JOURNAL OF CELLULAR PHYSIOLOGY 210:1–6 (2007)
Molecular Determinants of FGF-21 Activity—Synergy
and Cross-Talk With PPARg Signaling
JULIE S. MOYERS, TATIYANA L. SHIYANOVA, FARROKH MEHRBOD, JAMES D. DUNBAR,
TIMOTHY W. NOBLITT, KEITH A. OTTO, ANNE REIFEL-MILLER, AND ALEXEI KHARITONENKOV*
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana
and triglyceride lowering effects in rodent models of diabetes. The precise mechanisms whereby FGF-21 regulates metabolism
remain to be determined. Here we describe the early signaling events triggered by FGF-21 treatment of 3T3-L1 adipocytes and
reveal a functional interplay between FGF-21 and peroxisome proliferator-activated receptor gamma (PPARg) pathways that leads
agonist and anti-diabetic agent, rosiglitazone (BRL 49653), enhances FGF-21 action to induce tyrosine phosphorylation of FGF
of the GLUT1 glucose transporter and a marked synergy in stimulation of glucose transport. Together these results reveal a
novel synergy between two regulators of glucose homeostasis, FGF-21 and PPARg, and further define FGF-21 mechanism of
action.J. Cell. Physiol. 210: 1–6, 2007. ? 2006 Wiley-Liss, Inc.
Members of the fibroblast growth factor (FGF) family
play a wide variety of roles in cellular processes
including growth, angiogenesis, and development
(Bottcher and Niehrs, 2005; Grose and Dickson, 2005;
Presta et al., 2005). FGF proteins stimulate these
cellular signaling cascades by binding to single mem-
brane-spanning FGF receptor (FGFR) tyrosine kinases
(Dailey et al., 2005; Eswarakumar et al., 2005). Activa-
tion of the receptors induces dimerization and stimula-
tion of divergent downstream pathways mediated by
FRS-2, MAPK, SHP-2, PI3K and p70S6K, Raf, Stat and
other signaling molecules (Pelech et al., 1986; Carbal-
FGF-21 was identified as a novel FGF family member
(Nishimura et al., 2000). We have reported that FGF-21
cells that is mediated through FGF-21-dependent
increase in GLUT1 protein expression (Kharitonenkov
et al., 2005). FGF-21 induces early signaling events
including tyrosine phosphorylation of FGFR-1, -2 and
FRS-2, and transient MAPK activation. Exogenous
administration of FGF-21 to ob/ob mice results in
dose-dependent glucose and triglyceride lowering and
improved insulin sensitivity with decreased serum
glucagon and insulin levels. FGF-21 transgenic mice
levels and are resistant to weight gain and fat
accumulation on high fat, high carbohydrate diet
(Kharitonenkov et al., 2005). Based on structural
that is generally considered to support cell growth.
number of cell types, and no evidence of mitogenicity
was observed in FGF-21 transgenic mice or upon
rodents (Kharitonenkov et al., 2005). Coupled with its
metabolic activity, the non-mitogenic characteristic of
FGF-21 action makes this molecule an attractive
candidate to treat diabetes and related metabolic
21 signaling. In this study we report that FGF-21
ing events and more prolonged effects involving gene
expression. These studies led to the unexpected finding
that FGF-21 and the peroxisome proliferator-activated
receptor gamma (PPARg) ligand and anti-diabetic
agent, rosiglitazone, exhibit profound synergy in stimu-
lation of 2-deoxyglucose transport and GLUT1 expres-
sion in differentiated adipocytes.
MATERIALS AND METHODS
Expression and purification
FGF-21 was expressed and purified from Escherichia coli as
described (Kharitonenkov et al., 2005).
3T3-L1 cells were from American Type Culture Collection.
Cells were maintained at sub-confluence in Dulbecco’s mod-
ified Eagle’s medium (DMEM) with 10% calf serum. For
differentiation prior to glucose uptake and Western blotting
confluence in DMEM with 10% fetal bovine serum (FBS), 0.25
mM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine
(IBMX), and 5 mg/ml insulin for 48 h; then the media was
changed to DMEM/10% FBS/insulin (5 mg/ml) for 48 h. The
cells were incubated for an additional 9–20 days in DMEM/
10% FBS (changed every other day).
Immunoblotting and immunoprecipitation
3T3-L1 adipocytes were serum starved for 18 h in DMEM/
0.1% bovine serum albumin (BSA), and stimulated with FGF-
21 at the indicated doses and time duration. Cells were lysed
? 2006 WILEY-LISS, INC.
*Correspondence to: Alexei Kharitonenkov, Eli Lilly and Com-
pany, Lilly Corporate Center, Indianapolis, IN 46285.
Received 12 May 2006; Accepted 25 July 2006
and soluble fractions were analyzed by Western blotting with
anti-phospho-Akt (Ser473, Biosource International, Camarillo,
California) or the following antibodies from Cell Signaling
Technology (Beverly, MA): anti-phospho-GSK-3 (Ser21/9),
anti-phospho-MEK1/2, anti-phospho-SHP-2 (Tyr542), anti-SHP-
2, anti-phospho-p70S6K, anti-phospho-Stat3, anti-phospho-
Raf-1, and anti-Raf-1. For immunodetection, goat anti-mouse
and anti-rabbit HRP conjugates were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA) and the ECL detection
system from Amersham Biosciences (Piscataway, NJ) was
were treated for 72 h with FGF-21 (50 nM), rosiglitazone
(1 mM), or both. Cell lysates were immunoblotted with anti-
GLUT1 against the 29 C-terminal amino acids of the human
sequence, anti-GLUT4 (Slieker et al., 1992), anti-PPARg
(Santa Cruz) or anti-GAPDH (Sergienko et al., 1992). PPARg
protein bands were quantitated from film using a Molecular
Dynamics Personal Densitometer SI with ImageQuantTM
software (GE Healthcare, Piscataway, NJ). For detection of
FGFR, cells were treated for 72 h with media (control) or
rosiglitazone (1 mM), followed by treatment with FGF-21
(50 nM) for 10 min. Immunoprecipitation from lysates was
performed using anti-FGFR-2 (C-17, Santa Cruz) or anti-
phosphotyrosine (4G10, Upstate, Charlottesville, VA) anti-
bodies. Immune complexes or lysates were probed with
anti-phosphotyrosine or anti-FGFR-2 antibody.
T plates (Amersham) and subjected to the differentiation
protocol described above. Adipocytes were treated with FGF-
21 or rosiglitazone for 72 h at the indicated concentrations in
DMEM/0.1% BSA. Cells were washed twice with Krebs–
Ringer buffer (15 mM HEPES, pH 7.4, 118 mM NaCl, 4.8 mM
KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 1.2 mM KH2PO4, 0.1%
DOG) (0.1 mCi, 100 mM) was added to each well. Control wells
contained 100 ml of buffer with 2-DOG (0.1 mCi, 10 mM) to
monitor for non-specific transport. The uptake reaction was
carried out for 1 h at 378C, terminated by addition of
cytochalasin B (20 mM), and quantitated with a Wallac 1450
MicroBeta counter (Perkin Elmer, Boston, MA). Statistical
significance was evaluated by Student’s t-test.
3T3-L1 cells were plated at 10,000 cells/well in 96-well plate
in DMEM plus 10% calf serum. Cells were allowed to attach
dexamethasone, 0.5 mM IBMX in DMEM media containing
replaced with DMEM plus 10% FBS containing fresh rosigli-
cells were rinsed with PBS, lysed with 50 ml/well of 0.1%
IGEPAL in PBS for 10 min, and then 100 ml/well of Infinity
Triglyceride Reagent (Sigma-Aldrich, St. Louis, MO) was
added. Plates were incubated at 378C for 1 h and then the OD
quadruplicate. Statistical significance was evaluated by
RNA extraction, cDNA synthesis, quantitative PCR
3T3-L1 adipocytes were treated for 72 h with media
(control), FGF-21 (50 nM), or rosiglitazone (1 mM) alone or in
combination with FGF-21. Total RNA was isolated from
treated cells using TRIzol Reagent from Invitrogen Corpora-
Total RNA (2 mg) was reverse transcribed using SuperScript
First-Strand Synthesis System for RT-PCR (Invitrogen). A
Foster City, CA). The forward and reverse primer sequences
for GLUT1 were 50-GCCCCCAGAAGGTTATTGA-30and 50-
CGTGGTGAGTGTGGTGGATG-30, respectively. The probe
sequence was 50-TTCTACAATCAAACATGGAACCACCGCA-
30.The forward and reverse primer sequences forGLUT4were
GTGTTCCAGTCAC-30, respectively. The probe sequence was
50-ACGATGGGGAACCCCCTCGGCAGCG-30. For PPARg pri-
mer and probes, Assays on DemandTM(Mm00440945_m1)
from Applied Biosystems were used. To normalize for differ-
ences in the amount of total RNA added to each reaction, we
performed amplification of 18S ribosomal RNA as an endogen-
ous control. Statistical significant was evaluated by Student’s
3T3-L1 cells were differentiated in 96-well black walled
tissue culture plates. Cells were serum starved for 18 h in
DMEM/0.1% BSA. Analysis of intracellular Ca2þwas carried
out using Fluorometric Imaging Plate Reader (FLIPR) analy-
sis utilizing methods as described with some modifications
for 90 min at room temperature with 45 ml of DMEM/0.75%
BSA with 5 mM fluo-3 AM calcium-sensitive dye (Molecular
Probes, Eugene, OR). Following PBS washing, 90 ml of Hank’s
0.49 mM MgCl2, 0.41 mM MgSO4, 5.33 mM KCl, 0.44 mM
KH2PO4, 4.17 mM NaHCO3, 137.9 mM NaCl, 0.34 mM
Na2HPO4, and 5.56 mM D-glucose was added to each well.
Cells were treated with vehicle PBS with 0.75% BSA, 50 nM
FGF-21 or 5 mg/ml of the calcium ionophore, A23187 (Sigma-
Aldrich). Fluorescent signal was monitored over a period of
5 min using FLIPR analysis (Molecular Devices, Sunnyvale,
FGF-21 stimulates phosphorylation of signaling
molecules and induces calcium flux
treatment, we treated 3T3-L1 adipocytes with FGF-21
number of candidate signaling molecules. FGF-21
treatment for up to 30 min resulted in stimulation of
pAkt, pGSK-3, and pSHP-2 (Fig. 1A). Total SHP-2
remained unchanged, shown here as a loading control.
In a separate experiment, cells were treated with FGF-
21 from 5 to 30 min. Increased phosphorylation of
MEK1/2, p70S6K, Stat3, and Raf-1 was observed, with
total Raf-1 content shown as a loading control (Fig. 1A).
We also detected no increase in total Akt, p70S6K, GSK-
3b, or Stat3 protein in response to FGF-21 treatment,
indicating that increased phosphorylation was not due
to increased protein levels (not shown).
FGFs are known to stimulate Ca2þmobilization
(Merle et al., 1997). In further efforts to define early
signaling events stimulated by FGF-21, we assayed for
increases in intracellular Ca2þas assessed by the
relative fluorescence of fluo-3 AM calcium-sensitive
dye. Treatment of 3T3-L1 adipocytes with the calcium
ionophore, A23187, as a positive control, resulted in
robust stimulation of Ca2þflux. FGF-21 increased Ca2þ
within 2 min (Fig. 1B) with the effect being dose-
dependent (not shown). This effect appeared to be
specific to 3T3-L1 adipocytes since we were unable to
detect FGF-21 stimulation of Ca2þflux in undifferen-
tiated 3T3-L1 fibroblasts (not shown).
Adipocyte differentiation is increased by FGF-21
A role for FGF signaling in pre-adipocyte differentia-
tion has been demonstrated (Hutley et al., 2004; Patel
et al., 2005). We assessed whether FGF-21 could
stimulate adipogenesis in 3T3-L1 cells by measuring
triglyceride formation after 10 days of treatment with
FGF-21. Incubation of 3T3-L1 fibroblasts with FGF-21
modestly stimulated triglyceride accumulation in a
dose-dependent manner with an increase of 50%
MOYERS ET AL.
Journal of Cellular Physiology DOI 10.1002/jcp
observed at the highest concentration of FGF-21 tested
(Fig. 2A). Since rosiglitazone induces differentiation of
pre-adipocytes into adipocytes (Lehmann et al., 1995;
Hutley et al., 2003), we next determined whether FGF-
21 could augment rosiglitazone effects. A low sub-
maximal concentration (5 nM) of rosiglitazone alone
caused a modest but significant increase in triglyceride
of FGF-21 further increased triglyceride accumulation,
the highest concentration of FGF-21 tested (Fig. 2A).
Previously, we have reported a lack of FGF-21 action in
undifferentiated 3T3-L1 fibroblasts measured by sev-
eral functional readouts including FGFR-1 and -2
phosphorylation, proliferation, glucose uptake (Khar-
itonenkov et al., 2005). The modest effect of FGF-21 on
low level of FGF-21 receptor on these fibroblasts, not
evident by measurement of Ca2þflux (above) or our
previous functional studies.
Synergy of FGF-21 and rosiglitazone for glucose
transport and GLUT1 expression
Since we showed that both FGF-21 and rosiglitazone
stimulated differentiation of 3T3-L1 fibroblasts in
adipocytes, we next examined whether glucose uptake
could be modulated during simultaneous treatment of
differentiated 3T3-L1 adipocytes with both FGF-21 and
rosiglitazone. Stimulation of cells with increasing
concentrations of FGF-21 alone resulted in a dose-
dependent potentiation of glucose uptake with an EC50
of 1.7 nM and a 3.2-fold increase above basal upon
treatment with 50 nM FGF-21 (Fig. 2B). FGF-21
treatment in the presence of rosiglitazone resulted in a
marked increase in the magnitude of glucose uptake,
suggesting a functional synergy upon simultaneous
activation of both FGF-21 and PPARg pathways. A
observed with 50 nM FGF-21 in the presence of 1 mM
rosiglitazone with an EC50 for FGF-21 of 0.7 nM
(Fig. 2B). This effect was dose-dependent for rosiglita-
zone with an EC50 of 7 nM and the maximal effect
occurring at approximately 100 nM rosiglitazone
(Fig. 2C). Rosiglitazone alone had no effect on glucose
tional activation was involved. Based on our previous
findings that FGF-21 increases expression of GLUT1
mRNA and protein in adipocytes (Kharitonenkov et al.,
2005), we hypothesized that transcriptional activation
of GLUT1 played a role in the synergistic effect on
greater extent than that of FGF-21 alone, contributing
to the synergistic effect on glucose uptake. As expected,
FGF-21 alone increased GLUT1 mRNA and protein
levels (Fig. 3A,B). Upon simultaneous treatment with
mRNA and protein was observed compared to control
and either FGF-21 or rosiglitazone treatment alone
(Fig. 3A,B). In comparison, expression of GLUT4 at
mRNA and protein levels was relatively unchanged by
FGF-21 and/or rosiglitazone treatment (Fig. 3A,B).
Interestingly, rosiglitazone treatment for 72 h led to
an increase in GLUT1 mRNA levels compared to
controls (Fig. 3A), but had no effect on GLUT1 protein
levels under the same conditions (Fig. 3B). This is
consistent with the absence of glucose uptake-modulat-
ing activity by rosiglitazone alone (Fig. 2C) but is in
contrast to the 1.5-fold increase in GLUT1 protein and
by Nugent et al. (2001). It is possible that differences in
days of differentiation or treatment time and conditions
account for this disparity.
treatment of 3T3-L1 adipocytes with FGF-21 and
rosiglitazone may be explained at the molecular level
by a robust change in GLUT1 expression, the question
remained what mechanism within FGF-21 and PPARg
pathways underlies the effect and whether pathway
interaction may be a contributing factor leading to a
member of the FGF family, FGF-2, has been previously
demonstrated to stimulate adipogenesis and increase
PPARg in mesenchymal stem cells (Neubauer et al.,
2004) and in 3T3-L1 adipocytes treated with MEK
inhibitor (Prusty et al., 2002). Thus, we evaluated
whether FGF-21 may have an impact on rosiglitazone-
dependent machinery through modulation of PPARg
expression. To determine effects on protein expression,
PPARg antibody following treatment of 3T3-L1 adipo-
cytes with FGF-21. PPARg isoforms were robustly
elevated over the course of FGF-21 treatment in 3T3-
L1 adipocytes (Fig. 4A). Quantitation of the protein
L1 adipocytes were treated with vehicle or 50 nM FGF-21 for the
indicated time. Cell lysates were immunoblotted for phospho- or total
proteins as indicated. B: 3T3-L1 adipocytes were pre-loaded with the
calcium sensitive dye fluo-3 prior to treatment with vehicle, FGF-21 or
the calcium ionophore, A23187. Fluorescence intensity was deter-
mined over the time-course of 5 min after 18 sec of baseline
measurement was recorded. The results are expressed as total relative
fluorescent units (RFU).
FGF-21 stimulates phosphorylation and calcium flux. A: 3T3-
FGF-21 AND PPARg SYNERGY
Journal of Cellular Physiology DOI 10.1002/jcp
determine if this correlates with mRNA levels, PPARg
expression was analyzed by quantitative PCR analysis.
In contrast to protein levels, results showed a trend
(Fig. 4B). PPARg is required for differentiation of
adipocytes and plays a role in maintaining differentia-
tion (Rosen et al., 1999; Tamori et al., 2002). By
increasing PPARg protein levels, FGF-21 may contri-
bute to the maintenance of the mature adipocyte
To determine whether rosiglitazone could in turn
influence FGF-21 actions, we examined FGF-21-depen-
dent tyrosine phosphorylation of FGFR-2 in 3T3-L1
adipocytes that were pre-treated with or without
rosiglitazone. Immunoprecipitation with anti-FGFR-2
antibody, followed by Western blotting with anti-
phosphotyrosine antibody confirmed that FGF-21 alone
stimulated phosphorylation of this receptor (Fig. 5) as
we have shown previously (Kharitonenkov et al., 2005).
Notably, pre-treatment with rosiglitazone resulted in a
marked increase in FGF-21 action to stimulate tyrosine
phosphorylation of FGFR-2 to a greater extent than
tyrosine antibody, followed by Western blotting with
21-dependent signal for cells pre-treated with rosiglita-
zone compared to FGF-21 alone (Fig. 5). This was
without effects on FGFR-2 protein levels (Fig. 5).
Similar results were obtained in anti-phosphotyrosine
immunoprecipitates by Western blotting with anti-
FGFR-1 antibody (not shown).
Together, the results of this article describe the
signaling profile of FGF-21 in 3T3-L1 adipocytes and
demonstrate a novel and marked synergy for stimula-
FGF-21 and rosiglitazone that corresponds to a robust
increase in GLUT1 expression. Although we have
previously shown that FGF-21 stimulates phosphoryla-
was unclear the extent to which FGF-21 signaling
events share commonality with other members of the
glucose uptake. A: 3T3-L1 fibroblasts were treated with FBS media
containing IBMX and dexamethasone (control), sub-maximal rosigli-
tazone, FGF-21, or a combination of FGF-21 and rosiglitazone. Cells
were stained for triglyceride content using Oil Red O staining and
quantitated by absorbance detection at OD490. The graph shows
results expressed as mean?SD, n¼3. *P<0.05, different from
control,#P<0.05, different from rosiglitazone-alone (t-test). B: 3T3-
L1 adipocytes were treated with FGF-21 in the absence or presence of
1 mM rosiglitazone for 72 h and glucose transport was assessed as
FGF-21 and rosiglitazone stimulation of differentiation and
described in Methods Section. Results are expressed as CPM of14C-
deoxyglucose incorporation, mean?SD, n¼4. *P<0.05, significant
difference from control,#P<0.05, significant difference from rosigli-
tazone-alone (t-test). C: To determine the rosiglitazone dose response,
cells were assayed for glucose transport as in (B) following treatment
with vehicleor the indicated concentrations of rosiglitazone alone or in
the presence of FGF-21 (50 nM). Results are expressed as CPM of14C-
rosiglitazone alone. *P<0.05, significant difference from FGF-21
#P<0.05, significant difference from
MOYERS ET AL.
Journal of Cellular Physiology DOI 10.1002/jcp
FGF family. Our current results indicate that FGF-21
shares many signaling features in common with
structurally related ligands. Thus, treatment of cells
tion of calcium flux. At this time, the relative contribu-
tion of each of these signaling cascades to the
physiological events mediated by FGF-21 is not known
and is a subject of ongoing studies.
found that FGF-21 synergizes with rosiglitazone to
stimulate glucose transport. While many aspects of this
intriguing effect remain to be defined, it is unlikely that
the actions of FGF-21 and rosiglitazone to stimulate
glucose uptake in adipocytes are attributed to further
increase in the degree of differentiation of the cells.
FGF-21 alone or in combination with low rosiglitazone
promotes only modest differentiation of 3T3-L1 fibro-
blasts. Furthermore, treatment of differentiated adipo-
cytes with rosiglitazone alone, even at maximal
concentrations, did not increase glucose transport in
we observe no increase in protein expression of GLUT4
which is a marker of differentiation of fibroblasts into
adipocytes (Mora et al., 2002). Rather, FGF-21 alone
increases GLUT1 expression and rosiglitazone signifi-
cantly potentiates this effect, resulting in a synergistic
and marked increase in GLUT1 mRNA and protein
levels compared to either agent alone. Thus, our
observations suggest that the synergism on glucose
uptakemaybeprimarily mediatedby increasedGLUT1
Further examination of the FGF-21 and PPARg
synergy at the mechanistic level demonstrated a
marked functional interplay between FGF-21 and
PPARg pathways. First, we found that FGF-21 stimula-
tion of 3T3-L1 adipocytes elevates PPARg expression.
Since no robust effect of FGF-21 on PPARg mRNA was
of PPARg degradation or both. PPARg protein levels
have been shown to be regulated by ubiquitination and
SUMO-1 modification and this is modified by co-factor
Also, treatment of 3T3-L1 adipocytes with a MEK
inhibitor blocked the decay in PPARg protein in
expression. 3T3-L1 adipocytes were treated with FGF-21 or rosiglita-
zone, alone or in combination. A: RNA was extracted and quantitative
PCR was performed (see Methods Section). Results are expressed as
relative change normalized to control (mean?SD, n¼3). P<0.01,
significant difference from control (t-test). B: Lysates were immuno-
blotted with anti-GLUT1, anti-GLUT4, or anti-GAPDH as a control.
FGF-21 and rosiglitazone stimulation of glucose transporter
adipocytes were untreated or treated with FGF-21 for the indicated
times. Lysates were used for Western blotting with anti-PPARg
antibody and the resulting bands were quantitated by densitometry.
B: 3T3-L1 adipocytes were treated with media (control) or FGF-21 for
72 h. RNA was extracted and quantitative PCR was performed (see
Methods Section). Results are expressed as relative change normal-
ized to control.
FGF-21 increases PPARg protein expression. A: 3T3-L1
FGF-21 AND PPARg SYNERGY
Journal of Cellular Physiology DOI 10.1002/jcp
response to interferon g (Floyd and Stephens, 2002),
which implicates phosphorylation processes in the
degradation of PPARg protein. It remains to be
addressed if and how FGF-21 treatment regulates
PPARg post-translational modification to decrease
Second, we discovered that rosiglitazone also mod-
ulates FGF-21 action, sensitizing its ability to induce
tyrosine phosphorylation FGFR-2. One of the interest-
ing questions remaining to be answered is how this is
regulated since no effect on FGFR-2 protein levels was
observed. PPARg activation may increase the expres-
sion of a receptor co-factor that participates in ligand-
dependent activation. Alternatively, rosiglitazone may
decrease the expression of an inhibitory factor. Future
rosiglitazone on various downstream FGF-21 signaling
pathways. Overall, the results of this article reveal a
novel interaction of FGF-21 and PPARg, adding
mechanistic insights to the role of FGF-21. These
findings suggest that FGF-21, alone or in combination
a useful treatment for diabetes.
We thank A. B. Shanafelt, S. J. Jacobs, R. A. Owens,
and S. K. Karathanasis for helpful discussions, L. J.
Slieker for GLUT1 and GLUT4 antibody, and G.
Cardona for help with PCR analysis.
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FGF-21 for 10 min. Immunoprecipitation from lysates was performed
using anti-FGFR-2 or anti-phosphotyrosine antibody as indicated.
Immune complexes or lysates were probed with anti-phosphotyrosine
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MOYERS ET AL.
Journal of Cellular Physiology DOI 10.1002/jcp