THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 32, pp.

23307–23321, August 9, 2013

© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Anandamide-derived Prostamide F2␣ Negatively Regulates

Adipogenesis□ S

Received for publication, May 31, 2013 Published, JBC Papers in Press, June 25, 2013, DOI 10.1074/jbc.M113.489906
Cristoforo Silvestri‡1, Andrea Martella‡1, Neil J. Poloso§, Fabiana Piscitelli‡1, Raffaele Capasso¶, Angelo Izzo¶,
David F. Woodward§2, and Vincenzo Di Marzo‡1,3
From the ‡Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via Campi
Flegrei 34, 80078 Pozzuoli, Italy, §Allergan, Inc., Irvine, California 92612, and the ¶Endocannabinoid Research Group, Department
of Pharmacy, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy
Background: Adipogenesis is the process by which adipocytes are formed to maintain or expand fat depots.
Results: Prostaglandin F2␣ ethanolamide (PGF2␣EA) is produced from anandamide in preadipocytes and inhibits adipogenesis.
Conclusion: Conversion of proadipogenic anandamide to antiadipogenic PGF2␣EA is a novel mechanism for the regulation of
adipogenesis.
Significance: Discovering a PGF2␣EA-mediated negative regulatory mechanism over adipogenesis may lead to the development
of antiobesity therapies.

Lipid mediators variedly affect adipocyte differentiation. Excessive adiposity (especially in central depots) is a leading
Anandamide stimulates adipogenesis via CB1 receptors and per- factor for a host of complications, altogether termed metabolic
oxisome proliferator-activated receptor ␥. Anandamide may be syndrome, that not only lead to a decreased quality of life but
converted by PTGS2 (COX2) and prostaglandin F synthases, may also result in death (4). Adipose tissue mass is determined
such as prostamide/prostaglandin F synthase, to prostaglandin by the amount of fat stored in each adipocyte as well as the
F2␣ ethanolamide (PGF2␣EA), of which bimatoprost is a potent number of adipocytes, both of which can be modified through
synthetic analog. PGF2␣EA/bimatoprost act via prostaglandin the interplay between energy input and expenditure (5). Several
F2␣FP receptor/FP alt4 splicing variant heterodimers. We inves- species are able to produce new fat cells via adipogenesis
tigated whether prostamide signaling occurs in preadipocytes throughout their entire lifetime, and this process can be in-
and controls adipogenesis. Exposure of mouse 3T3-L1 or creased through high fat or carbohydrate consumption (6).
human preadipocytes to PGF2␣EA/bimatoprost during early Increased adipocyte number, both during development and in
differentiation inhibits adipogenesis. PGF2␣EA is produced adulthood, is associated with high levels of obesity (6 – 8). The
from anandamide in preadipocytes and much less so in differ- understanding of the genetic and molecular processes regulat-
entiating adipocytes, which express much less PTGS2, FP, and ing adipogenesis will, therefore, provide insight into the patho-
its alt4 splicing variant. Selective antagonism of PGF2␣EA logical dysregulation of adipose tissue accumulation and aid in
receptors counteracts prostamide effects on adipogenesis, as the development of therapeutic strategies for the prevention
does inhibition of ERK1/2 phosphorylation. Selective inhibition and/or treatment of obesity. The key players in the transcrip-
of PGF2␣EA versus prostaglandin F2␣ biosynthesis accelerates tional program that regulates adipogenesis have been eluci-
adipogenesis. PGF2␣EA levels are reduced in the white adipose dated. Pparg and Cebpa are key adipogenic regulators across
tissue of high fat diet-fed mice where there is a high requirement several species, and their expression is up-regulated within the
for new adipocytes. Prostamides also inhibit zebrafish larval first 2 days of the differentiation process (9 –11). Specific over-
adipogenesis in vivo. We propose that prostamide signaling in expression or activation of these proteins is able to promote the
preadipocytes is a novel anandamide-derived antiadipogenic differentiation of fibroblasts into adipocytes, and they are crit-
mechanism. ical in the control of adipocyte-specific genes such as Fabp4/
AP1, Retn, and Acrp30 (12–14).
The endocannabinoid N-arachidonoylethanolamine (AEA,4
Adipocytes, the main components of white adipose tissue, anandamide) is a lipid signaling molecule derived from arachi-
serve as energy stores through the accumulation and storage of donic acid that is important for adipocyte biology by regulating
triglycerides, which are metabolized upon an increased the adipogenic process and lipid metabolism within mature
requirement for higher energy levels (1) and produce an array of adipocytes. AEA levels rise and then stabilize or decrease with
factors (adipokines) with physiological roles ranging from the progression of adipogenesis (15) and are increased in the
immunological responses to the regulation of appetite (2, 3).
4
The abbreviations used are: AEA, N-arachidonoylethanolamine/anandamide;
PPAR␥, peroxisome proliferator-activated receptor ␥; FAAH, fatty acid
□
S
This article contains supplemental Tables S1 and S2. amide hydrolase; PGF2␣EA, prostaglandin F2␣ ethanolamide; PGF2␣, pros-
1
Recipients of an Allergan research grant. taglandin F2␣; QPCR, quantitative PCR; PTGFR/FP, prostaglandin F recep-
2
To whom correspondence may be addressed: E-mail: Woodward_David@ tor; SFD, standard-fat diet; HFD, high-fat diet; LC-APCI-TOF, LC-atmo-
Allergan.com. spheric pressure chemical ionization-TOF; LC-IT-TOF, LC-ion trap-TOF;
3
To whom correspondence may be addressed: E-mail: vincenzo.dimarzo@ DMSO, dimethyl sulfoxide; alt4, alternate 4; WAT, white adipose tissue; AA,
icb.cnr.it. arachidonic acid; PGH2, prostaglandin H2; d0, day 0.

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PGF2␣EA Inhibits Adipogenesis
plasma and adipose tissue under conditions of central adiposity/ were obtained from Lonza (catalog no. PT-5020) and were
obesity (16, 17). AEA stimulates adipogenesis through the G grown and differentiated in the recommended medium (Lonza,
protein-coupled cannabinoid CB1 receptor or, at higher con- catalog no. PT-8002) as described by the manufacturer. The
centrations, PPAR␥ (18, 19). In some cells, AEA, instead of drugs bimatoprost and AGN211335 (Allergan Inc., Irvine, CA),
being inactivated by fatty acid amide hydrolase (FAAH) (20), AL8810, PD98059, and R-flurbiprofen (Cayman Chemicals)
can be oxygenated by prostaglandin-endoperoxide synthase 2 were added to the medium, and cells were harvested at various
(PTGS2) (also known as COX 2), and this reaction, after the times as described below.
subsequent action of either prostamide/prostaglandin F syn- Quantitative and Non-quantitative PCR Analysis—Total
thase or prostaglandin F synthase (AKR1C3/prostaglandin F RNA was isolated from 3T3-L1 cells cultured in 24-well plates
synthase), results in the formation of the prostanoid prosta- using TRIzol (Invitrogen) and treated with DNase I (Ambion)
glandin F2␣ ethanolamide (prostamide F2␣ or PGF2␣EA). These and from human subcutaneous preadipocytes cultured in
enzymes (as well as COX1, which, however, does not recognize 96-well plates using TRIzol and Purelink RNA micro scale kits
AEA as substrate) are primarily responsible for the conversion (Invitrogen) and reverse-transcribed with the SuperScript III
of cell membrane-derived arachidonic acid into prostaglandin RT reaction kit (Invitrogen) according to the instructions of the
F2␣ (PGF2␣) (21–23). However, PGF2␣EA and PGF2␣, apart manufacturer. 10 to 20 ng of starting RNA was then used for
from deriving from different biosynthetic pathways, also dis- QPCR analysis using IQ SYBR Green Supermix (Bio-Rad) with
play distinct pharmacology. They are sensitive to specific primers designed with AlleleID (Premier Biosoft, supplemental
antagonists, and this is a result of the fact that the latter medi- Table S1) on a CFX 384 optical thermal cycler (Bio-Rad). Data
ator acts via the G protein-coupled FP receptor, whereas analysis was performed using CFX Manager software (Bio-
PGF2␣EA requires for its actions a heterodimer consisting of Rad) using either Hprt (for 3T3-L1 cells) or actin ␤ (for
wild-type and an alternately spliced FP (24 –28). Like endocan- human cells) as reference genes, and data are expressed as rel-
nabinoids, PGF2␣ and other prostaglandins also play numerous ative mRNA levels with standard errors of the mean of triplicate
physiological roles, including the regulation of adipogenesis, reactions. Statistical significance was determined with the
generally, but not uniquely, through the activation of G protein- REST 2009 software. For analysis of PTGFR splice variant
coupled receptors (29 –31). Prostaglandin J2 was the first expression in undifferentiated and differentiated human prea-
endogenous PPAR␥ ligand identified capable of inducing dipocytes, primer combinations for each splice variant were
this process (32, 33), whereas PGF2␣ inhibits adipocyte dif- designed by hand (supplemental Table S2) on the basis of
ferentiation. This activity is dependent on FP activation of sequences available in patent no. US 7,320,871 B2, used to
the ERK1/2 and calcium-calcineurin signaling pathways, amplify cDNA obtained as described above with Platinum TAQ
with the former being responsible for the phosphorylation (Invitrogen) using a C1000 thermal cycler (Bio-Rad), and visu-
and inactivation of PPAR␥ (34 –36). alized by agarose gel electrophoresis.
To date, it is unknown what, if any, effects PGF2␣EA has on Mouse Feeding and Purification and Quantification of AEA,
the process of adipogenesis, nor whether this putative lipid PGF2␣EA, and PGF2␣ from Adipose Tissues and Cells—Nine-
mediator is present in differentiating adipocytes. Interestingly, week-old male C57Bl/6J mice (Charles River Laboratories, Inc.)
however, bimatoprost (AGN 192024), a stable, synthetic, and were utilized in all studies. For standard-fat diet (SFD) versus
potent analog of PGF2␣EA approved for the treatment of ocular high-fat diet (HFD) studies mice were fed for 1 week with either
hypertension and also capable of activating the FP/FP splice standard chow or an HFD (Harlan, catalog no. 97366). After
variant heterodimers (28, 37), decreases periorbital fat deposits treatment, animals were sacrificed, and adipose tissues were
in glaucoma patients through an unknown mechanism (38 – removed, snap-frozen, and stored at ⫺80 °C. 3T3-L1 cells were
41). This observation may suggest that bimatoprost and, by grown in 100-mm plates as described above, washed once in
extension, PGF2␣EA inhibit adipogenesis, perhaps through the ice-cold PBS, collected, and stored at ⫺80 °C.
stimulation of the aforementioned heterodimers. We have For AEA or PGF2aEA, tissues or cells were Dounce-homog-
investigated this possibility in vitro and in vivo and report that enized and extracted with acetone containing internal deuter-
PGF2␣EA is a potent anandamide-derived antiadipogenic ated standards for AEA and PGF2aEA quantification by isotope
mediator in preadipocytes. dilution ([2H]8 AEA and [2H]4 PGF2␣EA). The lipid-containing
organic phase was dried, weighed, and prepurified by open-bed
EXPERIMENTAL PROCEDURES chromatography on silica gel. Fractions were obtained by elut-
Cell Culture—3T3-L1 cells (ATCC) were cultured in growth ing the column with 99:1, 90:10, 70:30, and 50:50 (v/v) chloro-
medium (DMEM, Lonza) supplemented with NaHCO3 (1.5 form/methanol. The 90:10 fraction was used for AEA quantifi-
g/liter), 10% NCS (Invitrogen) and penicillin/streptomycin cation by LC-APCI-MS and using selected ion monitoring at
(Invitrogen). To induce adipogenesis, cells were grown to con- M⫹1 values for AEA and its deuterated homologue, as
fluence and, after 1 day, switched to differentiation medium described previously (42). The 70:30 fraction was used for
(IDM; growth medium plus 1 ␮g/ml insulin (Sigma), 250 nM PGF2aEA quantification by LC-MS-MS using an LC20AB cou-
dexamethasone (Sigma), and 500 ␮M 3-isobutyl-1-methylxan- pled to a hybrid detector IT-TOF (Shimadzu Corp., Kyoto,
thine (Sigma)) for 2 days, followed by incubation in growth Japan) equipped with an electrospray ionization (ESI) interface.
medium with 1 ␮g/ml insulin for 2 days, and then cultured in LC analysis was performed in the isocratic mode using a Dis-
growth medium for the rest of the experiment with medium covery HC18 column (15 cm, 62.1 mm, 5 mm) and methanol/
changes every 2 days. Human subcutaneous preadipocytes water/acetic acid (53:47:0.05 by volume) as mobile phase with a

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 PGF2␣EA Inhibits Adipogenesis
flow rate of 0.15 ml/min. Identification of PFG2aEA was carried (1:200, Santa Cruz Biotechnology, catalog no. sc-1747), or actin
out using ESI ionization in the positive mode with a nebulizing ␤ (1:30000, Sigma, catalog no. A1978) overnight, followed by
gas flow of 1.5 ml/min and a curved desolvation line tempera- HRP-conjugated secondary antibodies (Bio-Rad), and then
ture of 250 °C. detected by ECL (Bio-Rad).
For PGF2␣, solid-phase extraction cartridges (CHROMABOND威 Zebrafish Larvae and Adipocyte Staining—Zebrafish (Danio
HR-X, 3 ml/200 mg, Macherey-Nagel) were used for extraction. rerio) larvae were obtained from a stable laboratory strain and
For cellular assays, 2 ml of culture medium was utilized. For raised at 28.5 °C on a 14 light:10 dark photoperiod according to
tissue extraction, epididymal adipose tissue was weighed, the Zebrafish Book. Embryos were raised in filtered egg water
homogenized with pestles in 800 ␮l of PBS, and centrifuged at until 8 days post-fertilization, and larvae were exposed to drug
15,000 ⫻ g for 15 min at 4 °C. 10 ␮l of 2[H]4 PGF2␣ (2.5 ␮M) solutions for a further 8 days. Bimatoprost was dissolved in
internal standard was added to the supernatant and the pH was DMSO, diluted with filtered egg water, and replaced daily. Lar-
adjusted to 3 with 1 M HCl. Media or tissue samples were then vae were then stained with Nile red (Sigma) to a final concen-
added to a solid-phase extraction cartridge that was precondi- tration of 0.5 ␮g/ml for 30 min. Stained larvae were anesthe-
tioned with 2 ml of methanol, 2 ml of water, and 2 ml of 95:5 tized with Tricaine (Sigma), photographed with a Tri-Red filter
water:methanol. PGF2␣ was eluted with 3 ml of methanol under on a Leica DMI 6000B fluorescence microscope under the same
vacuum, whereas most (⬃95%) of the PGF2␣EA was retained on optical conditions, and the signal intensity of adipocytes within
the column. The eluate was dried under a nitrogen gas stream at the abdominal cavity was analyzed with ImageJ 1.44p (National
25 °C, and the residue was reconstituted in 30 ␮l of mobile Institutes of Health). Staining of cultured 3T3-L1 adipocytes
phase for tissue samples or 2 ml of medium for cellular assays. was performed using Adipored (Lonza) according to the
The recovery of the extraction protocol was between 20 and instructions of the manufacturer and then either photographed
50%. as with zebrafish embryos or analyzed with a Genios Pro plate
For PGF2␣ analysis in tissue, an LC20AB coupled to a hybrid reader (Tecan) to quantify fluorescence intensity.
detector IT-TOF (Shimadzu Corp.) equipped with an ESI inter- Ethics Statement—All animal procedures were in conformity
face was used, and they were measured via multiple reaction with the principles of laboratory animal care (National Insti-
monitoring. LC analysis was performed using a Discovery威 C18 tutes of Health publication no. 86-23, revised 1985) and the
column (15 cm ⫻ 2.1 mm, 5 ␮m) at a flow rate of 200 ␮l/min. Italian Decreto Legge no. 116 of 27 January 1992 and associated
The column was equilibrated in solvent A (water-acetonitrile- guidelines in the European Communities Council Directive of
acetic acid (70:30:0.02, v/v/v)), and 10 ␮l of sample (100% of 24 November 1986 (86/609/ECC). All efforts were made to
medium and 33% of tissue) was injected using a 10-␮l injection minimize animal suffering and the number of animals used.
loop and eluted with 0% solvent B (acetonitrile-isopropyl alco-
hol (50:50, v/v)) between 0 and 1 min. Solvent B was increased RESULTS
in a linear gradient to 25% solvent B until 3 min, to 45% until 11 Prostamides Inhibit Early 3T3-L1 Cell Adipogenesis—Given
min, to 60% until 13 min, to 75% until 18 min, and to 90% until the well documented effects of prostaglandins on adipogenesis,
18.5 min. Solvent B was held at 90% until 20 min, dropped to 0% we set out to determine whether bimatoprost and PGF2␣EA
by 21 min, and held until 25 min (43). Identification of PGF2␣ affected the early stages of adipogenesis in vitro. We differenti-
was carried out using ESI ionization in the negative mode with ated 3T3-L1 cells in the presence of various concentrations of
a nebulizing gas flow of 1.5 ml/min and a curved desolvation either bimatoprost, PGF2␣EA, or PGF2␣ and assayed the
line temperature of 250 °C. Quantitative prostaglandin deter- expression of Pparg and Cebpa after 2 days. All compounds
mination was performed by the stable isotope dilution method dose-dependently reduced the expression of these targets to
as described previously (44). The limit of detection of this levels similar to those of undifferentiated cells, although, as
method was 100 fmol/sample. Because of sensitivity issues, expected from previous pharmacological data (37), bimato-
PGF2␣ analysis in solid-phase extraction cartridge-fractionated prost was approximately 10 times more potent than PGF2␣EA,
cell medium (unlike prostamides, which, being less polar, are yielding maximal effects at about 1 ␮M as compared with 10 ␮M
retained by cells, prostaglandins are known to be largely for PGF2␣EA (Fig. 1A). Long-term differentiation of 3T3-L1
released from cells into the culture medium), was instead per- cells for 8 days in the presence of PGF2␣EA or bimatoprost
formed with a PGF2␣EIA kit (Cayman Chemicals) according to resulted in the sustained inhibition of Pparg expression as well
the instructions of the manufacturer. as the inhibition of the up-regulation of the mature adipocyte
Western Blot Analysis—Cells grown in 6-well plates were dif- markers Fabp4, Retn, and Acrp30 (Fig. 1B). These data show
ferentiated as described above and lysed in 1⫻ TNE buffer (50 that prostamides are antiadipogenic agents.
mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA) with 0.5% Triton We then determined whether early inhibition of the adipo-
X-100 and protease and phosphatase inhibitor mixtures genic program alone by bimatoprost was sufficient to inhibit
(Sigma, catalog nos. P8340, P5726, and P0044). Proteins were the formation of mature adipocytes. We treated differentiating
analyzed via a Lowry protein assay (Bio-Rad) and resolved by 3T3-L1 cells at different time points for various periods of time
SDS-PAGE with a routine Tris-glycine buffering system. Pro- and observed that bimatoprost exerts its antiadipogenic activity
teins transferred onto PVDF membranes were then blocked in when the cells were exposed during the first 2 days of the dif-
5% skim milk in TBST (20 mM Tris, 137 mM NaCl, 0.1% Tween ferentiation protocol only (Fig. 2). This indicated that bimato-
20) and probed with anti phospho-ERK, ERK (1:1000, Cell Sig- prost inhibits the initial stages of differentiation by inhibiting
naling Technology, catalog nos. 9101 and 9102), PTGS2/Cox2 the early up-regulation of the key adipogenic factors Pparg and

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PGF2␣EA Inhibits Adipogenesis

FIGURE 1. Dose-dependent effects of prostamides on 3T3-L1 adipogenesis. A, mRNA levels of Pparg and Cebpa were determined by QPCR in undifferen-
tiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO (D), bimatoprost, PGF2␣EA, or PGF2␣ at the indicated concentrations.
B, mRNA levels of Pparg, Fabp4, Acrp30, and Retn were determined by QPCR in 3T3-L1 adipocytes after 8 days of differentiation in the absence (-) or presence
of DMSO, bimatoprost, or PGF2␣EA at the indicated concentrations. Relative mRNA values are expressed as mean ⫾ S.E. *, p ⬍ 0.05 versus DMSO control.

Cebpa. This inhibition was long-lasting because the subsequent (45). However, no evidence exists for the presence of PGF2␣EA
up-regulation of the mature adipocyte markers Fabp4, Retn, in preadipocytes or adipocytes. Utilizing LC-IT-TOF mass
and Acrp30 was also repressed. However, Fabp4 expression is spectrometry, we measured PGF2␣EA levels in lipid extracts
similarly inhibited at day 8 by bimatoprost regardless of from undifferentiated 3T3-L1 cells or cells undergoing adipo-
whether the cells are treated for only the first 2 days of or genesis. Undifferentiated 3T3-L1 cells produce significant lev-
throughout the entire differentiation program, whereas Retn els of PGF2␣EA and, by day 2 of differentiation, these levels drop
and Acrp30 gene expression is further reduced by 50% if the to barely detectable levels (Fig. 3A). This may suggest that
bimatoprost treatment is extended beyond 2 days. When we PGF2␣EA may act to maintain preadipocytes in an undifferen-
stained triglycerides in 3T3-L1 cells at day 8 of differentiation, tiated state and that its down-regulation, together with other
we confirmed that treatment during the first 2 days of differen- factors, is required for adipogenesis. Given that AEA is the only
tiation is sufficient for the inhibition of triglyceride accumula- biosynthetic precursor of PGF2␣EA via PTGS2/COX2, we
tion (Fig. 2B). hypothesized that PGF2␣EA levels in 3T3-L1 cells could be
PGF2␣EA Is Produced in 3T3-L1 Preadipocytes, and Its Lev- manipulated by incubation with AEA. Incubation of undiffer-
els and Those of Its Biosynthetic Enzymes Decrease during entiated cells with 10 ␮M AEA resulted in an almost 4-fold
Differentiation—Recently, we identified PGF2␣EA as an endog- increase in PGF2␣EA levels after 4 h, and coincubation with the
enously occurring eicosanoid/neutral lipid in the spinal cord PTGS2 inhibitor NS398 (10 ␮M) reduced PGF2␣EA to unde-

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 PGF2␣EA Inhibits Adipogenesis

FIGURE 2. Time course analysis of the effects of prostamide treatment on 3T3-L1 adipogenesis. A, mRNA levels of adipogenic (Pparg, Cebpa) and
mature adipocyte markers (Fabp4, Retn, Acrp30) in 3T3-L1 preadipocytes (d0) and mature adipocytes (d8) in the absence or presence of 1 ␮M bimato-
prost for various times (B0 – 8 days) were determined by QPCR. Relative mRNA values are expressed as mean ⫾ S.E. B, quantification (top panel, mean ⫾
S.D. of 64 independent fluorescence readings) and images (bottom panel) of Adipored-stained undifferentiated (Undiff.) 3T3-L1 cells or after 8 days of
differentiation treated in the absence (-) or presence of either DMSO, 1 ␮M bimatoprost (Bim.), 10 ␮M PGF2␣EA, or 0.1 ␮M PGF2␣ from day 0 –2. *, p ⬍ 0.05
versus DMSO control.

tectable levels (Fig. 3A). By day 2 of differentiation, although no required for PGF2␣ production in 3T3-L1 adipocytes (46))
endogenous PGF2␣EA was detected, incubation with AEA decrease significantly by the second day of differentiation as
resulted in 3-fold lower levels of PGF2␣EA compared with expected, whereas Pmpgfs (encoding for prostamide/prosta-
undifferentiated cells (Fig. 3A), indicating a marked decrease in glandin F synthase) is expressed in these cells at levels that do
the ability of differentiating cells to convert exogenous AEA to not change by this time point (Fig. 3B). These data indicate that
this compound. To surmise how levels of PGF2␣EA levels PTGS2 (COX2) and AKR1B3 may regulate PGF2␣EA levels in
change during differentiation, we assayed the expression of a adipocytes. However, because the formation of PGH2EA from
number of biosynthetic enzymes during this process. We found PTGS2 action is the rate-limiting step in PGF2␣EA biosynthe-
that both Ptgs2 (COX-2) and Akr1b3 (which was shown to be sis, we cannot rule out a role for prostamide/prostaglandin F

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 PGF2␣EA Inhibits Adipogenesis
synthase. Taken together, these data support the idea that dif- synthesis and action within differentiating adipocytes, thereby
ferentiating adipocytes down-regulate the pathway required for creating a self-amplificatory loop for their action.
AEA-derived PGF2␣EA biosynthesis. Surprisingly, when we Prostamide Receptor Antagonism Reverses Prostamide-medi-
measured Ptgs2 expression in differentiating 3T3-L1 cells ated Inhibition of Adipogenesis—We next tested the ability of
treated with bimatoprost or PGF2␣EA, we found that they bimatoprost to inhibit adipogenesis in 3T3-L1 preadipoctyes in
induced a strong up-regulation of Ptgs2 mRNA (Fig. 3C) and the absence or presence of the prostamide receptor antagonist
protein (see below) expression to levels above those observed in AGN211335, which specifically blocks bimatoprost/prosta-
undifferentiated cells. We also observed that PGF2␣ similarly mide activity at 1 ␮M in HEK293 cells and not PGF2␣ activity
up-regulated Ptgs2 expression. (28), or the prostaglandin FP receptor antagonist AL8810. We
Human Preadipocytes Are Sensitive to the Antiadipogenic found that AGN211335 (5 ␮M) reversed bimatoprost-mediated
Activity of Prostamides and Express PTGFR Alternative Splice (0.1 ␮M) inhibition of Pparg and Cebpa gene induction in cells
Transcripts Required for Their Activity—To determine whether differentiated for 2 days back to control levels, whereas AL8810
the antiadipogenic activity of bimatoprost also occurs in human (5 ␮M) was much less, if at all, effective (Fig. 4A). Further,
cells, we differentiated human preadipocytes in the presence bimatoprost-mediated Ptgs2 induction was completely reversed
of various doses of bimatoprost and assayed the expression by AGN211335 but only reduced by half by AL8810 (Fig. 4B). In
of PPARG and the mature adipocyte markers FABP4 and human preadipocytes, we found that bimatoprost-mediated
ADIPOQ. We observed dose-dependent inhibition of the up- (0.5 ␮M) inhibition of FABP4 and ADIPOQ expression was
regulation of all genes after 8 days of differentiation (Fig. 3D). completely reversed by the prostamide receptor antagonist
PGF2␣EA and bimatoprost signal through prostamide recep- (Fig. 4C). These data suggest that the antiadipogenic activity of
tors, which are splice variants of the FP receptor encoded by the prostamides is mediated primarily by the prostamide receptors
PTGFR gene responsible for PGF2␣ signaling (28). To date, in both murine and human cells.
there has been no report of the expression of these variants in Activation of MAPK Signaling Is Required for Prostamide
adipose tissue. We therefore performed RT-PCR on human Antiadipogenic Activity—As activation of the FP receptor by
premature and mature adipocytes using various primers that PGF2␣ activates MAPK signaling and ERK phosphorylation/
detect all the known splice variants of PTGFR. In both undiffer- activation, which negatively regulates adipogenesis, we next
entiated and differentiated cells we were able to detect all of the determined whether the antiadipogenic activity of bimatoprost
known PTGFR splice variants (supplemental Table S2), and and PGF2␣EA required the activation of MAPK signaling in
subsequent sequencing confirmed the expression of the PTGFR 3T3-L1 cells through the use of the MEK inhibitor PD98059.
alternate 4 (alt4) splice variant, reported to dimerize with the PD98059 alone resulted in a small up-regulation of Pparg and
wild-type FP receptor and mediate bimatoprost/PGF2␣EA sig- Cebpa expression after 2 days of differentiation per se and com-
naling. QPCR analysis of differentiating human adipocytes pletely abolished the bimatoprost- and PGF2␣EA-mediated
using primers common to all splice variants or just the alt4 inhibition of the expression of these genes (Fig. 5A). Interest-
variant (PTGFRv4) showed that these receptors are down-reg- ingly, the same concentration of PD98059 was only able to par-
ulated during adipogenesis, indicating that the cells would tially revert the PGF2␣-mediated inhibition of Cebpa expres-
likely be less sensitive to PGF2␣ and PGF2␣EA upon differenti- sion while fully restoring Pparg expression. As above (Fig. 2B),
ation (Fig. 3E). Searches of genome and expression databases bimatoprost, PGF2␣EA or PGF2␣ treatment for the first 2 days
failed to identify a potential murine homolog of the PTGFRv4 of differentiation results in significantly decreased accumula-
splice variant. However, we also assayed the expression of the tion of triglyceride levels at day 8, completely reversed by coin-
wild type murine Ptgfr gene during 3T3-L1 differentiation. As cubation with PD98059 (Fig. 5C). We then tested the require-
observed in the human cells, and in contrast to a previous ment of MAPK signaling for the bimatoprost-mediated
report, we found that, after 2 days of differentiation, Ptgfr inhibition of adipogenesis in primary human preadipocytes. As
expression decreased significantly (Fig. 3F). Further, similar to with 3T3-L1 cells, the ability of bimatoprost to inhibit adipogen-
Ptgs2 expression, bimatoprost, PGF2␣EA, and PGF2␣ all caused esis, as measured by expression of the mature adipocyte markers
a very large increase in Ptgfr expression to levels that were 3- to FABP4 or ADIPOQ, was completely dependent on MAPK sig-
4-fold higher than those observed in undifferentiated cells. naling (Fig. 5B).
Taken together, these data indicate that prostamides inhibit Given that bimatoprost and PGF2␣EA induce a large up-reg-
adipogenesis in both murine and human preadipocytes, possi- ulation of Ptgs2 in differentiating 3T3-L1 cells at day 2, we
bly through prostamide receptors/PTGFR splice variants, and tested whether this was also dependent on MAPK signaling.
reverse the down-regulation of prostamide/prostaglandin bio- PD98059 was able to significantly reverse the up-regulation

FIGURE 3. Regulation of prostamide levels during early adipogenesis in 3T3-L1 cells. A, quantification (mean ⫾ S.E.) of PGF2␣EA levels by LC-APCI-MS in
undifferentiated (d0) or differentiating (d2) 3T3-L1 cells alone (left panel) or in the presence of DMSO or 10 ␮M AEA without or with 10 ␮M NS398 (NS). Total
amounts of lipids were similar for all samples (not shown). Note that ⬃1 mg of lipid extract is normally obtained from 2–3 million cells. n ⫽ 3 for each point.
* and #, p ⬍ 0.05 versus DMSO and AEA at d0, respectively. B, C, and F) mRNA levels of Pparg, Ptgs2, Akr1b3, Pmpgfs, or Ptgfr were determined by QPCR in
undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence of DMSO, 1 ␮M bimatoprost (Bim.), 10 ␮M PGF2␣EA or 0.1 ␮M PGF2␣.
D, mRNA levels of PPARG, FABP4, or ADIPOQ were determined by QPCR in undifferentiated (d0) human preadipocytes or cells after 8 days of differentiation in
the absence or presence of DMSO or the indicated concentration of bimatoprost. E, mRNA levels of PPARG, PTGFR (FP receptor), and PTGFR alternate variant 4
(PTGFRv4) were determined by QPCR in undifferentiated (d0) or differentiating (d3) human subcutaneous preadipocytes. Relative mRNA values are expressed
as mean ⫾ S.E. *, p ⬍ 0.05 versus d0 (B and E) or DMSO control (C, D, and F).

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PGF2␣EA Inhibits Adipogenesis

FIGURE 4. Effect of prostamide receptor antagonism on prostamide antiadipogenic activity. A and B, mRNA levels of Pparg, Cebpa, and Ptgs2 were
determined by QPCR in differentiating 3T3-L1 cells at day 2 in the presence of DMSO, 0.1 ␮M bimatoprost, 5 ␮M AGN211335 (AGN), or 5 ␮M AL8810 (AL) as
indicated. C, mRNA levels of FABP4 and ADIPOQ were determined by QPCR in human preadipocytes undifferentiated (Undif.) or differentiated (d4) in the
absence or presence of DMSO, 0.5 ␮M bimatoprost, or 5 ␮M AGN211335 as indicated. Relative mRNA values are expressed as mean ⫾ S.E. * and #, p ⬍ 0.05 versus
DMSO control and bimatoprost-cotreated samples, respectively.

of Ptgs2 by bimatoprost, PGF2␣EA, and PGF2␣ (Fig. 5D). Bimatoprost Inhibits the Early Development of Adipose Tis-
Western blot analysis confirmed that treatment of 3T3-L1 sue in D. rerio in Vivo—Fish rely on the same transcriptional
cells with bimatoprost or PGF2␣EA during the first 2 days of cascade to regulate adipogenesis and lipid metabolism as mam-
differentiation increased ERK phosphorylation to levels sim- mals (47, 48). In zebrafish (D. rerio), adipogenesis initiates in
ilar to those observed in undifferentiated 3T3-L1 cells and larvae not sooner than 8 days post-fertilization. Adipocytes
that both compounds similarly result in the up-regulation of form as clusters of cells initially located along the pancreas and
PTGS2 protein levels in a MEK-dependent manner (Fig. 5E). posterior to the swim bladder along the peritoneal cavity (47).
Taken together, these data indicate that bimatoprost and This process is extremely labile and dependent not only on the
PGF2␣EA signal through the MAPK pathway in preadi- age but the size and nutritive state of the fish. As such, food
pocytes to inhibit adipogenesis and up-regulate PTGS2 deprivation inhibits the early development of adipocytes (48).
expression. To determine whether bimatoprost was able to inhibit fat for-

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PGF2␣EA Inhibits Adipogenesis

FIGURE 6. Specific role of prostamides in adipogenesis in vitro and in vivo. A, zebrafish larvae were treated from 8 to 16 days post-fertilization with DMSO
or bimatoprost (Bim.) and stained with Adipored in the final 24 h of treatment before analysis by fluorescent microscopy. Anterior is left, dorsal is up. Arrow,
adipocytes; asterisk, swim bladder; g, gall bladder. B, quantification of pixel intensity from stained adipocytes in the abdominal cavity. n ⫽ 16 for each
treatment. *, p ⬍ 8⫺7. C, quantification (mean ⫾ S.E.) of PGF2␣EA (n ⫽ 6) and PGF2␣ (n ⫽ 3) levels by LC-APCI-MS in epididymal fat of mice on an SFD or HFD for
1 week for each treatment. *, p ⬍ 1⫺6. D, quantification (mean ⫾ S.E.) of PGF2␣EA by LC-APCI-MS in undifferentiated 3T3-L1 cells in the presence of DMSO or 10
␮M AEA without or with 10 ␮M R-flurbiprofen (RF) for 4 h. Total amounts of lipids were similar for all samples (not shown). n ⫽ 3 for each point. * and #, p ⬍ 0.05
versus DMSO and AEA, respectively. E, quantification (mean ⫾ S.D.) of PGF2␣ levels by EIA in medium from undifferentiated (d0) or differentiating (d2) 3T3-L1
cells in the absence or presence of 100 ␮M AA and 10 ␮M R-flurbiprofen alone or in combination for 4 h. n ⫽ 3 for each point. * and #, p ⬍ 0.05 versus DMSO of
the same day and similar treatment at d0, respectively. Note that 10 pg of PGF2␣ corresponds to ⬃28.5 fmol and that 2 ml of medium from 2–3 million cells were
used for each data point. Therefore, the base-line amounts of PGF2␣ released from 2–3 million cells (i.e. ⬃57 fmol) was below the limit of detection of our
LC-IT-TOF MS method. Hence, the use of EIA. F, QPCR quantification of Pparg, Cebpa (both at day 1), Acrp30 (at day 3), and Fabp4 (at day 4) mRNA levels in
differentiating 3T3-L1 cells (relative to undifferentiated controls) treated with either DMSO (D) or the indicated concentration of R-flurbiprofen for 24 h prior to
the onset of the differentiation protocol. *, p ⬍ 0.05 versus DMSO control. G, quantification (mean ⫾ S.D.) of Adipored-stained 3T3-L1 cells after 4 days of
differentiation as in F. n ⫽ 6. *, p ⬍ 0.05 versus DMSO control.

mation in vivo, we utilized zebrafish as a developmental model triglyceride accumulation (Fig. 6A, arrows). Accordingly, quan-
of adipogenesis. Although bimatoprost-treated fish had identi- tification of pixel intensity within the abdominal region where
fiable adipocytes, the adipose mass was not only smaller but adipocytes developed showed that bimatoprost resulted in sig-
much less intensely stained as well, indicating lower levels of nificantly less average fluorescence in this region (Fig. 6B).

FIGURE 5. Effect of MEK inhibition on prostamide antiadipogenic activity. A and D, mRNA levels of Pparg, Cebpa, or Ptgs2 were determined by QPCR in
differentiating (d2) 3T3-L1 cells in the absence or presence of DMSO, 1 ␮M bimatoprost (Bim.), 10 ␮M PGF2␣EA, 0.1 ␮M PGF2␣, and/or 25 ␮M PD98059 (PD) as
indicated. B, mRNA levels of FABP4 and ADIPOQ were determined by QPCR in human preadipocytes undifferentiated (Undif.) or differentiated (d4) in the
absence or presence of DMSO (D), 1 ␮M bimatoprost, and/or 25 ␮M PD98059 as indicated. Relative mRNA values are expressed as mean ⫾ S.E. C, quantification
(left panel, mean ⫾ S.D. of 16 independent fluorescence readings) and images (right panel) of Adipored-stained 3T3-L1 cells after 8 days of differentiation
treated in the absence (-) or presence of either DMSO, 1 ␮M bimatoprost, 10 ␮M PGF2␣EA, or 0.1 ␮M PGF2␣ from day 0 –2. E, Western blot analysis of phospho-ERK,
ERK, PTGS2, and actin ␤ (ACTB) in lysates from undifferentiated (d0) or differentiating (d2) 3T3-L1 cells in the absence (-) or presence (⫹) of DMSO, 1 ␮M
bimatoprost, 10 ␮M PGF2␣EA, 0.1 ␮M PGF2␣, or 25 ␮M PD98059 in the indicated combinations. * and #, p ⬍ 0.05 versus DMSO control and bimatoprost or
PGF2␣EA or PGF2␣-cotreated samples, respectively.

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 PGF2␣EA Inhibits Adipogenesis
Staining of the gall bladder was observed periodically, as point to PGF2␣EA as an anandamide-derived, endogenous
reported previously. No toxicity was observed within the fish mediator of adipogenesis that is distinct from PGF2␣.
throughout the course of the experiment, nor were there any
observable differences in size between control and treated spec- DISCUSSION
imens. Control unfed fish were found to not have any positively Prostaglandin ethanolamides (or prostamides) are a distinct
stained adipocytes (Fig. 6A). class of lipid signaling molecules related to prostaglandins and
PGF2␣EA Levels Are Reduced in WAT of Mice under Dietary derived from the oxygenation of the endocannabinoid anand-
Conditions That Favor Adipocyte Mass Expansion—Exposure amide to PGH2-EA by PTGS2 (COX-2, encoded by Ptgs2) and
to an HFD is able to induce adipose hyperplasia (6, 49, 50). We the subsequent reduction of PGH2-EA by prostaglandin syn-
therefore tested whether, as compared with an ad libitum SFD, thases. Either AKR1C3/prostaglandin F synthase or prosta-
PGF2␣EA levels were altered in the WAT of mice on a short mide/prostaglandin F synthase (PMPGFS) catalyze the conver-
(5-day) period of ad libitum HFD, insufficient to increase body sion of PGH2-EA to PGF2␣EA (21–23). Likewise, PGF2␣ is
weight more than the SFD. We were able to detect levels of produced from the PTGS2-catalyzed oxidation of AA, followed
PGF2␣EA in the epididymal WAT of SFD mice. However these by reduction catalyzed by AKR1C3 or AKR1B3 (46, 53, 54).
were reduced to undetectable levels in HFD mice (Fig. 6C). By However, although the biosynthesis of AEA, and, hence, ulti-
contrast, epididymal WAT PGF2␣ levels, which were lower than mately, PGF2␣EA, relies on AA from the sn-1 position of phos-
PGF2␣EA levels, were not reduced following the short period of pholipids, AA serving as a PGF2␣ precursor is released from the
HFD (Fig. 6C). These data suggest that PGF2␣EA is down-reg- sn-2 position of phospholipids (55). Thus, divergent biosyn-
ulated in adipose tissue under dietary conditions that favor adi- thetic pathways lead to PGF2␣EA and PGF2␣. Furthermore,
pocyte mass expansion, implicating PGF2␣EA as a novel, despite their structural similarity, significant differences in the
endogenous regulator of adipogenesis and bimatoprost as a pharmacology of PGF2␣EA and PGF2␣ exist in various cell
potential tool for studying this process. types, including the dependence of the biological responses to
Selective Inhibition of Prostamide versus Prostaglandin F2␣ the former on the formation of heterodimers between the wild-
type FP receptor and one of its splice variants (both encoded by
Biosynthesis Enhances Adipogenesis—To distinguish the endoge-
Ptgfr). Although PGF2␣ does not activate such heterodimers,
nous role of PGF2␣EA from PGF2␣ in adipogenesis, we utilized
PGF2␣EA is nearly inactive at the wild-type FP receptor
R-flurbiprofen, a substrate-specific inhibitor of PTGS2 target-
homodimers (24 –28). PGF2␣EA pharmacology has been
ing AEA and 2-arachidonoylglycerol but not arachidonic acid
mostly investigated through the use of its synthetic structural
oxygenation (51). We found that pretreatment of 3T3-L1 prea-
analog bimatoprost (37), which has been approved for the treat-
dipocytes at day 0 with 10 ␮M R-flurbiprofen for 15 min reduced
ment of glaucoma and eyelash hypotrichosis (56, 57), and of
basal levels of PGF2␣EA to below detectable levels after 4 h and
antagonists specific for the FP/FP splice variant heterodimer
totally blocked the up-regulation of PGF2␣EA production
versus FP homodimers, such as AGN211335 (28).
induced by exposure of the cells to AEA (Fig. 6D). Analysis of
Patients treated with PGF2␣ prodrugs such as travoprost may
PGF2␣ production from the media of similarly treated undiffer-
develop a reversible reduction in periorbital fat pads, leading to
entiated 3T3-L1 cells revealed that R-flurbiprofen treatment the theory that these compounds result in the atrophy of fat in
did not modulate basal PGF2␣ levels nor prevent its increase in the vicinity of topical administration (38 – 41, 58). These results
response to treatment with 100 ␮M arachidonic acid (Fig. 6E). are not surprising because PGF2␣ is a well documented potent
At day 2 of the differentiation protocol, the addition of arachi- antiadipogenic agent (59, 60). Several hypotheses have been put
donic acid resulted in significantly less PGF2␣ production, as forward in the search of the mechanism of action of PGF2␣ on
expected from PTGS2 down-regulation at this time point, and adipogenesis, including activation of the MEK-ERK MAPK cas-
R-flurbiprofen did not reduce this production and only slightly cade, hypoxia-inducible factor 1, Ca2⫹/calmodulin-dependent
affected basal PGF2␣ levels (Fig. 6E). protein kinase, and calcineurin via increases in intracellular cal-
We then went on to determine whether the selective inhibi- cium (34 –36, 61). Differentiation assays of human orbital adi-
tion of PGF2␣EA with R-flurbiprofen (1, 3, and 10 ␮M) at day 0 pose precursors showed that, like PGF2␣, commercially avail-
in 3T3-L1 cells could affect the subsequent adipogenesis. When able prodrugs of this mediator inhibit adipogenesis in vitro (62).
we assayed the expression of adipogenic and mature adipocyte Surprisingly, however, bimatoprost, which is not hydrolyzed to
markers at 1, 2, and 4 days of the differentiation protocol, we PGF2␣, inhibits periorbital fat formation, thus suggesting that
found that, although R-flurbiprofen did not change Pparg lev- PGF2␣EA too might inhibit adipogenesis. To date, no known
els, it did result in significantly increased Cebpa expression at role for PGF2␣EA in the regulation of adipogenesis exists, nor is
day 1. Furthermore, we observed that the onset of the mature there any information on the mechanisms behind the antiadi-
adipocyte marker Acrp30 expression was enhanced markedly at pogenic activity of bimatoprost. We have addressed these open
day 2 of the differentiation protocol, whereas Fapb4 expression questions by utilizing well characterized in vitro model systems
was increased significantly by day 4 (Fig. 6F), indicating that and have found that PGF2␣EA and bimatoprost (prostamides)
R-flurbiprofen-mediated inhibition of PGF2␣EA production dose-dependently inhibit the up-regulation of the early adipo-
positively modulates adipogenesis. Indeed, triglyceride levels in genic program, resulting in a near complete blockade of the
3T3-L1 cells differentiated in the presence of R-flurbiprofen maturation of 3T3-L1 adipocytes and human adipocytes, as
were increased significantly by about 10% as compared with measured by the expression of mature adipocyte markers and
controls after 4 days of differentiation (Fig. 6G). These data the production of triglyceride stores. Bimatoprost appeared to

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PGF2␣EA Inhibits Adipogenesis
be about 10 times more potent than PGF2␣EA, which may be cade for its antiadipogenic activity (36). The requirement for
due to its higher stability (24). This property furthers the use- this latter signaling cascade feature was not observed with
fulness of bimatoprost as a tool for the study of prostamide- bimatoprost (data not shown), indicating, in this case, a diver-
mediated regulation of adipogenesis, which we have taken gence of the bimatoprost/PGF2␣EA and PGF2␣ signaling
advantage of in these studies. cascades.
We also showed here that preadipocytes produce significant To further examine the potential of prostamides to regulate
levels of PGF2␣EA and that these levels quickly decrease with the process of adipogenesis in vivo, we opted to utilize a devel-
the onset of differentiation, as has been observed with PGF2␣ opmental model of adipogenesis within juvenile zebrafish
(46), along with the concomitant down-regulation of Ptgs2 (D. rerio) (47). Zebrafish produce PGF2␣ and express homo-
expression. Furthermore, in mice under a regime of high-fat logues of the FP receptor (64), PTGS2 and prostamide/prosta-
feeding, which eventually results in increased adiposity, glandin F synthase (65 and Zebrafish Information Network,
PGF2␣EA levels in the white adipose tissue are reduced to unde- curation of protein database links, automated data submission),
tectable levels. We, like others, were unable to detect the as well as producing AEA and expressing biosynthetic and cat-
expression of Akr1c18 in 3T3-L1 cells (data not shown and Ref. abolic enzymes for this endocannabinoid,5 indicating that they
63)), the homolog of human AKR1C3 that regulates prostamide may also be capable of producing prostamides. We found that
production (21), but found that these cells do express the pros- exposing larvae to bimatoprost dissolved in water resulted in a
tamide synthase Pmpgfs. However, the expression of this marked decrease in the number of adipocytes that were observ-
enzyme did not decrease during early adipogenesis. In contrast, able by microscopy. These data indicate that prostamides are
we confirm the results of Fujimori et al. (46) by finding that the able to inhibit developmental adipogenesis in vivo and point to
PGF2␣ synthase Akr1b3 does decrease during adipogenesis, an evolutionarily conserved role for PGF2␣EA in the regulation
indicating that either the decrease in Ptgs2 expression alone of adipogenesis.
results in the observed decrease in PGF2␣EA levels or that Bimatoprost, PGF2␣EA, and PGF2␣ all induced a large
AKR1B3, like AKR1C3, may have prostamide as well as prosta- increase in Ptgs2 expression in 3T3-L1 cells treated with IDM
glandin synthase activity, a possibility that remains to be exam- adipogenic medium, as has been observed also with the FP
ined. We also show that the expression of the FP receptor receptor agonist fluprostenol in undifferentiated 3T3-L1 cells,
(Ptgfr) is decreased during adipogenesis in 3T3-L1 cells and resulting in increased de novo PGF2␣ production (52). The flu-
human subcutaneous preadipocytes. In the latter cells, the prostenol-mediated induction, however, was transient, being
PTGFRv4 (alt4) splice variant responsible for PGF2␣EA/bi- observed for only 1 h, after which Ptgs2 transcript levels
matoprost signaling is also down-regulated. Taken together, decreased significantly below base line. These results differ sig-
these data indicate that not only PGF2␣EA production nificantly from ours in that we not only demonstrated a reversal
decreases in differentiating adipocytes but that the ability of of the decrease in Ptgs2 expression normally associated with
these cells to respond to this compound also diminishes, point- adipogenesis (46, 67) by bimatoprost, PGF2␣EA, and PGF2␣ but
ing to a potential endogenous role for PGF2␣EA as a negative also observed that these compounds greatly increase Ptgs2
regulator of adipogenesis. expression beyond the levels observed in undifferentiated cells
Although we were unable to identify through Basic Local and do so in a long-lasting manner, the effect still being present
Alignment Search Tool analysis mouse splice variants similar to after 2 days. We suspect, therefore, that bimatoprost and
those of the human Ptgfr gene, the fact that 3T3-L1 cells PGF2␣EA also elevate PTGS2 activity and, thus, prostamide/
responded to bimatoprost in a manner and concentration sim- prostaglandin levels, indicating that PGF2␣EA induces a feed-
ilar to those that inhibit adipogenesis in human cells, which do forward loop that may work to keep prostamide/prostaglandin
express FP receptor splice variants, suggests that murine prea- signaling high within preadipocytes. Self-propagation of pros-
dipocytes also express prostamide receptors. This notion is taglandin signaling is not limited to cells of the adipocyte line-
supported by evidence that, like in human cells, pharmacolog- age because a positive feedback loop for PGF2␣ production/
ical antagonism of PGF2␣EA receptors with AGN211335 signaling via Ptgs2 up-regulation was reported in endometrial
reversed PGF2␣EA/bimatoprost-mediated inhibition of adipo- adenocarcinoma cells as well as in the corpus luteum (68, 69).
genesis to a much greater degree than the FP receptor antago- Our data also indicate that this feed-forward loop is not lim-
nist AL8810. These data are in line with ocular hypertension ited to Ptgs2 up-regulation alone because we also found that the
studies indicating that bimatoprost and PGF2␣EA signal expression of FP (PTGFR) and FP alternative transcript 4 (PTG-
through receptors different from FP. However, as has been FRv4), the proposed prostaglandin and PGF2␣EA receptors,
shown for PGF2␣ (35), we observed that PGF2␣EA and bimato- respectively, are down-regulated during adipogenesis in
prost treatment stimulate the phosphorylation of ERK1/2 in 3T3-L1 and human preadipocytes and that PGF2␣EA and
3T3-L1 cells downstream of MEK activation and that this is PGF2␣, along with bimatoprost, not only inhibit this down-reg-
required for the antiadipogenic activity of PGF2␣EA and ulation but also significantly stimulate the expression of these
bimatoprost, indicating some convergence of the signaling mRNAs. These data differ from results published previously in
pathways downstream of PGF2␣EA and PGF2␣ in preadi- which it was shown that FP receptor expression increases with
pocytes. Interestingly, the MEK antagonist PD98059 we uti- 3T3-L1 adipogenesis (46) and that FP receptor stimulation by
lized appeared to be more efficacious at inhibiting the antiadi-
pogenic activities of PGF2␣EA and bimatoprost than of PGF2␣,
which was also shown to require the calcium-calcineurin cas- 5
A. Martella, C. Silvestri, and V. Di Marzo, unpublished observations.

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 PGF2␣EA Inhibits Adipogenesis
fluprostenol decreases FP receptor expression in preadipocytes
(52). These differences may be due to clonal differences in the
3T3-L1 cells used and the fact that activation of FP by different
agonists might produce different effects. However, we must
point out that we observed receptor down-regulation not only
in 3T3-L1 adipocytes but also in human primary adipocytes and
that this phenomenon is consistent with that observed in
murine mesenchymal stem cells with adipogenic capacity (63).
We speculate that activation of the FP prostaglandin and pros-
FIGURE 7. Model of AEA- and PGF2␣EA- mediated adipocyte-preadi-
tamide receptors by PGF2␣ and PGF2␣EA or bimatoprost, pocyte communication during adipogenesis or adaptation to increased
respectively, during the initial phases of adipogenesis, inhibits energy intake. During developmental adipogenesis, adipocyte differentia-
tion starts and self-amplifies when the levels of PTGS2, FAAH, PGF2␣EA, and
the entry of preadipocytes into the differentiation pathway in prostamide receptors (as well as PGF2␣ and FP receptors) start decreasing and
vivo in part by up-regulating not only the production of prosta- those of AEA, CB1, and PPARG (as well as of other proadipogenic signals) start
mides/prostaglandins within preadipocytes but also by sensi- increasing. Adipogenesis and excessive lipogenesis (red dots) are then kept
under control when, in the presence of down-regulated FAAH, adipocyte-
tizing them to paracrine and/or autocrine PGF2␣ and PGF2␣EA produced AEA is oxidized by COX-2 in neighboring preadipocytes. This neg-
signaling within a microenvironment such as that existing ative control is strengthened by PGF2␣EA via further up-regulation of PTGS2
around the adipose vasculature, which provides a niche for adi- and FP expression in preadipocytes and early differentiating adipocytes but
might be blunted in the presence of high FAAH in adipocytes, such as during
pocyte progenitors and adipogenesis (70, 71). In support of this short term high-fat diet-induced hyperinsulinemia and hyperleptinemia.
concept, it has been shown that human microvascular adipose
endothelial cells promote the proliferation of preadipocytes in needs to adapt to changes in dietary energy intake and its stor-
vitro (72) and that adipose capillary endothelial cells produce age as fat, as we have shown here for the WAT of HFD- versus
PGF2␣, which is able to increase the rate at which Swiss 3T3 STD-fed mice. During adipocyte differentiation, adipocytes
cells enter the S phase (73, 74). Thus, prostamide/prostaglandin start producing more and more AEA that, rather than being
signaling may be part of an endogenous mechanism to regulate hydrolyzed in these cells, may be converted by neighboring
preadipocyte number and adipocyte turnover. The loss of peri- undifferentiated preadipocytes into PGF2␣EA to terminate a
orbital fat observed in patients taking bimatoprost for glau- prolipogenic signal and at the same time keep the formation of
coma treatment may be a result of the bimatoprost-mediated further adipocytes on “standby” (Fig. 7). In support of this
down-regulation of this process rather than an induction of hypothesis, it was shown that FAAH, the main hydrolytic
atrophy of adipose tissue, as has been speculated (39). Given the enzyme for AEA, is significantly less expressed in differentiat-
conservation of the adipogenic program between different fat ing 3T3-L1 adipocytes than in preadipocytes (17), thus creating
depots, bimatoprost and other synthetic PGF2␣EA analogs may the ideal conditions to convert AEA into PGF2␣EA because
provide a unique opportunity for the treatment of localized tissue prostamide levels are significantly higher when FAAH
hyperadiposity. Our in vivo data in a vertebrate species phylo- expression is inactivated (as in Faah-null mice) (76). However,
genetically distant from mammals but still capable of producing both leptin and insulin up-regulate the expression of FAAH (15,
prostaglandins, endocannabinoids, and their respective recep- 17, 66). Therefore, the above mechanism for adipogenesis sup-
tors suggest that the phenomenon described here might, in fact, pression should be reversed following food deprivation/refeed-
be relevant not only to adipogenesis in adults but also during ing or when the organism needs to cope with high energy
development. intake, such as during a HFD but before the development of
Perhaps the most novel and intriguing implication of the data insulin or leptin resistance. Under these conditions, the levels of
described here (and, in particular, of our finding that PGF2␣EA, the two hormones and, hence, those of adipocyte FAAH, would
via its own receptor, can exert similar, albeit not identical, be highest, and subsequently AEA would be processed in adi-
effects as PGF2␣, while at the same time amplifying not only its pocytes and not in neighboring preadipocytes, thereby stop-
own production and action but also that of the more studied ping the production of PGF2␣EA formation (as suggested by
cognate compound) lies in the fact that these two prostanoids our data in mice) and disinhibiting adipogenesis. Clearly, such a
originate from two divergent biosynthetic pathways and that mechanism is not a mere replica of the antiadipogenic action of
PGF2␣EA, unlike PGF2␣, is produced at the expense of a well PGF2␣ that, in fact, unlike PGF2␣EA, was shown not to be
established proadipogenic mediator, i.e. AEA. In fact, we have down-regulated in vivo following a short period of HFD.
shown here that PGF2␣EA is the product of AEA metabolism by Accordingly, we also report that the selective inhibition of the
PTGS2 in preadipocytes and, much less so, in differentiating biosynthesis of PGF2␣EA versus that of PGF2␣ with R-flurbipro-
adipocytes. Further, the pattern of both AEA production and fen (51) in preadipocytes enhances their subsequent differenti-
expression of its CB1 receptor during adipogenesis is the oppo- ation into adipocytes.
site to that observed here for PGF2␣EA because AEA levels and In conclusion, we have provided evidence for the presence,
CB1 expression rise with differentiation (15, 75). PGF2␣EA for- biosynthesis from AEA, and mechanism of action in preadi-
mation, as mentioned above, may represent an endogenous pocytes of PGF2␣EA, a novel antiadipogenic mediator. Prosta-
“stop” signal to adipogenesis that keeps under control the “go” mide signaling or its inactivation might represent a novel
signal of this process afforded by AEA activation of CB1 and “switch” mechanism from inhibition to stimulation of adipo-
PPAR-␥ (18, 19). This stop signal may be terminated not only at genesis or vice versa that becomes necessary during develop-
the onset of WAT development but also when the adult WAT ment and/or to comply with alterations in the nutritional state.

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PGF2␣EA Inhibits Adipogenesis
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