface of intracellular lipid droplets (3-5). The
perilipins, a family of phosphoproteins, are also specifically located
at the surface of the lipid droplet (6, 7). Perilipins are believed to
inhibit the actions of lipases on intracellular lipid, perhaps acting
as a barrier to lipase access (8-11). Upon catecholamine stimulation,
Peri A is hyperphosphorylated by PKA (7, 12), and this
hyperphosphorylation has been suggested to facilitate lipase access to
the lipid droplet (10, 13). Analysis of the predicted protein sequence
of Peri A, the most abundant perilipin isoform in adipocytes (7, 14) indicates the presence of six consensus PKA phosphorylation sites (RRXSX; RXSX) (15, 16).
To dissect the relative roles of Peri A, HSL, and PKA in regulating
intracellular lipid hydrolysis, we used ACS1/FATP1, a previously
established cell line of NIH 3T3 fibroblasts stably transfected with
long chain ACS1 and FATP1 (17). Both of these proteins have been shown
to increase long chain fatty acid (FA) import when expressed in
mammalian cells (17, 18). Although the exact mechanism of FA transport
remains the subject of active investigation, ACS1 and possibly FATP1
contribute to formation of acyl-CoA esters upon cellular FA import (19,
20). These esterified fatty acids can then be directed to diverse
metabolic fates such as TG synthesis. The present study demonstrates
that in the ACS1/FATP1 cells, the exogenous fatty acids are stored within lipid droplets as TG. This accumulation of TG makes the ACS1/FATP1 cells a useful model for elucidating the relative roles of
adenovirally expressed Peri A and HSL in constitutive and PKA-mediated lipolysis.
Materials--
[1-14C]oleic acids (50 mCi/mmol)
were purchased from Amersham Biosciences, Inc. NIH 3T3 cells were
obtained from the American Type Culture Collection (Manassas, VA).
Silica gel 60 plates were from Merck Research Laboratory. A specific
polyclonal anti-rabbit Peri A antibody (PREK antibody) was generated
using the peptide PREKPARRVSDSFFRPSVC. A polyclonal anti-HSL antibody
was generated using a peptide based upon the rat HSL sequence
KDLSFKGNSEPSDSPEM. Antibodies were subsequently affinity-purified and
used for Western blotting (1:1500). A polyclonal anti-adipocyte
differentiation-related protein (ADRP) antibody was generated using a
peptide based upon the mouse ADRP sequence ATEVNKASLKVQQSEVKAQ.
Antibodies were subsequently affinity-purified and used for Western
blotting (1:1000). Phosphoserine antibody (1:1000) was purchased from
Zymed Laboratories Inc. All other chemicals were
purchased from Sigma. Tissue culture media was purchased from Invitrogen.
Cell Culture and FA Loading--
The NIH 3T3 ACS1/FATP1 stable
cell line was generated as described previously (17). For FA uptake
assays, cells were seeded in 12-well plates, grown to confluence in
Dulbecco's modified Eagle's medium and 45 mM glucose
supplemented with 10% calf serum, and transduced with adenoviruses (as
described below), followed by incubation for 48 h with 0.4 µCi/ml [14C]oleic acid in Dulbecco's modified Eagle's
medium + 5 mM glucose containing 10% calf serum and 1%
fatty acid-free albumin bound to palmitic and oleic acid (120 µM of each). Incubation was terminated by discarding the
labeling media and washing the cells with phosphate-buffered saline.
Mutagenesis and Generation of Recombinant
Adenovirus--
Mutagenesis of putative PKA recognition sites was
carried out using Muta-Gene Phagemid in Vitro Mutagenesis
version 2 (Bio-Rad). Primers used for mutagenesis of serine to alanine
residue for the first three amino-terminal PKA recognition sites in
Peri A were as follows: serine 81, PKA1
(5'-GTCCGTCGGCTGGCCACCCAGTTCACAGC); serine 222, PKA2
(5'-TTTTGAGGAGGGTCGCCACCCTGGCCAACACTC); and serine 276, PKA3
(5'-CCCGGCGGCAGGCTGAGGTGC). The presence of the three mutations (Peri
A Lipid Analysis--
Cells were washed with cold
phosphate-buffered saline, and lipids were extracted by two 15-min
incubations with 250 µl of hexane:isopropanol (3:2, v/v) at 4 °C.
Solvent-containing lipid was dried at room temperature, and lipids were
dissolved in 50 µl of chloroform. [14C]Triacylglycerol
oleate was resolved by TLC using heptane:diethyl ether:glacial acetic
acid (90:30:1, v/v/v) and visualized by iodine (23).
14C-labeled TG spots were cut into vials and counted in a
liquid scintillation counter. TG is expressed in nmol/mg protein and represents the average of triplicate values.
Lipolysis--
NIH 3T3 ACS1/FATP1 cells were transduced with Ad
control or Ad GFPHSL as described above. The next day, cells were
transduced with Ad control or Ad Peri A, followed by incubation with
FAs. 48 h later, cells were washed with phosphate-buffered
saline and treated for 4 h in the absence or presence of 20 µM forskolin in Dulbecco's modified Eagle's medium + 5 mM glucose + 2% FA-free bovine serum albumin. Media were
collected, and glycerol was measured as an index of lipolysis. Glycerol
content of the incubation medium was determined using a colorimetric
assay (GPO-Trinder; Sigma).
Western Analysis--
After lipid extraction, proteins were
extracted as described previously (21) and quantified using the
BCA protein assay (Pierce). Total lysates (20 µg/sample) were
separated in 10% SDS-PAGE, transferred electrophoretically to
nitrocellulose membranes, and blotted as described previously (21).
Immunofluorescence--
For determination of Peri A
immunofluorescence, cells were cultured in 35-mm coverslip-bottomed
dishes (MatTek Corp., Ashland, MA) and infected as described above.
After treatment, cells were fixed in 2% paraformaldehyde for 10 min,
washed, and treated with anti-Peri A polyclonal antibody (PREK
antibody; 1:100 dilution) and a donkey anti-rabbit Cy-5-labeled
antibody (Jackson Immunoresearch). Neutral lipids were stained with
Nile red (1 µM) (Molecular Probes) (24). Cy-5
fluorescence imaging was assessed as described previously (25).
Statistical Analysis--
Data are expressed as the mean + S.E.
Data were analyzed by using three-way analysis of variance with the
presence or absence of forskolin, Ad GFPHSL, and Ad Peri A as study
factors. Treatment effects were considered statistically significant if
the attained significance levels (p values) were Fatty Acid Loading Increases TG Accumulation in NIH 3T3
ACS1/FATP1 Cells--
Previous studies demonstrated
that overexpression of both ACS1 and FATP1 in NIH 3T3 cells resulted in
a 25-fold increase in FA uptake (17). To determine the fate of the FAs
inside the cell, NIH 3T3 and NIH 3T3 ACS1/FATP1 cells were incubated
with 14C-labeled FAs for 48 h. Analysis of the neutral
lipids formed demonstrated that the vast majority of the incubated
oleate and palmitate was incorporated into TG (>95%; data not shown)
in both cell lines and that ACS1/FATP1 cells accumulated 5-fold more TG than parental NIH 3T3 cells (Fig. 1).
These results demonstrate that FA loading increased TG accumulation in
ACS1/FATP1 cells.
Fatty Acid Loading Increases ADRP and Adenovirally Expressed Peri
A--
To understand the mechanisms involved in TG accumulation, we
examined the endogenous expression of the perilipins and ADRP, a
ubiquitous lipid droplet marker. ADRP localizes to the surface of lipid
droplets and is thought to have a role in regulating lipid droplet
metabolism (26-29). Immunoblot analysis revealed that ACS1/FATP1 cells
expressed ADRP at low levels in the absence of FAs and at ~10-fold
greater levels when cells were incubated with FAs for 48 h (Fig.
2, lanes 1 and 2).
In contrast, ACS1/FATP1 cells did not express endogenous Peri A (Fig.
2, lanes 1 and 2) or Peri B (data not shown) in
the presence or absence of FAs. ACS1/FATP1 cells were therefore
transduced with Ad Peri A. Western blotting of protein lysates revealed
that in the absence of FA loading, Peri A expression was easily
detectable as compared with nontransduced cells (Fig. 2, lane 1 versus lane 3). An additional ~4-fold increase in the level of
Peri A expression was observed in cells loaded with FAs (Fig. 2,
lane 3 versus lane 4). Peri A expression was associated with
decreased expression of ADRP to levels comparable with those observed
in the absence of FAs. Although FA loading and Peri A expression
altered ADRP protein expression, Northern analyses showed that neither
incubation with FAs for 48 h nor Peri A expression altered ADRP
mRNA (data not shown). Our observation of decreased ADRP protein
levels with Peri A expression is the converse of increased ADRP
expression in adipocytes of mice with a targeted disruption of the
perilipin gene (11). The above observations demonstrate that ACS1/FATP1 cells are a suitable model to investigate the role of Peri A in lipolysis.
Adenovirus Expression of Peri A Increases TG Accumulation--
To
determine the effects of Peri A expression on lipid content, ACS1/FATP1
cells were transduced with Ad control or Ad Peri A. After infection,
cells were incubated in media with albumin-bound FAs for 48 h,
followed by 48 h without FAs. TLC analyses of cellular lipids
extracted from cells demonstrated a ~2-fold increase in TG
accumulation in cells expressing Peri A (p < 0.001) as
compared with control cells (Fig.
3A). Western blotting analyses
of cell lysates demonstrated that Peri A expression was present in Ad Peri A-transduced cells as compared with Ad control-transduced cells
(Fig. 3B). ADRP levels decreased (~70%) with expression of Peri A. Therefore, Peri A expression in ACS1/FATP1 cells results in
increased TG accumulation.
Co-localization of Peri A and Neutral Lipids in
ACS1/FATP1 Cells--
To determine the subcellular
localization of expressed Peri A, ACS1/FATP1 cells were transduced with
either Ad control or Ad Peri A and loaded with FAs for 48 h (Fig.
4). Simultaneous fluorescent detection of
Peri A and neutral lipids, using a specific Peri A antiserum
(green fluorescence) and Nile red (red
fluorescence) to stain neutral lipids, allowed us to correlate
immunoreactivity with intracellular structure. These studies
demonstrated that Peri A is located at the surface of the lipid
droplet. Cells expressing Peri A had more Nile red fluorescence located
in bigger and more numerous droplets than in control cells, consistent
with the increased TG demonstrated biochemically. The clusters of
droplets appeared to be distributed throughout the cell. In the
majority of cells, Peri A immunostaining around the droplet did not
appear to be evenly distributed in a ring-like fashion on the droplet
surface; in fact, Peri A immunoreactivity often appeared somewhat
patchy in its distribution. In summary, Peri A expression increased
lipid droplet size and the number of droplets.
Interactions between Peri A, HSL, and cAMP Activation to Regulate
Lipid Hydrolysis--
To further characterize the role of Peri A in
regulating lipid hydrolysis, ACS1/FATP1 cells were transduced with
either Ad control, Ad GFPHSL, Ad Peri A, or combinations of these
different adenoviruses (see "Experimental Procedures"). These cells
were then incubated with FAs for 48 h. The FAs were then removed
from the media, and the cells were incubated for an additional 4 h in the absence or presence of 20 µM forskolin. Glycerol
in the media was assayed to quantitate cellular lipolysis. Western
blotting analyses of cell lysates (Fig.
5A) using HSL antiserum
demonstrated that ACS1/FATP1 cells do not express endogenous HSL,
whereas cells transduced with Ad GFPHSL showed a band of 111 kDa,
consistent with the predicted size of the GFPHSL fusion protein (Fig.
5A, lanes 3, 4, 7, and
8). Likewise, using Peri-A antisera, endogenous Peri A
expression was not observed in ACS1/FATP1 cells, but Ad Peri
A-transduced cells expressed a 62-kDa band that increased to 65 kDa
with forskolin treatment (Fig. 5A, lanes 5 and 7 versus lanes 6 and 8). The
alterations in Peri A migration in SDS-PAGE have been previously
demonstrated to be due to hyperphosphorylation by PKA (7, 12).
Ad Peri A-transduced cells incubated with FAs accumulated ~45% more
TG than Ad control-transduced cells (p = 0.03, analysis of variance), consistent with the notion that Peri A can impede the
hydrolysis of TG by non-HSL lipases (Fig. 5B). Expression of
GFPHSL reduced TG accumulation by ~40% as compared with control cells (p = 0.03, analysis of variance). However, the
combined expression of Peri A and GFPHSL significantly increased TG
accumulation as compared with GFPHSL alone (~72%; p = 0.0002, analysis of variance). It is likely that Peri A expression
significantly blocks the effects of the unknown, endogenous lipase in
ACS1/FATP1 cells and the effects of HSL to reduce TG accumulation.
The extent of lipolysis in ACS1/FATP1 cells was determined by measuring
glycerol release after the removal of the FAs from the media. Infection
with Ad Peri A lowered basal (absence of forskolin) glycerol release by
38% as compared with cells infected with Ad control (p = 0.03) (Fig. 5C). Expression of Ad GFPHSL increased basal
lipolysis by 32% as compared with control cells (p = 0.03). The combination of Peri A and GFPHSL reduced the rate of
lipolysis by ~45% as compared with GFPHSL alone (p = 0.0002). We did not detect any significant effect of forskolin
treatment on glycerol release in Ad control- or Ad GFPHSL-transduced
cells. Forskolin-stimulated lipolysis was increased as compared with non-stimulated cells in ACS1/FATP1 cells expressing Peri A
(p < 0.0001). The forskolin-mediated increase in
lipolysis was more robust in cells expressing both Peri A and GFPHSL as
compared with cells expressing Peri A alone (242% versus
200%, respectively; p = 0.03). Essentially, forskolin
abrogated the ability of Peri A to block the lipolytic effects of the
endogenous lipase(s) and GFPHSL. In summary, expression of Peri A
significantly blocked lipolysis in ACSI/FATP1 cells, and PKA reversed
the actions of Peri A on lipolysis. In addition, a significant effect
of forskolin on lipolysis was observed in ACS1/FATP1 cells only when
Peri A was expressed.
Mutation of the Three Amino-terminal PKA Recognition Sites of Peri
A Decreases Forskolin-stimulated Lipolysis--
To further
characterize the role of Peri A in PKA-mediated lipolysis, we mutated
the amino-terminal PKA sites (16) (Fig. 6A; Ser-81, Ser-222, and
Ser-276) in Peri A from serines to alanines (Peri A Stimulation of adipocyte lipolysis by PKA is the critical pathway
by which the body increases fatty acids in physiologic states such as
fasting (31, 32). PKA phosphorylates HSL, resulting in modest
(~2-fold) increases in HSL activity (33). Perhaps more importantly,
phosphorylated HSL translocates to the surface of intracellular lipid
droplets (3-5), where perilipins reside (6, 7). Peri A is the major
PKA substrate in adipocytes (7, 12). Using adenovirally engineered
ACS1/FATP1 cells, we are able for the first time to directly assess the
individual contributions of Peri A, HSL, and PKA to modulation of TG
accumulation and breakdown. Our data demonstrate the importance
of Peri A in regulating constitutive and PKA-stimulated lipolysis in
ACS1/FATP1 cells. Moreover, our studies elucidate the critical role of
Peri A phosphorylation state in PKA-stimulated lipolysis.
In the ACS1/FATP1 cell model, Peri A expression increased TG
accumulation and reduced lipolysis. Our data in ACS1/FATP1 cells are
consistent with and extend the report by Brasaemle et al. (8) that expression of Peri A in 3T3-L1 preadipocytes increased TG
accumulation. Although they did not directly measure lipolysis, Brasaemle et al. (8) found that Peri A decreased the rate of TG turnover, suggesting that perilipins block the actions of lipases. Preadipocytes, like ACS1/FATP1 cells, do not express HSL, and in both
cell lines, the non-HSL lipase(s) that hydrolyzes TG is unknown.
Consistent with our results, other laboratories have suggested that a
neutral long chain lipase is present in fibroblasts and is not HSL
(34). The identity of the lipase in fibroblasts is not known at the
present time. We now find that expression of Peri A reduces the
lipolytic actions of both HSL and non-HSL lipases, resulting in reduced
glycerol release and increased TG accumulation. Importantly, when
GFPHSL and Peri A were co-expressed, Peri A expression did not reduce
GFPHSL protein expression, thus supporting the argument that the
effects of Peri A are mediated by its ability to reduce TG hydrolysis.
Expression of Peri A was necessary to demonstrate a statistically
significant lipolytic response to forskolin treatment in ACS1/FATP1
cells. PKA increased lipolysis in cells expressing Peri A because it
abrogated the inhibitory actions of Peri A on lipolysis. Specifically,
forskolin-stimulated hyperphosphorylation of Peri A reduced its ability
to block the lipolytic actions of both HSL and non-HSL lipases. In
contrast, forskolin did not significantly increase lipolysis in
ACS1/FATP1 cells expressing GFPHSL or untagged HSL (data not shown) in
the absence of Peri A. Consistent with this observation, PKA
stimulation did not increase lipolysis (glycerol/mg protein) in
adipocytes from a perilipin knockout mouse (9). Thus, in both our cell
model and adipocytes from Peri-null mice, perilipin was necessary to
demonstrate an effect of PKA on lipolysis.
Perilipins have been proposed to modulate lipolysis by two
distinct mechanisms: (i) regulation of perilipin protein expression (8-11, 21), and (ii) PKA-dependent hyperphosphorylation of
perilipins (4, 7, 10, 13). In support of the former, we demonstrated previously (10, 21, 35) that a reduction in perilipin protein expression in response to tumor necrosis factor It is unknown how perilipins modulate lipase hydrolysis of TG.
Perilipin localizes specifically to the surface of intracellular lipid
droplets (6-8, 10), an observation that suggests that perilipins block
(either directly or indirectly) lipase access. At the present time, it
is unclear whether the perilipins physically block lipases from
accessing the lipid droplet surface or alter the biophysical properties
of the phospholipid monolayer surrounding lipid droplets. PKA
phosphorylation of Peri A may facilitate lipase access to the lipid
droplet by altering Peri A conformation and/or by causing Peri A to
translocate off the lipid droplet. Studies have been presented that
support both of these hypotheses (4, 10, 13, 36). Future studies will
be aimed at understanding the interaction of Peri A and Peri A We thank Dr. Lina Moitoso de Vargas for
assistance in construction of adenoviruses. Mouse Peri A cDNA was a
generous gift of Drs. C. Londos, A. R. Kimmel, and J. Gruia-Gray.
We are grateful to Dr. C. Londos for suggesting that phosphorylation of
PKA consensus sites Ser-81, Ser-222 and Ser-276 facilitates
catecholamine-stimulated lipolysis.
*
Portions of this work were presented at the 60th
(2000) and 61st (2001) meetings of the American Diabetes
Association. This work was supported in part by the United States
Department of Agriculture, under agreement No. 581950-9-001 and DK
50647 (to A. S. G.) and DK 46942 (to F. B. K.), the Research
Service of the Department of Veterans Administration (to F. B. K.)
and research awards from the American Diabetes Association (to
A. S. G.). This work was supported in part by the Molecular Biology
Core of the Gastroenterology Research on Absorptive and Secretory
Processes, Grant P30 DK 34928.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.
¶¶
To whom correspondence should be addressed: JM-USDA
Human Nutrition Research on Aging at Tufts University, 711 Washington St., Boston, MA 02111. Tel.: 617-556-3144; Fax: 617-556-3224; E-mail:
agreenberg@hnrc.tufts.edu.
Published, JBC Papers in Press, December 20, 2001, DOI 10.1074/jbc.M108329200
The abbreviations used are:
TG, triacylglycerol;
PKA, cyclic AMP-dependent protein kinase;
HSL, hormone-sensitive lipase;
Peri, perilipin;
ACS1, acyl-CoA synthetase 1;
FATP1, fatty acid transport protein 1;
FA, fatty acid;
Ad, adenovirus;
GFPHSL, hormone-sensitive lipase tagged at its amino-terminal with
green fluorescent protein;
ADRP, adipocyte differentiation
related-protein.
Modulation of Hormone-sensitive Lipase and Protein Kinase
A-mediated Lipolysis by Perilipin A in an Adenoviral Reconstituted
System*
,,,,,
,,, and§§¶¶
Jean Meyer United States Department
of Agriculture Human Nutrition Research Center on Aging at Tufts
University, Boston, Massachusetts 02111, § Department of
Physiology, Tufts University School of Medicine, Boston, Massachusetts
02111, ¶ Departments of Medicine, Molecular Biology, and
Pharmacology, Washington University, St. Louis, Missouri 63110, Nessel Gene Therapy Center and Department of Medicine and
Molecular Biology, Massachusetts General Hospital, Boston,
Massachusetts 02114, ** Division of Endocrinology, Department
of Medicine, Stanford University, Stanford, California,
VA Palo Alto Health Care System, Palo Alto,
California 94305, and §§ Division of
Endocrinology, Metabolism, and Molecular Medicine, New England Medical
Center, Boston, Massachusetts 02111
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222,
and Ser-276) reduced Peri A hyperphosphorylation and blocked
constitutive and forskolin-stimulated lipolysis. Thus, perilipin
expression and phosphorylation state are critical regulators of
lipid storage and hydrolysis in ACS1/FATP1 cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3) was confirmed by sequencing, and the cDNA was used to
generate adenovirus Peri A3. Adenoviruses 
-galactosidase and Peri
A (Ad Peri A) were generated as described previously (21). Adenoviruses
expressing the Aequoria victoria green fluorescent protein
and GFPHSL were generated by the two-cosmid system (22). Briefly,
plasmid pEGFP-HSL was constructed by inserting a
HindIII-KpnI fragment containing rat HSL cDNA
into HindIII-KpnI opened pEGFPC1 (Ad GFPHSL)
(CLONTECH). An
EcoIII47-KpnI-blunted fragment containing the
sequence for GFPHSL was subcloned into SmaI-treated
pLEP-hCMV, a derivative of pLEP3 (22). Adenoviruses were amplified in
HEK 293 cells and purified and concentrated to 1012
plaque-forming units/ml by CsCl ultracentrifugation. Cells were transduced by incubation with LipofectAMINE Plus (Invitrogen) for
3 h at a multiplicity of infection of 1000 plaque-forming units/cell. Adenoviruses expressing either green fluorescent protein or
-galactosidase were used as controls (Ad control). All adenoviruses used contained a cytomegalovirus promoter to express the cloned cDNA.
0.05.
When there were no statistically significant interactions between
forskolin, Ad GFPHSL, and Ad PeriA, the overall (main) effects of each
treatment were examined. When interactions were detected, two
approaches were considered. First, the effect of one factor was
examined in the presence and absence of the other factors. Second,
different combinations of factors were regarded as distinct treatments
in a one-way analysis of variance, and differences between these
treatment means were evaluated post hoc by using the
Tukey's honestly significant differences procedure for multiple
comparisons. All analyses were performed by using SYSTAT version 9.01 (SPSS Inc., Chicago, IL).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FA loading increases accumulation of TG in
NIH 3T3 ACS1/FATP1 cells. NIH 3T3 and NIH 3T3 ACS1/FATP1 cells
were incubated for 48 h with 0.4 µCi of [14C]oleic
acid and 1% FA-free bovine serum albumin bound to palmitic and oleic
acid (120 µM of each). TG mass was determined by total
lipid extraction of cells followed by TLC analyses. Data are expressed
as the means + S.E. (n = 4 experiments performed in
triplicate).
View larger version (34K):
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Fig. 2.
Expression of Peri A and ADRP in ACS1/FATP1
cells. ACS1/FATP1 cells (lanes 1 and 2) and
ACS1/FATP1 cells transduced with Ad Peri A (lanes 3 and
4) were incubated in the presence (lanes 2 and
4) or absence (lanes 1 and 3) of
albumin-coupled palmitic and oleic acid (120 µM of each).
After 48 h, cell lysates were collected and immunoblotted using
antibodies against perilipins or ADRP. Data shown are representative of
four experiments.
View larger version (20K):
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Fig. 3.
Adenovirus expression of Peri A increases TG
accumulation. ACS1/FATP1 cells were transduced with Ad control or
Ad Peri A. After infection, cells were incubated with 0.4 µCi of
[14C]oleic acid and albumin-bound palmitic and oleic acid
(120 µM of each) for 48 h, followed by a 48-h
incubation in media without FAs. A, TG mass was determined
by total lipid extraction of cells followed by TLC analyses normalized
by protein levels. Data are expressed as the means + S.E.
(n = 7 experiments performed in triplicate). *,
p < 0.001. B, immunoblots of the cell
lysates described above, using antibodies against Peri A or ADRP. Data
shown are representative of seven experiments.
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Fig. 4.
Immunofluorescence of Ad Peri A-transduced
cells demonstrates the localization of Peri A to the lipid
droplet. ACS1/FATP1 cells transduced with Ad control or Ad
Peri A were incubated in media with albumin-bound FAs for 48 h.
Cells were fixed, neutral lipids were stained with Nile red
(red), and immunofluorescence analyses were performed using
a specific Peri A antibody (green fluorescence). Data are
representative of five experiments.
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Fig. 5.
Peri A interacts with HSL and forskolin to
regulate TG accumulation and lipolysis. ACS1/FATP1 cells were
transduced with Ad control, Ad GFPHSL, Ad Peri A, or Ad Peri A + Ad
GFPHSL followed by incubation with 0.4 µCi of
[14C]oleic acid and palmitic acid (120 µM
of each) for 48 h. A, immunoblots of cell lysates using
antibodies against HSL, Peri A, and ADRP. Data are representative of
four experiments. B, TG content of the ACS1/FATP1 cells was
determined by TLC as described under "Experimental Procedures."
Data are expressed as the mean + S.E. (n = 4 experiments performed in quadruplicate). *, Ad Peri A
versus Ad control, p = 0. 03; **, Ad
GFPHSL versus Ad control, p = 0.03;
***, Ad GFPHSL versus Ad Peri A + Ad GFPHSL,
p = 0.0002. C, lipolysis. Glycerol
accumulation in the media was assayed in the absence and presence of a
4-h forskolin treatment (n = 9-12 experiments, each
performed in triplicate). For details, see "Experimental
Procedures." In the absence of forskolin: *, p = 0.03, Ad Peri A versus Ad control and Ad Peri A + Ad GFPHSL
versus Ad control; **, p = 0.03, Ad
GFPHSL versus Ad control; and ***, p = 0.0002, Ad GFPHSL + Ad Peri A versus Ad GFPHSL. ,
p < 0.0001, forskolin-stimulated increase in lipolysis
versus nonstimulated cells.
3). These three
PKA sites are common to both Peri A and Peri B, the major forms of
perilipin expressed in adipocytes. Peri A3 was cloned into an
adenovirus and expressed in ACS1/FATP1 cells. Cells co-expressing
GFPHSL and Peri A or Peri A3 accumulated similar levels of TG in
response to FA loading (Peri A + GFPHSL, 6.2 + 1.3 nmol TG/mg protein;
Peri A3 + GFPHSL, 6.5 + 1.8 nmol TG/mg protein; n = 4 in triplicate). As observed earlier with Peri A, expression of Peri
A3 down-regulated ADRP protein expression, and confocal microscopy
demonstrated that the Peri A3 protein localized to the surface of
lipid droplets (data not shown). We next compared lipolysis in cells
expressing GFPHSL and Peri A versus the rate of lipolysis in
cells expressing GFPHSL and Peri A3. The rate of basal (without
forskolin) lipolysis was not significantly different in ACS1/FATP1
cells expressing GFPHSL and Peri A as compared with cells expressing
GFPHSL and Peri A3 (Fig. 6B). Again we observed that
forskolin significantly increased lipolysis, as compared with basal
lipolysis, in cells expressing both Peri A and GFPHSL (Fig.
6B; p < 0.0001). However, when we
co-expressed Peri A3 protein and GFPHSL in ACS1/FATP1 cells,
forskolin treatment did not significantly increase lipolysis as
compared with basal lipolysis. Previously, it was hypothesized that
phosphorylation of the PKA consensus sites in carboxyl-terminal Peri A
(Ser-433, Ser-494, and Ser-517) (Fig. 6A) caused Peri A to
migrate slower in SDS-PAGE as a 65-kDa protein (15). Both Peri A and
Peri A3 migrated to 65 kDa with forskolin treatment, consistent with
activation of PKA-stimulated lipolysis and the fact that that the
carboxyl-terminal PKA sites were not mutated in either protein (Fig.
6C). We next examined the effects of PKA activation on
phosphoserine content in Peri A and Peri A3 when co-expressed
with GFPHSL in ACS1/FATP1 cells. Experiments demonstrated that in the
absence of Peri A expression, we could not detect any significant
amount of phosphoserine immunoreactivity in the 60-65-kDa regions of
ACS1/FATP1 cells (data not shown). When Peri A was expressed and cells
were treated with forskolin, phosphoserine immunoreactivity was
significantly increased at 65 kDa, similar to prior observations in
adipocytes (30) (Fig. 6C). In contrast with forskolin
treatment, the phosphoserine content of the Peri A3 protein was
significantly reduced as compared with that of Peri A. The reduction in
phosphoserine content was consistent with substitution of alanine for
serine in the three amino-terminal PKA sites in Peri A3 (Fig.
6C). The slight increase in Peri A3 phosphoserine
content, as compared with untreated cells, corresponds to
phosphorylation of the remaining three PKA recognition sites. In this
study, we were able to demonstrate that PKA phosphorylation of Peri A
regulates the protein's actions to modulate lipolysis in response to
PKA activation.
View larger version (13K):
[in a new window]
Fig. 6.
PKA-mediated lipolysis in ACS1/FATP1 cells
expressing Peri A, Peri A 3, and GFPHSL. A,
schematic representation of consensus PKA phosphorylation sites in Peri
A protein. Gray box represents 405 amino acids common to
both perilipin A and B proteins. Arrows indicate the
location of serine residues for the six PKA recognition motifs.
B and C, ACS1/FATP1 cells were transduced with
either Ad Peri A and Ad GFPHSL or Ad Peri A3 and Ad GFPHSL. Cells
were loaded with FAs for 48 h, FAs were removed, and cells were
incubated in the absence or presence of forksolin for 4 h
(n = 4). B, lipolysis was assayed by
determining glycerol release in the media over a 4-h period in the
absence and presence of forskolin. Asterisk indicates
significantly different as compared with all other conditions,
p < 0.0001. C, top panel,
immunoblot using phosphoserine antisera; bottom panel,
immunoblot using antisera against Peri A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
increased adipocyte lipolysis. Furthermore, maintenance of Peri A or Peri B protein levels
with adenovirus overexpression blocked tumor necrosis factor -stimulated lipolysis (10). However, overexpression of either perilipin isoform did not block PKA-stimulated lipolysis in these studies. We now demonstrate the role of PKA-mediated
hyperphosphorylation of perilipin in regulating lipolysis. In this
study, we mutated the three consensus PKA sites in Peri A that are
common to both Peri A and Peri B (Fig. 6; Ser-81, Ser-222, and Ser-276)
(15). When the mutant protein (Peri A3) was expressed in ACS1/FATP1 cells, like Peri A, it targeted itself to the surface of
intracellular lipid droplets, increased TG accumulation, and decreased
basal lipolysis. However, unlike Peri A, Peri A3 blocked
PKA-stimulated lipolysis in ACS1/FATP1 cells expressing GFPHSL. Our
observations with Peri A3 demonstrate that PKA-stimulated
phosphorylation of Peri A is necessary to abrogate Peri A's inhibitory
actions on lipolysis. Because Peri A and Peri B share the same PKA
phosphorylation sites that were mutated in this study, we speculate
that Peri B may also modulate PKA-stimulated lipolysis.
3 with
the lipid droplet surface and HSL.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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D. L. Brasaemle, G. Dolios, L. Shapiro, and R. Wang Proteomic Analysis of Proteins Associated with Lipid Droplets of Basal and Lipolytically Stimulated 3T3-L1 Adipocytes J. Biol. Chem., November 5, 2004; 279(45): 46835 - 46842. [Abstract] [Full Text] [PDF]
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V. Subramanian, A. Garcia, A. Sekowski, and D. L. Brasaemle Hydrophobic sequences target and anchor perilipin A to lipid droplets J. Lipid Res., November 1, 2004; 45(11): 1983 - 1991. [Abstract] [Full Text] [PDF]
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V. Subramanian, A. Rothenberg, C. Gomez, A. W. Cohen, A. Garcia, S. Bhattacharyya, L. Shapiro, G. Dolios, R. Wang, M. P. Lisanti, et al. Perilipin A Mediates the Reversible Binding of CGI-58 to Lipid Droplets in 3T3-L1 Adipocytes J. Biol. Chem., October 1, 2004; 279(40): 42062 - 42071. [Abstract] [Full Text] [PDF]
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P. K. Saha, H. Kojima, J. Martinez-Botas, A. L. Sunehag, and L. Chan Metabolic Adaptations in the Absence of Perilipin: INCREASED {beta}-OXIDATION AND DECREASED HEPATIC GLUCOSE PRODUCTION ASSOCIATED WITH PERIPHERAL INSULIN RESISTANCE BUT NORMAL GLUCOSE TOLERANCE IN PERILIPIN-NULL MICE J. Biol. Chem., August 20, 2004; 279(34): 35150 - 35158. [Abstract] [Full Text] [PDF]
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T. Yamaguchi, N. Omatsu, S. Matsushita, and T. Osumi CGI-58 Interacts with Perilipin and Is Localized to Lipid Droplets: POSSIBLE INVOLVEMENT OF CGI-58 MISLOCALIZATION IN CHANARIN-DORFMAN SYNDROME J. Biol. Chem., July 16, 2004; 279(29): 30490 - 30497. [Abstract] [Full Text] [PDF]
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A. W. Cohen, B. Razani, W. Schubert, T. M. Williams, X. B. Wang, P. Iyengar, D. L. Brasaemle, P. E. Scherer, and M. P. Lisanti Role of Caveolin-1 in the Modulation of Lipolysis and Lipid Droplet Formation Diabetes, May 1, 2004; 53(5): 1261 - 1270. [Abstract] [Full Text] [PDF]
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N. Arimura, T. Horiba, M. Imagawa, M. Shimizu, and R. Sato The Peroxisome Proliferator-activated Receptor {gamma} Regulates Expression of the Perilipin Gene in Adipocytes J. Biol. Chem., March 12, 2004; 279(11): 10070 - 10076. [Abstract] [Full Text] [PDF]
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 P. A. Kern, G. Di Gregorio, T. Lu, N. Rassouli, and G. Ranganathan Perilipin Expression in Human Adipose Tissue Is Elevated with Obesity J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1352 - 1358. [Abstract] [Full Text] [PDF]
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A. Garcia, V. Subramanian, A. Sekowski, S. Bhattacharyya, M. W. Love, and D. L. Brasaemle The Amino and Carboxyl Termini of Perilipin A Facilitate the Storage of Triacylglycerols J. Biol. Chem., February 27, 2004; 279(9): 8409 - 8416. [Abstract] [Full Text] [PDF]
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M. J. Watt, P. Krustrup, N. H. Secher, B. Saltin, B. K. Pedersen, and M. A. Febbraio Glucose ingestion blunts hormone-sensitive lipase activity in contracting human skeletal muscle Am J Physiol Endocrinol Metab, January 1, 2004; 286(1): E144 - E150. [Abstract] [Full Text]
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H. H. Zhang, S. C. Souza, K. V. Muliro, F. B. Kraemer, M. S. Obin, and A. S. Greenberg Lipase-selective Functional Domains of Perilipin A Differentially Regulate Constitutive and Protein Kinase A-stimulated Lipolysis J. Biol. Chem., December 19, 2003; 278(51): 51535 - 51542. [Abstract] [Full Text] [PDF]
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A. W. Cohen, T. P. Combs, P. E. Scherer, and M. P. Lisanti Role of caveolin and caveolae in insulin signaling and diabetes Am J Physiol Endocrinol Metab, December 1, 2003; 285(6): E1151 - E1160. [Abstract] [Full Text] [PDF]
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N. E. Wolins, J. R. Skinner, M. J. Schoenfish, A. Tzekov, K. G. Bensch, and P. E. Bickel Adipocyte Protein S3-12 Coats Nascent Lipid Droplets J. Biol. Chem., September 26, 2003; 278(39): 37713 - 37721. [Abstract] [Full Text] [PDF]
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 C. Sztalryd, G. Xu, H. Dorward, J. T. Tansey, J. A. Contreras, A. R. Kimmel, and C. Londos Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation J. Cell Biol., June 23, 2003; 161(6): 1093 - 1103. [Abstract] [Full Text] [PDF]
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 D. DiDonato and D. L. Brasaemle Fixation Methods for the Study of Lipid Droplets by Immunofluorescence Microscopy J. Histochem. Cytochem., June 1, 2003; 51(6): 773 - 780. [Abstract] [Full Text] [PDF]
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M. L. Misso, Y. Murata, W. C. Boon, M. E. E. Jones, K. L. Britt, and E. R. Simpson Cellular and Molecular Characterization of the Adipose Phenotype of the Aromatase-Deficient Mouse Endocrinology, April 1, 2003; 144(4): 1474 - 1480. [Abstract] [Full Text] [PDF]
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J. T. Tansey, A. M. Huml, R. Vogt, K. E. Davis, J. M. Jones, K. A. Fraser, D. L. Brasaemle, A. R. Kimmel, and C. Londos Functional Studies on Native and Mutated Forms of Perilipins. A ROLE IN PROTEIN KINASE A-MEDIATED LIPOLYSIS OF TRIACYLGLYCEROLS IN CHINESE HAMSTER OVARY CELLS J. Biol. Chem., February 28, 2003; 278(10): 8401 - 8406. [Abstract] [Full Text] [PDF]
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 M. J Watt, G. J F Heigenhauser, and L. L Spriet Effects of dynamic exercise intensity on the activation of hormone-sensitive lipase in human skeletal muscle J. Physiol., February 15, 2003; 547(1): 301 - 308. [Abstract] [Full Text] [PDF]
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A. Garcia, A. Sekowski, V. Subramanian, and D. L. Brasaemle The Central Domain Is Required to Target and Anchor Perilipin A to Lipid Droplets J. Biol. Chem., January 3, 2003; 278(1): 625 - 635. [Abstract] [Full Text] [PDF]
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S. Lucas, G. Tavernier, C. Tiraby, A. Mairal, and D. Langin Expression of human hormone-sensitive lipase in white adipose tissue of transgenic mice increases lipase activity but does not enhance in vitro lipolysis J. Lipid Res., January 1, 2003; 44(1): 154 - 163. [Abstract] [Full Text] [PDF]
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H. H. Zhang, M. Halbleib, F. Ahmad, V. C. Manganiello, and A. S. Greenberg Tumor Necrosis Factor-{alpha} Stimulates Lipolysis in Differentiated Human Adipocytes Through Activation of Extracellular Signal-Related Kinase and Elevation of Intracellular cAMP Diabetes, October 1, 2002; 51(10): 2929 - 2935. [Abstract] [Full Text] [PDF]
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G. M. Hatch, A. J. Smith, F. Y. Xu, A. M. Hall, and D. A. Bernlohr FATP1 channels exogenous FA into 1,2,3-triacyl-sn-glycerol and down-regulates sphingomyelin and cholesterol metabolism in growing 293 cells J. Lipid Res., September 1, 2002; 43(9): 1380 - 1389. [Abstract] [Full Text] [PDF]
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S. Miura, J.-W. Gan, J. Brzostowski, M. J. Parisi, C. J. Schultz, C. Londos, B. Oliver, and A. R. Kimmel Functional Conservation for Lipid Storage Droplet Association among Perilipin, ADRP, and TIP47 (PAT)-related Proteins in Mammals, Drosophila, and Dictyostelium J. Biol. Chem., August 23, 2002; 277(35): 32253 - 32257. [Abstract] [Full Text] [PDF]
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