Pterostilbene, a new agonist for the peroxisome proliferator-activated receptor α-isoform, lowers plasma lipoproteins and cholesterol in hypercholesterolemic hamsters

Article abstract of DOI:10.1021/jf0580364

Resveratrol, a stilbenoid antioxidant found in grapes, wine, peanuts and other berries, has been reported to have hypolipidemic properties. We investigated whether resveratrol and its three analogues (pterostilbene, piceatannol, and resveratrol trimethyl ether) would activate the peroxisome proliferator-activated receptor α (PPARα) isoform. This nuclear receptor is proposed to mediate the activity of lipid-lowering drugs such as the fibrates. The four stilbenes were evaluated at 1, 10, 100, and 300 μM along with ciprofibrate (positive control), for the activation of endogenous PPARα in H4IIEC3 cells. Cells were transfected with a peroxisome proliferator response element-AB (rat fatty acyl CoA β-oxidase response element)-luciferase gene reporter construct. Pterostilbene demonstrated the highest induction of PPARα showing 8- and 14-fold increases in luciferase activity at 100 and 300 μM, respectively, relative to the control. The maximal luciferase activity responses to pterostilbene were higher than those obtained with the hypolipidemic drug, ciprofibrate (33910 and 19460 relative luciferase units, respectively), at 100 μM. Hypercholesterolemic hamsters fed with pterostilbene at 25 ppm of the diet showed 29% lower plasma low density lipoprotein (LDL) cholesterol, 7% higher plasma high density lipoprotein (HDL) cholesterol, and 14% lower plasma glucose as compared to the control group. The LDL/HDL ratio was also statistically significantly lower for pterostilbene, as compared to results for the control animals, at this diet concentration. Results from in vitro studies showed that pterostilbene acts as a PPARα agonist and may be a more effective PPARα agonist and hypolipidemic agent than resveratrol. In vivo studies demonstrate that pterostilbene possesses lipid and glucose lowering effects.

Full text of DOI:10.1021/jf0580364

J. Agric. Food Chem. 2005, 53, 3403−3407 3403
Pterostilbene, a New Agonist for the Peroxisome
Proliferator-Activated Receptor -Isoform, Lowers Plasma
r
Lipoproteins and Cholesterol in Hypercholesterolemic Hamsters
AGNES M. RIMANDO,*,† RANGASWAMY NAGMANI,‡ DENNIS R. FELLER,§ AND
|
WALLACE YOKOYAMA
Natural Products Utilization Research Unit, ARS, U.S. Department of Agriculture, P.O. Box 8048,
University, Mississippi 38677, Department of Pharmacology, School of Pharmacy, University of
Mississippi, University, Mississippi 38677, and Western Regional Research Center, ARS,
U.S. Department of Agriculture, 800 Buchanan St., Albany, California 94710
Resveratrol, a stilbenoid antioxidant found in grapes, wine, peanuts and other berries, has been
reported to have hypolipidemic properties. We investigated whether resveratrol and its three analogues
(pterostilbene, piceatannol, and resveratrol trimethyl ether) would activate the peroxisome proliferator-
activated receptor R (PPARR) isoform. This nuclear receptor is proposed to mediate the activity of
lipid-lowering drugs such as the fibrates. The four stilbenes were evaluated at 1, 10, 100, and 300
µM along with ciprofibrate (positive control), for the activation of endogenous PPARR in H4IIEC3
cells. Cells were transfected with a peroxisome proliferator response element-AB (rat fatty acyl CoA
â-oxidase response element)sluciferase gene reporter construct. Pterostilbene demonstrated the
highest induction of PPARR showing 8- and 14-fold increases in luciferase activity at 100 and 300
µM, respectively, relative to the control. The maximal luciferase activity responses to pterostilbene
were higher than those obtained with the hypolipidemic drug, ciprofibrate (33910 and 19460 relative
luciferase units, respectively), at 100 µM. Hypercholesterolemic hamsters fed with pterostilbene at
25 ppm of the diet showed 29% lower plasma low density lipoprotein (LDL) cholesterol, 7% higher
plasma high density lipoprotein (HDL) cholesterol, and 14% lower plasma glucose as compared to
the control group. The LDL/HDL ratio was also statistically significantly lower for pterostilbene, as
compared to results for the control animals, at this diet concentration. Results from in vitro studies
showed that pterostilbene acts as a PPARR agonist and may be a more effective PPARR agonist
and hypolipidemic agent than resveratrol. In vivo studies demonstrate that pterostilbene possesses
lipid and glucose lowering effects.
KEYWORDS: Pterostilbene; PPARr; cholesterol; lipoprotein
INTRODUCTION
role in carbohydrate and lipid homeostasis and govern other
biological processes such as energy metabolism, cell prolifera-
tion and differentiation, and inflammation (3-5). The PPARR
isoform is predominantly involved in fatty acid and lipid
catabolism and import and activation of genes involved in fatty
acid oxidation in the liver, heart, kidney, and skeletal muscles
(6, 7). In the liver, activation of PPARR leads to increased
â-oxidation of fatty acids and decreased triglyceride and very
low density lipoprotein (VLDL) synthesis (8). Activation of
PPARR also leads to reduction of triglyceride because of
repression of hepatic apolipoprotein C-III and an increase in
lipoprotein lipase gene expression (9). Furthermore, PPARR
activation causes induction of hepatic apoliporotein A-I and A-II
expression, in humans, leading to increased plasma HDL
cholesterol. PPARR agonists are also known to slow the
progression of premature coronary atherosclerosis (6) and have
The peroxisome proliferator-activated receptor (PPAR) iso-
forms are members of the nuclear receptor superfamily of
ligand-activated transcription factors. They were first identified
in Xenopus frogs as receptors that induce the proliferation of
peroxisomes (1). Three PPAR isoforms are known as follows:
PPARR, PPARγ, and PPARδ. The PPARs control gene expres-
sion by interaction with specific response elements in the
promoter region of target genes (2). The PPARs play a central
* To whom correspondence should be addressed. Tel: 662-915-1037.
Fax: 662-915-1035. E-mail: arimando@msa-oxford.ars.usda.gov.
† Natural Products Utilization Research Unit, ARS, U.S. Department of
Agriculture.
‡ Current address: Enzymatic Therapy, Inc., Product Development, 825
Challenger Drive, Green Bay, WI 54311.
§ University of Mississippi.
| Western Regional Research Center, ARS, U.S. Department of Agri-
culture.
10.1021/jf0580364 CCC: $30.25
© 2005 American Chemical Society
Published on Web 03/31/2005

3404 J. Agric. Food Chem., Vol. 53, No. 9, 2005
Rimando et al.
When the cultures were 75% confluent, the cells were detached and
transfected with the firefly LUC reporter gene construct containing
PPRE-AB (peroxisome proliferator response element with rat fatty acyl
CoA â-oxidase AB promoter region sequence). Detached cells were
electroporated in 400 µL of medium containing the plasmid at 190 V
and a single 70 ms pulse using BTX disposable cuvettes (model 640;
4-mm gap) with a BTX model T 820 electroporator (BTX Instrument
Division, Harvard Apparatus, Inc., Holliston, MA). The transfected cells
were plated at a density of 100000 cells/well in 96 well microtiter plates
(Cultureplate, Packard) and allowed to grow for 24 h. After 24 h, the
cells were treated with ciprofibrate at 10 and 100 µM and resveratrol
and its three analogues at 1, 10, 100, and 300 µM concentrations. After
incubation with the test compounds for 30 h, the cells were lysed and
the LUC activity was measured using a LucLite assay kit (Packard).
Light output was detected by a TopCount microplate scintillation
counter in the single photon counting mode luminometer (Packard
Instrument Company, Meriden, CT).
been demonstrated to regulate metabolism of amino acids in
the liver (10).
Resveratrol is a well-known antioxidant (11, 12) and cancer
chemopreventive (13) compound present in grapes and wine.
Its occurrence in wine has been linked to a low incidence of
fatal coronary heart disease among populations consuming wine
moderately (14, 15). Dietary resveratrol at 50 parts per million
(ppm) suppressed blood serum lipid peroxidase levels in rats
and dose-dependently suppressed serum triglyceride and VLDL
and low density lipoprotein (LDL) cholesterol levels (16).
Pterostilbene is another grape compound that was also found
to have antioxidant (11, 17) and cancer chemopreventive
properties similar to resveratrol (17). Pterostilbene has antidia-
betic (18) properties and is cytotoxic to a number of cancer
cell lines in vitro (19). A cardiotonic Ayurvedic preparation,
Darakchasava, which has Vitis Vinifera as the main ingredient,
was shown to contain resveratrol and pterostilbene (20). Like
resveratrol and pterostilbene, piceatannol has cancer chemo-
preventive properties (21) and is a stronger antioxidant than
resveratrol and a potent antiarrhythmic agent (22, 23). Piceat-
annol showed antiallergic effects in experimental models of type
I allergy (24). Resveratrol trimethyl ether was found to be more
cytotoxic than resveratrol in cultured human lung and colon
cancer cells (25).
In view of the above-mentioned reports on the hypolipidemic
property and a recent report on the activation of PPAR isoforms
by resveratrol (26) and cardioprotective effects of drinks and
preparations containing resveratrol and pterostilbene, we propose
that these structurally related compounds may be activators of
PPARR. Piceatannol and resveratrol trimethyl ether were of
interest because their biological activity profiles are similar to
that of resveratrol and pterostilbene and in some assays are
reported to be more potent. We therefore also investigated
whether these analogues would activate PPARR isoform.
Data Analysis. Data are expressed as relative light or LUC units,
and the changes in LUC response (counts per minute) are plotted vs
the analogue concentration. Results are also expressed as fold-induction
over the corresponding control value. Each analogue treatment was
done in triplicate or quadruplicate. Data were analyzed using GraphPad
Prism 3 software.
Hamster Feeding. Male golden Syrian hamsters (Charles River,
Wilmington, MA), 7-8 weeks old and 34-41 g, were housed
individually in wire bottom cages in a room kept at 20-22°C, 60%
relative humidity, and 12 h light and dark cycle. The hamsters were
fed a powdered stock diet (Rodent Lab Chow 5001, Purina Mills, St.
Louis, MO) for 7 days. Following the initial 7 day period, 8-10 animals
were randomly assigned from a weight-sorted list to each of two test
diets (R-cellulose control and pterostilbene-fortified diet). The diet
consisted of 80 g of anhydrous butterfat, 100 g of corn oil, 20 g of
Menhaden fish oil, 1.5 g of cholesterol, 50 g of microcrystalline
cellulose, 200 g of casein, 497.5 corn starch, 3 g of methionine, 3 g of
choline bitartrate, 35 g of mineral mix, and 10 g of vitamin mix. The
diets were prepared by dissolving powdered cholesterol into warmed
butterfat, followed by the addition of corn and fish oil. The liquid fat
was also added to the dry ingredients while stirring. The diet for the
test group was fortified with pterostilbene at 25 mg/kg of diet.
Pterostilbene was dissolved in about 50 mL of ethanol and added to
the stirring dry ingredients.
Food intake was measured twice each week, and body weights were
monitored once a week. After 21 days, the animals were anesthesized
with isofluorane, and then, 4-5 mL of blood was taken by heart
puncture. Blood was analyzed for total cholesterol by the cholesterol
oxidase method and lipoprotein cholesterol by size exclusion chroma-
tography as previously described (28). All animal procedures were
approved by the Animal Care and Use Committee, Western Regional
Research Center, U.S. Department of Agriculture (Albany, CA) and
conformed to the principles in Guide for the Care and Use of Laboratory
Animals (Committee on Care and Use of Laboratory Animals 1985).
Plasma blood glucose was determined using a blood glucose meter
(Fast Glucose Meter-Precision Xtra, MediSense, Bedford, MA).
EXPERIMENTAL SECTION
General Experimental Procedures. Resveratrol and piceatannol
were commercial samples obtained from Sigma-Aldrich (St. Louis, MO)
and Calbiochem (San Diego, CA), respectively. H4IIEC3 cells were
purchased from the American Type Culture Collection (Rockville, MD).
The PPRE-AB luciferase (LUC) gene reporter construct was provided
by Dr. Daniel J. Noonan (Department of Biochemistry, University of
Kentucky, Lexington, KY). The LUC assay kit was obtained from
Promega Corporation (Madison, WI). NMR experiments were carried
out on a Bruker Avance DRX (500 MHz) instrument. For thin-layer
chromatography (TLC), aluminum-backed silica gel F254 plates, 10 cm
× 20 cm, 0.2 mm thick (EM Science, Gibbstown, NJ) were used. For
preparative layer chromatography (PLC), Merck silica gel F254 20 cm
× 20 cm, 1.0 mm thick plates (VWR Scientific, Atlanta, GA) were
used. All solvents used were high-performance liquid chromatography
grade (Fisher Scientific, Suwanee, GA).
RESULTS AND DISCUSSION
Preparation of Pterostilbene and Resveratrol Trimethyl Ether.
Pterostilbene and resveratrol trimethyl ether were prepared by partial
methylation of trans-resveratrol. To a solution of trans-resveratrol (150
mg in 3.0 mL of MeOH), diazomethane was added dropwise, and the
reaction was monitored by TLC for the methylated products. The
reaction solution was dried under vacuum. The partially methylated
products were purified by PLC (developing solvent, hexane:EtOAc,
8:2; Rf 0.6 and 0.8 for pterostilbene and resveratrol trimethyl ether,
respectively). The identity and structure of these compounds were
confirmed by comparison with published spectroscopic data.
Activation of PPARr in Rat Liver Cells. The conditions for these
experiments are essentially as described (27). Briefly, H4IIEC3 (rat
hepatoma cell line) cells were grown in 150 mm × 25 mm Petri dishes
with 1:1 mixture of Dulbecco’s modified Eagle’s medium:Ham’s F12
medium (DMEM/F12) supplemented with 10% (v/v) fetal bovine serum
and penicillin G sodium (100 U/mL) or streptomycin (100 µg/mL).
Of the four stilbene analogues (Figure 1) tested, pterostilbene
demonstrated the highest induction of LUC activity at 100 and
300 µM in H4IIEC3 cells transfected with the PPRE-AB-LUC
reporter gene construct (Figure 2). The maximal LUC responses
to pterostilbene at 100 and 300 µM were about 8- and 14-fold,
respectively, when compared to the corresponding control
values. The maximal LUC activity responses to 100 µM
ciprofibrate were about 5-fold as compared to control. These
results showed that PPARR activation by pterostilbene (33910
( 788 relative LUC units) is higher than that of ciprofibrate
(19460 ( 1466 relative LUC units) at 100 µM. However, at a
lower concentration (10 µM), PPARR activation by pterostilbene
was lower than that of ciprofibrate. These results indicate that
ciprofibrate, a potent hypolipidemic agent of the fibrate class,

Pterostilbene as a New PPAR
R
Agonist
J. Agric. Food Chem., Vol. 53, No. 9, 2005 3405
Table 1. Effect of Pterostilbene on Plasma Cholesterol and Glucose
Levels and Liver and Body Weights in Hamsters
control
pterostilbene
plasma lipoprotein cholesterol (mg/dL)a
VLDL
LDL
HDL
total
99.3
320.9
127.4
547.6
2.6
±
±
±
±
±
15.3
4.9
1.1
6.7
0.37
82.7
228.1
137.0
447.8
1.8
±
±
±
±
±
15.7
4.2
2.1
5.6
0.39
LDL/HDL ratio
final weights (g)a
body weight
liver weight
liver/body weight ratio
plasma glucose (mg/dL)a
animals per group (n)
100.9
6.6
6.6
216.5
10
±
±
±
±
2.0
110.5
7.9
7.1
±
±
±
±
2.7
0.18
0.09
10.1
0.28
0.12
8.7
185.1
8
a Values are expressed as the means
± SEM.
resveratrol and the three other stilbenes investigated in this study
did not activate PPARγ using a MCF-7 breast tumor cell line
(data not shown). The difference in responses to resveratrol may
be due to the difference in experimental protocols and conditions
under which the tests were performed. In our study, endogenous
PPARR population was used and the rat hepatoma cells were
transfected with only the PPRE-AB-LUC reporter gene plasmid.
In the published study, the PPARR receptor was cotransfected
with the PPRE-AB response elements resulting in a higher
density of PPARR receptor in the cell-based assays. The
difference in cell type and species may also explain the
discrepancy between our result and those of Inoue’s group. It
has been shown that clofibrate induced PPARR expression
greater than linoleic acid in a murine hepatoma cell line;
however, in a human hepatoma cell line, linoleic acid was the
most effective ligand (29). In their study, Inoue et al. (26) tested
resveratrol for the activation of PPARR in the absence of a
positive control. In our study, we have determined the activity
of resveratrol together with its three analogues and ciprofibrate
as a positive control. Our results indicate that pterostilbene, like
ciprofibrate, acts as an agonist of PPARR in H4IIEC3 cells,
whereas the three other stilbene analogues are not activators of
PPARR.
Figure 1. Structures of compounds tested for activation of PPAR
liver cells.
R in rat
Figure 2. Effect of stilbenes on PPAR
R in H4IIEC3 cells transfected with
PPRE-AB LUC reporter gene plasmid. Cip, ciprofibrate; Res, resveratrol;
Pic, piceatannol; Pte, pterostilbene; and Rte, resveratrol trimethyl ether.
**Significantly different from control, p
)
0.001; ***highly significantly
Several PPARR specific agonists have been shown to reduce
hyperlipidemia and insulin resistance in hamsters fed normal
chow, high fat chow, or high fructose and fat augmented chow
diets (30-32, respectively). We conducted an animal feeding
study on high fat fed hamsters to investigate the effect of
pterostilbene on plasma levels of cholesterol and lipoproteins,
having found this compound to activate PPARR in our in vitro
assay. Hamsters were fed a single dose of 25 mg pterostilbene
per kg of diet. This dose was decided based upon published
studies on resveratrol feeding using rats, i.e., 10 and 50 ppm of
the diet (16), and is much lower than the dose of methylclofena-
pate used in a study, i.e., 25 mg/kg animal body weight (30),
or of fenofibrate, 30-300 mg/kg (31), fed to hamsters. Ptero-
stilbene intake is estimated to be about 2.5 mg/kg body weight
of animal based on approximately 10 g/day feed intake in a
100 g animal. As compared to the control group, plasma LDL
cholesterol was 29% lower and plasma high density lipoprotein
(HDL) cholesterol was 7% higher in the pterostilbene-fed group
(Table 1). The total plasma cholesterol was 18% lower as
compared with control animals. The LDL cholesterol and total
plasma cholesterol values for the pterostilbene-fed hamsters are
statistically significantly different from those of the control
animals (as analyzed by two-tailed t-test; p < ) 0.05). The
total cholesterol was lowered about 25% by fenofibrate in the
high fructose- and fat-fed hamster model and lowered by about
different from control, p 0.0001; n
is solvent control without test compounds.
)
) 4. Control is media only; DMSO
is more potent than pterostilbene and that higher concentrations
of pterostilbene (>100 µM) are required to produce a concen-
tration-dependent increase in PPARR activation as measured
by increased LUC activities. Piceatannol and resveratrol tri-
methyl ether did not significantly increase LUC activity at all
of the concentrations tested. Pterostilbene, as well as resveratrol,
at 1 and 10 µM, produced no significant change in LUC activity,
when compared to the control value. Resveratrol was toxic to
H4IIEC3 cells at 100 and 300 µM. In summary, it is noteworthy
that the presence of two methoxy groups (as in pterostilbene)
but not three methoxy groups (as in resveratrol trimethyl ether)
or three or four unmasked hydroxyl groups (as in resveratrol
and piceatannol, respectively) was important as a structural
requirement for PPARR activation in H4IIEC3 cells within this
series of compounds.
Our result on resveratrol differs from that of a published study
(26), where resveratrol was shown to activate PPARR in assays
using five different cell lines: monkey kidney CV-1 cells,
murine primary cortical cultures, bovine brain microvessel
vascular endothelial cells, human umbilical venous endothelial
cells, and bovine arterial endothelial cells. Inoue’s group also
found resveratrol to activate PPARγ in these cells. In contrast,

3406 J. Agric. Food Chem., Vol. 53, No. 9, 2005
Rimando et al.
30% in the high fat chow-fed hamsters. Methylclofenapate had
no effect on plasma cholesterol in hamsters fed chow (30). The
LDL/HDL ratio was also statistically significantly lower for
pterostilbene, as compared to results for the control animals at
this diet concentration. Weight gain was similar for both groups.
The plasma glucose was 14% lower in the pterostilbene-fed
group as compared to the control group. This result is in
agreement with an earlier study that showed pterostilbene to
lower plasma blood glucose in hyperglycemic rats (18) and
fenofibrate in high fat-fed hamsters (31, 32). It is worth
mentioning that at the same time the feeding experiment with
pterostilbene was done, feeding studies with two tomato varieties
were also conducted. Freeze-dried tomatoes were fed at 10%
of the total diet. Plasma cholesterol lowering was greater for
pterostilbene (results not presented). The plasma lipid profile
of the pterostilbene-fed hamsters showed improved cholesterol
levels after 21 days in this diet, as compared to the hypercho-
lesterolemic (control) animals. The plasma glucose levels were
also lowered. These results are similar to results obtained with
fenofibrate, a known PPARR specific agonist, in hamsters fed
high fat diets. While results are from a single dose feeding study
only, the data on lipid and glucose lowering effects appear to
support the results obtained in vitro on pterostilbene’s activation
of PPARR. In summary, the comparable potency of pterostilbene
to the known PPARR specific agonist, fenofibrate, at about 1/10
the dose warrants further studies of pterostilbene in animal
models of hyperlipidemia and diabetes.
experiments will be required to determine if pterostilbene is
acting primarily as a PPARR agonist.
ACKNOWLEDGMENT
We thank Dale Nagle and Flor Mora of the Department of
Pharmacognosy, School of Pharmacy, University of Mississippi,
for performing studies of the stilbenes in the PPARγ assay.
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Pterostilbene as a New PPAR
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Agonist
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Received for review February 2, 2005. Accepted February 15, 2005.
The PPARr studies were supported in part by the National Center for
Natural Products Research and the Department of Pharmacology at
the University of Mississippi.
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