Hydroxytyrosol promotes mitochondrial biogenesis and mitochondrial function in 3T3-L1 adipocytes
Hydroxytyrosol (HT) in extra-virgin olive oil is considered one of the most important polyphenolic compounds responsible for the health benefits of the Mediterranean diet for lowering incidence of cardiovascular disease, the most common and most serious complication of diabetes. We propose that HT may prevent these diseases by a stimulation of mitochondrial biogenesis that leads to enhancement of mitochondrial function and cellular defense systems. In the present study, we investigated effects of HT that stimulate mitochondrial biogenesis and promote mitochondrial function in 3T3-L1 adipocytes. HT over the concentration range of 0.1-10 micromol/L stimulated the promoter transcriptional activation and protein expression of peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha (PPARGC1 alpha, the central factor for mitochondrial biogenesis) and its downstream targets; these included nuclear respiration factors 1 and 2 and mitochondrial transcription factor A, which leads to an increase in mitochondrial DNA (mtDNA) and in the number of mitochondria. Knockdown of Ppargc1 alpha by siRNA blocked HT's stimulating effect on Complex I expression and mtDNA copy number. The HT treatment resulted in an enhancement of mitochondrial function, including an increase in activity and protein expression of Mitochondrial Complexes I, II, III and V; increased oxygen consumption; and a decrease in free fatty acid contents in the adipocytes. The mechanistic study of the PPARGC1 alpha activation signaling pathway demonstrated that HT is an activator of 5'AMP-activated protein kinase and also up-regulates gene expression of PPAR alpha, CPT-1 and PPAR gamma. These data suggest that HT is able to promote mitochondrial function by stimulating mitochondrial biogenesis.
Hydroxytyrosol promotes mitochondrial biogenesis and mitochondrial
function in 3T3-L1 adipocytes
, Weili Shen
, Guangli Yu
, Haiqun Jia
, Xuesen Li
, Zhihui Feng
, Ying Wang
, Karin Wertz
, Edward Sharman
, Jiankang Liu
Institute for Nutritional Science, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
Graduate School of the Chinese Academy of Sciences, Beijing, China
School of Medicine and Pharmacy, Ocean University of China, China
DSM Nutritional Products, R&D Human Nutrition and Health, Basel, Switzerland
Department of Neurology, University of California, Irvine, CA 92697-4292, USA
Institute of Mitochondrial Biology and Medicine, Department of Biology and Engineering, The Key Laboratory of Biomedical Information Engineering of Ministry of Education,
Xi’an Jiaotong University School of Life Science and Technology, Xi’an 710049, China
Received 13 July 2008; received in revised form 20 March 2009; accepted 27 March 2009
Hydroxytyrosol (HT) in extra-virgin olive oil is considered one of the most important polyphenolic compounds responsible for the health benefits of the
Mediterranean diet for lowering incidence of cardiovascular disease, the most common and most serious complication of diabetes. We propose that HT may
prevent these diseases by a stimulation of mitochondrial biogenesis that leads to enhancement of mitochondrial function and cellular defense systems. In the
present study, we investigated effects of HT that stimulate mitochondrial biogenesis and promote mitochondrial function in 3T3-L1 adipocytes. HT over the
concentration range of 0.1–10 μmol/L stimulated the promoter transcriptional activation and protein expression of peroxisome proliferator-activated receptor
(PPAR) coactivator 1 alpha (PPARGC1α, the central factor for mitochondrial biogenesis) and its downstream targets; these included nuclear respiration factors 1
and 2 and mitochondrial transcription factor A, which leads to an increase in mitochondrial DNA (mtDNA) and in the number of mitochondria. Knockdown of
Ppargc1α by siRNA blocked HT's stimulating effect on Complex I expression and mtDNA copy number. The HT treatment resulted in an enhancement of
mitochondrial function, including an increase in activity and protein expression of Mitochondrial Complexes I, II, III and V; increased oxygen consumption; and a
decrease in free fatty acid contents in the adipocytes. The mechanistic study of the PPARGC1α activation signaling pathway demonstrated that HT is an activator
of 5′AMP-activated protein kinase and also up-regulates gene expression of PPARα, CPT-1 and PPARγ. These data suggest that HT is able to promote
mitochondrial function by stimulating mitochondrial biogenesis.
© 2009 Elsevier Inc. All rights reserved.
Keywords: 5′AMP-activated protein kinase (AMPK); Fatty acid oxidation; Mitochondrial transcription factor A (Tfam); Mitochondrial DNA (mtDNA); Nuclear
respiration factors 1 and 2 (Nrf1 and Nrf2); Peroxisome proliferator-activated receptor coactivator 1 alpha (PPARGC1α)
Mitochondrial dysfunction plays a central role in a wide range of
age-associated disorders and various forms of cancer , as well as
type 2 diabetes . Increasing evidence shows that mitochondrial
metabolism and ATP synthesis decline in concert with a reduction of
key factors regulating mitochondrial biogenesis in patients with
insulin resistance, type 2 diabetes and obesity [3–6]. Key factors
regulating this process include peroxisome proliferator-activated
receptor (PPAR) coactivator 1 alpha (PPARGC1α) and the nuclear
respiratory factors (Nrfs). It also has been shown that a reduction of
mitochondrial DNA (mtDNA) copy number in adipose tissue from
diabetic volunteers and treatment with thiazolidinedione (TZD), an
insulin-sensitizing drug currently used in treating type 2 diabetes,
restored diminished mtDNA content and expression of genes involved
in mitochondrial biogenesis and fatty acid oxidation [6,7]. Therefore,
with the emerging evidence that mitochondrial dysfunction is
associated with various diseases, it has been suggested that promot-
ing mitochondrial biogenesis, just as improving adipocyte metabo-
lism, could be a strategy for preventing and reversing various diseases,
including cardiovascular disease, cancer, insulin resistance, obesity
and diabetes [2,8–10].
The Mediterranean diet has been associated with a lower
incidence of certain cancers and of cardiovascular disease, which is
the most common and serious complication of diabetes [11–13].
Olive oil is the principal source of fats in the Mediterranean diet, and
hydroxytyrosol (HT), a polyphenolic constituent of extra-virgin olive
oil, is considered to be one of the most potent determinants of its
efficacy [14–17]. Studies on the mechanism of HT's action have
focused on its antioxidant properties so far [14,18,19].Wehave
vailable online at www.sciencedirect.com
Journal of Nutritional Biochemistry xx (2009) xxx – xxx
Corresponding author. Tel.: +86 29 82664232.
E-mail address: email@example.com (J. Liu).
0955-2863/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
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recently shown that HT protects retinal pigment epithelial cells from
acrolein-induced oxidative damage and mitochondrial dysfunction
by inducing detoxifying Phase II enzymes . Based on our
hypothesis and recent results [21,22] that mitochondrial nutrients
can improve mitochondrial function through stimulating mitochon-
drial biogenesis, we hypothesize that the Mediterranean diet or
supplementation with HT could stimulate mitochondrial function
and prevent diabetes and obesity-related mitochondrial dysfunction,
thus reducing the risk of cardiovascular disease. Therefore, in the
present study, we determined whether treatment o f 3T3-L1
adipo cytes with HT could improve mitochondrial function by
stimulating mitochondrial biogenesis.
It has been observed that the expression of regulatory factors for
mitochondrial biogenesis was reduced in adipose tissues of diabetic
and obese subjects [3,6]. Therefore, we used adipocytes as a model to
study the effects of HT. In the present study, we first examined the
effect of HT on the protein expression of PPARGC1α, the key regulator
of mitochondrial biogenesis, and of its downstream targets, nuclear
respiration factors 1 and 2 (Nrf1 and Nrf2) and mitochondrial
transcription factor A (Tfam). Second, we examined mtDNA; protein
expression of Mitochondrial Complexes I, II, III, IV and V; and
mitochondrial mass/numbers. Third, we monitored the expression
of proteins or genes related to fatty acid oxidation, adipogenesis and
mitochondrial function, including activities of Mitochondrial Com-
plexes I, II, III, IV and V; oxygen consumption; and free fatty acid (FFA)
content. Fourth, we investigated the effects of HT on signaling
pathways involving phosphorylation of 5′AMP-activated protein
kinase (AMPK) and acetyl-CoA carboxylase (ACC).
2. Materials and methods
The AMPK activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), was
from Sigma (St. Louis, MO); anti-phospho-(Thr 172)-AMPK (pAMPK), total AMPK
(tAMPK), anti-phospho-ACC (Ser79) (pACC) and total ACC (tACC) were from Cell
Signaling Technology, Inc. (Beverly, MA); anti-OxPhos Complexes I, II, III, IV and V were
from Invitrogen (Carlsbad, CA); anti- α-tubulin and anti-β-actin were from Sigma; anti-
PPARGC1α (Santa Cruz, Heidelberg, Germany); the Reverse Transcription System Kit
was from Promega (Mannheim, Germany); HotStarTaq was from Takara (Otsu, Shiga,
Japan); primers were synthesized by Bioasia Biotech (Shanghai, China); and TRIzol and
other reagents for cell culture were from Invitrogen. HT was from DSM Nutritional
Products Ltd., Switzerland, and used in all experiments.
2.2. Cell culture and differentiation
Cell culture and differentiation of 3T3-L1 cells have been extensively used as a
model of adipogenic differentiation and insulin action. 3T3-L1 cells undergo growth
arrest and initiate a program of differentiation manifested by large lipid droplet
accumulation upon hormonal stimulation. In parallel, these cells become sensitive to
insulin, express Glut4 and display insulin-induced activation of glucose uptake
comparable to that seen in primary adipose cells . In the present study, murine
3T3-L1 pre-adipocytes (American Type Culture Collection, Manassas, VA) were
cultured in DMEM supplemented with 10% (v/v) fetal bovine serum and allowed to
reach confluence. Differentiation of pre-adipocytes was initiated with 1.0 μmol/L
insulin, 0.25 μmol/L dexamethasone and 0.5 mmol/L 3-isobutyl-1-methylxanthine in
DMEM supplemented with 10% (v/v) fetal bovine serum. After 48 h, the culture
medium was replaced with DMEM supplemented with 10% fetal bovine serum and 1.0
μmol/L insulin. The culture medium was changed every other day with DMEM
containing 10% (v/v) fetal bovine serum. Cells were used at 9 to 10 days following
induction of differentiation and when 90% exhibited the adipocyte phenotype.
2.3. Transient transfection and promoter activity assay
A 2-kb Ppargc1α promoter in pGL3-basic luciferase reporter construct was a gift
from Dr. X. Ge (Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences,
Shanghai, China). Cells were fully differentiated as described above and then seeded in
24-well plates at about 80% confluence and grown overnight. The cells were transiently
transfected with pGL3-Ppargc1α or pGL3-basic plasmid using the Cell Line Nucleofector
Kit from Amaxa (Gaithersburg, MD) following the manufacturer's instructions. The
Renilla vector was used to monitor the transfection efficiency. The transfected cells
were cultured for 18–20 h and then incubated with HT (1 μmol/L) for 24 h. Cells were
lysed, and the reporter activity was measured by a luciferase assay kit (KenReal,
Shanghai, China) with a luminometer (Berthold Technologies, Bad Wildbad, Germany).
The relative Luc activity was calculated as the ratio of firefly Luc activity to Renilla luc
activity. Transfections were performed in duplicate and repeated at least three times.
2.4. Western blot analysis
After treatment with HT, cells were washed twice with ice-cold phosphate-
buffered saline (PBS), lysed in sample buffer (62.5 mmol/L Tris–Cl, pH 6.8, 2% SDS and 5
mmol/L dithiothreitol) at room temperature and vortexed. Cell lysates were then
boiled for 5 min and cleared by centrifugation (13,000 rpm, 10 min at 4°C). Protein
concentrations were determined using the Bio-Rad DC protein assay. The soluble
lysates (10 μg per lane) were subjected to 10% SDS-PAGE; proteins were then
transferred to nitrocellulose membranes and blocked with 5% nonfat milk/TBST for 1 h
at room temperature. Membranes were incubated with primary antibodies directed
against anti-α-tubulin (1:5000), PPARGC1α (1:1000), phospho-(Thr 172)-AMPK
(1:1000), tAMPK (1:1000), phospho-ACC (Ser79) (1:1000), tACC (1:1000), anti-
OxPhos Complex I (NADH ubiquinone oxidoreductase 39-kDa subunit, 1:2000), anti-
OxPhos Complex II (succinate–ubiquinone oxidoreductase 70-kDa subunit, 1:2000),
anti-OxPhos Complex III (ubiquinol–cytochrome c oxidoreductase core II 50 kDa,
1:2000) or anti-OxPhos Complex V (ATP synthase, 53 kDa) in 5% milk/TBST at 4°C
overnight. After washing membranes wi th TBST three times, membranes were
incubated with horseradish-peroxidase-conjugated secondary antibody for 1 h at
room temperature. Western blots were developed using ECL (Roche, Mannheim,
Germany) and quantified by scanning densitometry .
2.5. RNA isolation and reverse transcription polymerase chain reaction
After incubation, cells were washed twice with ice-cold PBS. Total RNA was isolated
using the single-step TRI reagent, and 1 μg RNA was reverse transcribed into cDNA. In
brief, the isolated RNA was dissolved in sterile water and 2.5 mmol/L Mg
, 1 mmol/L
dNTPs, 0.5 μg oligodT15, 25 U AMV reverse transcriptase and 10× RT buffer to give a
final volume of 20 μl. The sample was incubated at 25°C (10 min), 42°C (60 min) and
99°C (5 min). cDNA was diluted in DNase-free water (1:25) before quantification by
real-time PCR. The primers for quantification of mRNA by real-time quantitative PCR for
Nrf1, Nrf2, Tfam, Cpt1a, Ppara, Pparg and 18S rRNA mRNAs were the same as those
published previously . Quantitative PCR was performed using Mx300 0P (see
above). Each quantitative PCR was performed in triplicate. The mouse 18S rRNA gene
served as the endogenous reference gene. The evaluation of relative differences of PCR
product among the treatment groups was carried out using the ΔΔCT method. The
reciprocal of 2
(using CT as a base 2 exponent) for each target gene was normalized to
that for 18S rRNA, followed by comparison with the relative value in control cells. Final
results are presented as percentage of control.
2.6. RT-PCR for mtDNA
Total DNA and mtDNA were extracted using a kit (QIAamp DNA Mini Kit; Qiagen,
Hilden, Germany), and quantitative PCR was done using 18S rRNA primers for a nuclear
target sequence and primers for the mitochondrial D-loop as an mtDNA target .
Quantitative PCR was performed using a real-time PCR system (Mx3000P; Stratagene,
Amsterdam, the Netherlands). Reactions were performed with 12.5 μl SYBR Green
Master Mix (ABI, Warrington, UK), 0.5 μl of each primer (10 μmol/L) and 100 ng
template (DNA) or no template (NTC), with RNase-free water being added to a final
volume of 25 μl. The cycling conditions were as follows: 50°C for 2 min, initial
denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 1 min
and 72°C for 30 s. Each quantitative PCR was performed in triplicate. The following
primers were used: mitochondrial D-loop: forward, 5′-AATCTACCATCCTCCGTG-3′;
reverse, 5′-GACTAATGATTCTTCACCGT; 18S rRNA: forward, 5′-CATTCGAACGTCTGCCC-
TATC-3′; reverse, 5′-CCTGCTGCCTTCCTTGGA-3′. The mouse 18S rRNA gene served as an
endogenous reference gene. Melting curves were performed to ensure specific
amplification. The standard curve method was used for relative quantification. The
ratio of mitochondrial D-loop to 18S rRNA was then calculated. Final results are
presented as percentage of control.
2.7. Cell respiration test
Oxygen consumption by intact cells was measured as an indication of mitochon-
drial respiration activity . The BD Oxygen Biosensor System utilizes an oxygen-
sensitive fluorescent compound [tris 1,7-diphenyl-1,10 phenanthroline ruthenium(II)
chloride] embedded in a gas-permeable and hydrophobic matrix permanently attached
to the bottom of a multiwell plate. The concentration of oxygen in the vicinity of the dye
is in equilibrium with that in the liquid media. Oxygen quenches the dye in a
concentration-dependent manner. The fluorescence correlates directly to oxygen
consumption in the well. This unique technology allows homogenous instantaneous
detection of oxygen levels. After treatment, adipocytes were washed in Krebs–Ringer
solution buffered with HEPES (KRH) buffer plus 0.1% bovine serum albumin (BSA). Cells
from each condition were divided into aliquots in a BD Oxygen Biosensor System plate
(BD Biosciences) in triplicate. The number of cells contained in equal volumes did not
differ statistically among samples treated with various nutrients and nutrient
concentrations. Plates were sealed and “read” on a fluorescence spectrometer
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(Molecular Probes, Sunnyvale, CA) at 1-min intervals for 60 min at an excitation of 485
nm and emission of 630 nm . Results are expressed as the slope of time-varying
2.8. Mitochondrial mass analysis
The fluorescent probe MitoTracker Green FM (Molecular Probes, Eugene, OR) was
used to determine the mitochondrial mass of adipocytes . In brief, adipocytes
treated with HT for 48 h were trypsinized and centrifuged at 3000×g at 4°C for 5 min,
resuspended in KRH buffer and 0.1% BSA (w/v) and then incubated with 0.1 μmol/L
MitoTracker Green FM in KRH buffer for 30 min at 37°C. Cells were centrifuged at
3000×g at 4°C for 5 min and resuspended in 400 μl of fresh KRH buffer. Fluorescence
was analyzed by flow cytometry (FACS Calibur, Becton Dickinson, Mountain View, CA).
2.9. Electron microscopic observation
3T3-L1 adipocytes on Day 8 of differentiation were seeded on glass coverslips.
On Day 9, cells were treated with HT (1.0 μmol/L) for 48 h. On Day 10, adipocytes
were fixed overnight with 2.5% (v/v) glutaraldehyde in 0.1 mol/L sodium phosphate
buffer (pH 7.3).
They were postfixed with 2% (w/v) OsO
in the same buffer, followed by block
staining with 1% (w/v) uranyl acetate. After dehydration with a graded ethanol series,
they were washed with propylene oxide and embedded in Spurr's low viscosity resin.
Silver to gold sections were cut and examined using a Philips CM 10 (Eindhoven, the
Netherlands) transmission electron microscope at a 60-kV accelerating voltage .
Measurements were made on six individual adipocytes treated with or without HT. For
each individual adipocyte in each image, the number of mitochondria and the total
mitochondrial sectional area were determined. All electron microscopic photographs
were analyzed by observers blind with respect to treatments .
2.10. Mitochondrial isolation
Following addition of trypsin, the cells were pelleted by centrifugation at 300×g for
5 min at 4°C. All of the subsequent steps were performed on ice. The resulting pellet
was then resuspended in 0.5 ml of mitochondrial isolation buffer (215 mmol/L
mannitol, 75 mmol/L sucrose, 0.1% BSA, 1 mmol/L EGTA and 20 mmol/L HEPES, pH 7.2)
and homogenized on ice with a 2-ml glass homogenizer (Dounce, Fisher Scientific,
Pittsburgh, PA). The mitochondria were then purified by differential centrifugation at
1300×g for 5 min to pellet unbroken cells and the nuclei. The supernatant was then
centrifuged at 13,000×g for 10 min to pellet the mitochondria. The pellet was
resuspended in EGTA-free isolation buffer .
2.11. Assays for activities of Mitochondrial Complexes I, II, III, IV and V
Adipocytes were cultured in 100-mm plates, washed in PBS, resuspended in an
appropriate isotonic buffer (0.25 M sucrose, 5 mM Tris–HCl, pH 7.5, and 0.1 mM
phenylmethylsulfonyl fluoride) and homogenized. Mitochondria were isolated by
differential centrifu gation of the cell homogenates. NADH–CoQ oxidoreductase
(Complex I) activity was tested by monitoring the reduction of 2,6-dichlorophenol
indophenol at 600 nm upon addition of assay buffer (finally, 0.05 M Tris–HCl, pH 8.1,
0.1% BSA (w/v), 1 mM antimycin A , 0.2 mM NaN
and 0.05 mM coenzyme Q1) .
Assays of succinate–CoQ oxidoreductase (Complex II), CoQ–cytochrome c reductase
(Complex III) and cytochrome c oxidase (Complex IV) were performed spectro-
metrically using conventional assays  with minor modifications. Complex V (ATP
synthase) activity was measured as oligomycin-sensitive, Mg
-ATPase activity .
2.12. Determination of FFA and glycerol content in the cell culture supernatant
Adipocytes cultured in six-well plates were stimulated with 1.0 μM HT for 72 h;
then, the FFA content of the supernatant was estimated by a commercially available FFA
kit (Jiancheng Biochemical Inc.). The test is based on the reaction of FFAs with Cu
form copper salt, which is detected photometrically by absorbance at 440 nm. The
glycerol content of the supernatant was determined by a free glycerol reagent kit from
Sigma (Cat #F6428). In brief, 10-μl samples were added to wells of 96-well plates, 200
μl of glycerol reagent was pipetted into each well and all samples were incubated for 5
Fig. 2. Effect of HT treatments on mRNA levels of Tfam, Nrf1 and Nrf2 in adipocytes. 3T3-L1 adipocytes were treated for 48 h with HT at 0.1, 1.0, 10 and 50 μmol/L. mRNA levels of Tfam,
Nrf1 and Nrf2 were analyzed by quantitative RT-PCR with gene-specific oligonucleotide probes in adipocytes. The cycle number at which the various transcripts were detectable was
compared with that of 18S rRNA as an internal control. Results were expressed as percentage of untreated control cells. Values are mean±S.E. of the results from at least four
independent experiments. *Pb.05 versus control cells without HT treatment.
Fig. 1. Effect of HT treatments on protein expression and transcriptional activation of
PPARGC1α. 3T3-L1 adipocytes were treated for 48 h with HT at concentrations of
0.1, 1, 10 and 50 μmol/L. Cells were subsequently solubilized into SDS sample buffer
and analyzed by Western blotting with an tib odie s agai nst α-tubulin and
PPARGC1α. (A) Upper panel: Immunoblots for representative samples of steady-
state levels of proteins; lower panel: Quantitative values (in percentage) were
tabulated for PPARGC1α:α-tubulin ratios determined by densitometry. (B) Relative
luciferase reporter activity. Values are mean±S.E. of the results from four
independent experiments. *Pb.05 versus control (without HT treatment).
3J. Hao et al. / Journal of Nutritional Biochemistry xx (2009) xxx–xxx
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min at 37°C. The absor bance of each sample was spectrophotometrically measured at
540 nm, with water as reference, with three samples per condition.
2.13. Determination of triglycerides using Oil Red O staining
After the induction of differentiation, adipocytes cultured in 24-well plates were
stimulated with HT (1.0 μM) for 72 h. Cells were washed twice with PBS and fixed
with 10% formalin in PBS for 1 h. After being washed three times with PBS, cells
were stained with Oil Red O (six parts of 0.6% Oil Red O dye in isopropanol and four
parts of water) for 1 h, and the excess of the stain was removed by washing with
water. Then, stained cells were put in a fume cabinet until they are dried, and the
stained oil droplets were dissolved in isopropanol containing 4% Nonidet P-40
overnight. The triglycerides in adipocytes were quantified by measuring the
absorbance at 520 nm.
2.14. Construction of RNAi adenovirus and transient transfection
The siRNA targeting of Ppargc1α has been described previously . The selected
sequence was screened using a BLAST search to ensure that only the Ppargc1α gene was
targeted. The double-strand oligonucleotides used in the present study were as follows:
TAGtctcttgaaCTACACCACT TCAATCCACCG-3′. 3T3-L1 adipocytes were seeded into six-
well plates at about 80% confluence the day before virus infection. Cells were then
incubated with recombinant virus (Ad-siRNA-Ppargc1α or Ad-siRNA-control) at a
concentration of 2×10
virus particles per cell. After incubation for 2 h, fresh growth
medium was added and cells were further cultured for 4 h and then stimulated with HT
(1.0 μmol/L) for 48 h.
2.15. Statistical analysis
All data are representative of at least three independent experiments. Data are
presented as means±S.E.M. Statistical significance was calculated by SPSS 10.0
software using one-way ANOVA, with P values b.05 considered significant.
Fig. 3. Effect of HT treatments on expression of mtDNA. 3T3-L1 adipocytes were treated
for 48 h with HT at 0.1, 1.0, 10 and 50 μmol/L. DNA was isolated and PCR products were
quantified using SYBR Green fluorescence. Quantitative values (in percentage) were
tabulated for D-loop:18S rRNA ratios. Values are mean±S.E. of the results from seven
independent experiments. *Pb.05 versus control without HT taken as 100%.
Fig. 4. Effect of HT treatments on protein expression of mitochondrial complexes in
adipocytes. 3T3-L1 adipocytes were treated for 48 h with HT at 0.1, 1.0, 10 and 50 μmol/L.
Cells were subsequently solubilized into SDS sample buffer and analyzed by Western
blotting with antibodies against α-tubulin and complex antibodies. (A) Anti-OxPhos
Complex I (NADH ubiquinone oxidoreductase 39-kDa subunit, 1:2000); (B) anti-
OxPhos Complex II (succinate–ubiquinone oxidoreductase 70-kDa subunit, 1:2000);
(C) anti-OxPhos Complex III (ubiquinol–cytochrome c oxidoreductase core II 50 kDa,
1:20 0 0); (D) anti-OxPhos Complex V (ATP synthase, 53 kDa). Representative
immunoblots of steady-state levels of proteins are shown for Complexes I, II, III and V.
Quantitative values (in percentage) were tabulated for complex:α-tubulin ratios
determined by densitometry for Complexes I (A), II (B), III (C) and V (D). (E) Time-
dependent effect of HT at 1 μmol/L on Complex I expression. (F) Time-dependent effect
of HT at 1 μmol/L on Complex I activity. Values are mean±S.E. of the results from four
independent experiments. *Pb.05 and **Pb.01 versus control cells without HT treatment.
4 J. Hao et al. / Journal of Nutritional Biochemistry xx (2009) xxx–xxx
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3.1. HT stimulated transcriptional activity and protein expression
PPARGC1α is a key factor that drives mitochondrial biogenesis,
which also plays an important stimulatory role in thermogenesis and
fatty acid oxidation in muscle and adipose tissues [29–31]. Treatment
of adipocytes with HT at 0.1–50 μmol/L resulted in a dose-dependent
curve of stimulation of expression of PPARGC1α by Western blot and
its promoter transcription by luciferase reporter assay. Both assays
showed that the most significant stimulation occurred at 0.1 and
10 μmol/L (Fig. 1A and B).
3.2. HT up-regulated genes involved in mitochondrial biogenesis and
fatty acid oxidation
Ppargc1α autoregulates its gene expression, along with the
expression of Nrf1 and Nrf2, which are mitochondrial transcription
factors encoded by nuclear genes. Nrf1 also induces the expression
of Tfam [29–31]. Therefore, we examined the effects of HT on the
mRNA expression of Nrf1, Nrf2 and Tfam. Treatment of HT at 0.1–50
μmol/L resulted in bell-shaped response curves of mRNA expression
of Nrf1, Nrf2 and Tfam, similar to that of PPARGC1α expression.
However, the increase was significant only at 1 μmol/L of HT for all
three factors (Fig. 2).
3.3. HT treatment increased mtDNA
mtDNA content decreases age-dependently and may be one of the
causal factors in age-related type 2 diabetes . Tfam is involved in
regulating expression of nuclear genes encoding some major
mitochondr ial protein s that regulate mtDNA transc ription and
replication. Its level is proportional to that of mtDNA [29–31]. Because
HT stimulated the mRNA expression of Tfam, it is expected that
mtDNA copy number should be increased. mtDNA expression was
quantified by real-time PCR measuring the ratio of D-loop to 18s rRNA
levels. The D-loop region is known as the major site of transcription
initiation on both the heavy and light strands of mtDNA. As shown in
Fig. 3, the HT treatment at 1 μmol/L resulted in a significant increase
in the ratio of mitochondrial D-loop/18s rRNA.
3.4. HT promoted the protein expression of OxPhos Complexes I, II,
III and V
Tfam, along with other nuclear-encoded mitochondrial proteins, is
imported into mitochondria by the protein import machinery and
regulates the expression of the 13 mtDNA-encoded proteins, which are
components of Respiratory Chain Complexes I (ND1–6
and 4L) and III
(Cyt b — cytochrome b) and ATP synthase (A8 and A6). The nuclear
DNA-encoded mitochondrial proteins and the mtDNA-encoded pro-
teins are assembled to form multisubunit enzyme complexes required
for oxygen consumption and ATP synthesis [29–31]. As shown in Fig. 4,
treatment of HT (48 h) significantly increased the expression of
Complex I (Fig. 4A) and Complex II (Fig. 4B) at 0.1, 1 and 10 μmol/L and
Complex III (Fig. 4C) and Complex V (Fig. 4D) at 1 and 10 μmol/L,
respectively. The increases in protein expression were about 1.5- to 1.7-
fold compared with control samples. The rationale for choosing the 48-
h treatment for all experiments was based on the time-dependent
effects of HT treatment: as shown in Fig. 4E and F, HT treatment
effected a dose-dependent increase of Complex I expression and
activity after periods varying from 24 to 72 h, with significant
stimulation beginning at 48 h.
Fig. 5. Effect of HT treatments on mitochondrial mass and ultrastructural changes under
the electron microscope. Adipocytes were stimulated with HT at 1.0 μM for 48 h. (A)
Mitochondrial mass was estimated by MitoTracker (100 nmol/L) staining with flow
cytometry. Results were expressed as fold increase of the fluorescence intensity over
untreated control cells. Values are mean±S.E. of the results from four independent
experiments. *Pb.05 versus control cells without HT treatment. (B) Morphometric
analysis of surface area and density of mitochondria under the electron microscope.
Values are means±S.E. of data from six cells. **Pb.01 versus control cells without HT
treatment. (C) Representative illustrations of mitochondrial profiles under the electron
microscope (magnification, ×2110 and ×11,000).
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ARTICLE IN PRESS
3.5. HT increased adipocyte mitochondrial mass
Mitochondrial formation is dependent on the assembly of large
hetero-oligomeric complexes, and this assembly requires coordina-
tion betwee n the nuclear and mitochondrial genomes [29–31].We
examined whether H T-induced activation of PPARGC1α and its
downstream signaling leads to an increase in mitochondrial
numbers. First, we used a specific dye, MitoTracker Green FM,
which accumulates inside mitochondria, to label and quantify
mitochondria in cells. As shown in Fig. 5A, treatment of HT at 1.0
μmol/L resulted in a significant increase in fluorescence inten sity,
suggesting an increase in mitochondrial mass. Mitochondrial
morpholog y was also examined under the electron microscope. As
shown in Fig. 5B, quantitative analysis (six cells were analyzed)
demonstrated that the treatment with HT at 1.0 μmol/L for 48 h
significa ntly increased mitochondrial section area and d ensity. A
representative control adipocyte and a 1.0-μmol/L HT-treated
adipocyte are shown in Fig. 5C.
3.6. HT augmented oxygen consumption and activities of Mitochondrial
Complexes I, II, III, IV and V
An increase in mitochondrial formation should be accompanied by
an increase in mitochondrial function [29–31]. We examined oxygen
consumption and activities of Mitochondrial Complexes I, II, III, IV and
V. As shown in Fig. 6A and B, the basal rate of oxygen consumption
was significantly increased in adipocytes by treatment with HT at
concentrations between 1 and 10 μmol/L; the optimal increase was
found to be at 1 μmol/L.
As shown in Fig. 7, treatment with HT (48 h) significant ly
increased the activities of Complex I (Fig. 7A) at 1 μmol/L HT;
Complex II (Fig. 7A) at 0.1, 1 and 10 μmol/L; Complex III (Fig. 7B) at 1
and 10 μmol/L; and Complex IV (Fig. 7B) and Complex V (Fig. 7B) at
3.7. HT up-regulated fatty-acid-oxidation-related expressions of Ppara,
Cpt1 and Pparg genes
PPARα is an important regulator of mitochondrial biogenesis and
β-oxidation. CPT-1 is the gatekeeper of mitochondrial fatty acid
oxidation because it regulates long-chain fatty acid transport across
the mitochondrial membrane by converting acyl-CoA into acylcarni-
tine. PPARγ plays an important role not only in adipogenesis but also
in regulating lipid metabolism in mature adipocytes. To study the
effects and mechanism of HT on mitochondrial biogenesis and fatty
acid oxidation, the effects of HT on Ppara, Cpt1 and Pparg were
studied by quantitative RT-PCR. As shown in Fig. 8
, HT showed dose-
nt increases in mRNA expression of Cpt1, Ppara and Pparg
with significant increases at 1.0 μmol/L HT for Cpt1 and Ppara and at
1.0 and 10 μmol/L HT for Pparg. However, HT did not increase PPAR
induction of the reporter gene in PPARα or PPARγ transactivation
assays (data not shown).
Fig. 6. Effect of HT treatments on oxygen consumption in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated for 48 h with various concentrations of HT as indicated. (A) Representative
oxygen consumption curves. Cells were trypsinized and equal volumes of cells were separated into aliquots in wells of a 96-well BD Oxygen Biosensor plate. Plates were covered and
fluorescence in each well was recorded over time with a fluorescence microplate spectrophotometer. (B) Quantitative changes in the respiratory rate of adipocytes under each
condition were calculated by determining the kinetic parameters. V
=maximum oxygen consumption rate. Final results are presented as percentage of control. Values are mean±S.
E. of the results from three independent experiments. ** Pb.01 versus control cells without HT treatment taken as 100%.
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3.8. HT decreased the FFA content in the supernatant of
A chronic, high concentration of plasma FFAs is one of the factors
that contributes to the underlying pathophysiology of type 2 diabetes,
including development of insulin resistance. FFA treatment impaired
insulin-receptor-mediated signal transduction and decreased insulin-
stimulated GLUT4 translocation and glucose transport. In one study,
FFAs activated the stress/inflammatory kinases JNK, IKKb and SOCS-3;
increased secretion of the inflammatory cytokine TNF-α;and
decreased secretion of adiponectin into the medium . Pharmaco-
logical agents that effectively lower FFA (such as TZD) are likely to
have a significant effect in reducing fasting plasma glucose. Therefore,
reducing FFA might be a target for treating obesity and type 2
diabetes. Therefore, we examined whether HT can target FFA levels. As
shown in Fig. 9, treatment of adipocytes with HT (1.0 μmol/L) for 72 h
significantly reduced the FFA content of the supernatant (Pb.01),
although the glycerol and triglyceride content did not show distinct
change compared with nontreated controls.
3.9. HT-activated phosphorylation of AMPK and ACC
One of the important pathways for activating PPARGC1α involves
AMPK . AMPK phosphorylates a number of targets, resulting in
increases in glucose transport, fatty acid oxidation and gene
transcription. One example of these targets is ACC. ACC is a biotin-
dependent enzyme that catalyses the irreversible carboxylation of
acetyl-CoA to produce malonyl-CoA. The carboxylation inhibits β-
oxidation, but when ACC is phosphorylated by AMPK, its activity is
decreased . AICAR, a pharmaceutical AMPK activator, has been
shown to cause a time- and concentration-dependent increase in
phosphorylation of AMPK and ACC . As shown in Fig. 10C and D,
HT affec ted ACC pho sphorylation and AMPK phospho rylatio n
Fig. 7. Effect of HT treatments on activities of mitochondrial complexes in adipocytes. 3T3-L1 adipocytes were treated for 48 h with HT at 0.1, 1.0, 10 and 50 μmol/L. Cells were then
washed; mitochondria were isolated, and the complex activities were assayed. (A) Complexes I and II; (B) Complexes III, IV and V. Final results are presented as percentage of control.
Values are mean±S.E. of the results from at least four independent experiments. *Pb.05 and **Pb.01 versus control cells without HT treatment.
Fig. 8. Effect of HT treatments on expression of Cpt1a and Ppara mRNA. 3T3-L1 adipocytes were treated for 48 h with HT at 0.1, 1.0, 10 and 50 μmol/L, and total RNA was isolated. PCR
fluorescence products were quantified using SYBR Green. The cycle number at which the various transcripts were detectable was compared with that of 18S rRNA as an internal control.
Results are expressed as percentage of control. Values are mean±S.E. of the results from at least four independent experiments. *Pb.05 versus control without HT treatment.
7J. Hao et al. / Journal of Nutritional Biochemistry xx (2009) xxx–xxx
ARTICLE IN PRESS
similarly. We studied the effects of HT on the phosphorylation of
AMPK and ACC, using AICAR as positive control. HT treatment (1.0
μmol/L) of 3T3-L1 adipocytes caused a time-dependent increase in
the phosphorylation of AMPK (Fig. 10A and B) over the time range of
5, 15, 30 and 60 min, with the maximum increase at 30 min. Both HT
(1.0 μmol/L) and AICAR (0.5 mmol/L) significantly affected AMPK
phosphorylation (Fig. 10C and D). That HT, at about 1/500th the
concentration, induced a similar degree of AMPK and ACC phosphor-
ylation as AICAR suggests that HT is the more potent AMPK activator.
3.10. Knockdown of Ppargc1α blocked the effects of HT
To better determine how important Ppargc1α is in producing the
stimulatory effects of HT on mitochondrial function and biogenesis in
3T3-L1 adipocytes, we knocked down Ppargc1α by siRNA. As shown in
Fig. 11, introducing Ad-siRNA-Ppargc1α to t he cells decre ased
PPARGC1α protein levels at the 24-h and 48-h time points. The Ad-
siRNA-control did not affect PPARGC1α expression (Fig. 11A). Next,
we determined the effects of Ppargc1α on the mtDNA quantity and
Complex I expression. Knockdown of Ppargc1α suppressed them
significantly (Fig. 11B), indicating the important role of Ppargc1α.
These results also suggest that Ppargc1α is an important target in the
process by which HT is able to stimulate mitochondrial function and
biogenesis in adipocytes. No big difference was observed between
control and Ad-siRNA-control groups, and HT stimulated both mtDNA
and Complex I in Ad-siRNA-control cells, but for Ad-siRNA-Ppargc1α
cells, mtDNA and Complex I were dramatically inhibited and HT
treatment could not reverse this inhibition. These data suggest a
crucial role of Ppargc1α in the effects of HT. Both mtDNA and Complex
I levels tended to be higher in the Ad-siRNA-Ppargc1α cells treated
with HT compared to untreated cells, but the increases were not
Identifying the mitochondrial dysfunction mechanis ms and
developing mitochondrial targeting drugs/nutrients have formed a
new discipline of mitochondrial medicine and opened up avenues
for manipulating mitochondrial function and health [21,37–39].Itis
well reported that mitochondrial biogenesis could, in part, underlie
the central role of adipose tissue in the control of whole-body
metabolism and the actions of some insulin sensitizers  and that
mitochondrial dysfunction might be an important contributing
factor in type 2 diabetes . Mitochondrial loss in adipose tissue
is correlated with the development of type 2 diabetes . Hence, it
is possible that stimulating mitochondrial biogenesis may reduce
Fig. 9. Effect of HT treatments on levels of supernatant FFAs, glycerol and triglycerides in
3T3-L1 adipocytes. Adipocytes were stimulated with HT at 1.0 μmol/L for 72 h. FFA and
glycerol contents in the supernatants of cells were detected. Results are expressed as a
percentage fold increase of the metabolite concentration over untreated control cells.
Values are mean±S.E. of the results from six independent experiments. **Pb.01 versus
control without HT treatment.
Fig. 10. Effect of HT treatments on AMPK and ACC phosphorylation in 3T3-L1
adipocytes. AMPK phosphorylation and ACC phosphorylation were determined by
Western blotting using lysates of 3T3-L1 adipocytes that had been cultured for 5, 15,
30 or 60 min with 1.0 μM HT and for 30 min with AMPK activator AICAR at 0.5 mM.
Proteins were prepared, applied (20 μg/lane) and separated. After transfer to a
nitrocellulose membrane, polyclonal sera specific for phospho-AMPK (pAMPK),
tAMPK, phospho-ACC (pACC) and tACC were applied overnight, and detection was
carried out as described in Materials and Methods. (A) A representative image of
AMPK phosphorylation. (B) Quantitative values (in percentage) are tabulated for
pAMPK:tAMPK ratios. (C) A representative image of ACC phosphorylation. (D)
Quantitative values (in percentage) are tabulated for pACC:tACC ratios. Values are
mean±S.E. of the results from four independent experiments. *Pb.05 versus control
cells without HT treatment.
8 J. Hao et al. / Journal of Nutritional Biochemistry xx (2009) xxx–xxx
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the symptoms of metabolic syndrome. In the present study, we
showed that HT over the concentration range of 0.1–10 μmol/L
stimulated the protein expression of PPARGC1α — the central factor
for mitochondrial biogenesis — and the mRNA of its downstream
targets, Nrf1, Nrf2 and Tfam. HT increased the quantity of mtDNA
and the protein expression of Mitochondrial Complexes I, II, III and
V; consequently, mitochondrial numbers increased. This increase in
mitochondrial biogenesis was accompanied by an enhancement of
mitochondrial function, including an increase in the activity and
protein expression of Mitochondrial Complexes I, II, III and V and
oxygen consumption, as well as a decrease in FFAs. Using siRNA, we
further demonstrated that a key factor for H T to target is
PPARGC1α. These data suggest that HT, as a mitochondrial targeting
nutrient, is able to promote mitochondrial function by stimulating
Mitochondrial fatty acid oxidation is one of the key processes in
ATP production. Mitochondrial biogenesis and remodeling in white
adipocyte tissue enhance fatty acid uptake and oxidation, indicated by
increased oxygen consumption. The increase in oxygen consumption
is accompanied by an increase in expression of Cpt1 and Ppara,
suggesting that HT stimulates mitochondrial biogenesis, leading to
increased fatty acid oxidation.
We hypothesized that the protective effects of mitochondrial
antioxidants and nutrients on mitochondria may i nclude (a)
protecting mitochondria from oxidative damage and thus slowing
down the loss of mitochondria, (b) stimulating repair of damaged
mitochondria, (c) stimulating degradation of damaged mitochondria
(by lysosomes) and (d) stimulating de novo mitochondrial biogen-
esis . α-Lipoic acid and acetyl-
L-carnitine are two examples of
mitochondrial nutrients. In our previous experiments, we  have
demonstrated that treatments using a combination of R-α-lipoic acid
L-carnitine at concentrations of 0.1, 1 and 10 μM for 24 h
significantly increased mitochondrial mass, expression of mtDNA,
mitochond rial complexes, oxygen consumption and fatty acid
oxidation in 3T3-L1 adipocytes. These changes were accompanied
by an increase in the mRNA expression of Cpt1a and expression of
several transcription factors involved in mitochondrial biogenesis,
including Ppargc1α, Tfam, Nrf1 and Nrf2. We concluded that the
combination of R-α-lipoic acid and acetyl-
L-carnitine may act as
Pparg and Ppara dual ligands to complementarily promote mitochon-
drial synthesis and adipocyte metabolism.
HT has long been considered as a potent antioxidant polyphenol
[14,15,17]. However, its effect on mitochondrial biogenesis has never
been studied. Therefore, this is the first study to show that HT is
able to act as a mitochondrial targeting nutrient and provides a
new mechanism of the efficacy of the Mediterranean diet on
lowering the risk of various diseases, including cardiovascular
disease, cancer, diabetes and obesity. As we know, cardiovascular
disease is the most common and most serious complication of
diabetes and obesity. Because mitochondrial respiration plays a
critical role in glucose metabolism, mitochondrial dysfunction has
been shown to be associated with diabetes and obesity. The
Mediterranean diet, including a high intake of HT, may stimulate
mitochondrial biogenesis and function (enhancement of fatty acid
oxidation) and, thus, reduce the risk of obesity and diabetes,
leading to a lowered risk of cardiovascular disease. It seems that
mitochondrial biogenesis and the phase II antioxidant system are
closely related or coupled because the transcriptional coactivator
PPARGC1α was shown to suppress ROS and neurodegeneration
. Therefore, it is possible that HT, a potent antioxidant and
Phase II enzyme inducer, may enhance mitochondrial biogenesis
and improve mitochondrial function by suppressing ROS and
stimulating the Phase II antioxidant system to strengthen the cell's
antioxidant defenses, in addition to its direct effect on mitochon-
drial assembly as demonstrated here.
Mitochondrial biogenesis is a complicated process. The activation
of PPARGC1α is associated with a number of signaling pathways
involving the activation of AMPK , intracellular calcium and the
Fig. 11. Effect of PPARGC1a (Pgc1a) silencing on HT-stimulated mtDNA and Complex I expression. Cells were infected with Ad-siRNA-Ppargc1α or Ad-siRNA-control as described. DNA
and protein were isolated and detected by RT-PCR and Western immunoblotting. (A) PPARGC1α protein expression after 12, 24 and 48 h of adenovirus infection. (B) Complex I
expression in adipocytes treated with or without HT (1.0 μmol/L) and mtDNA quantity in adipocytes treated with or without HT (1.0 μmol/L). Values are mean±S.E. of the results from
three independent experiments. *Pb.05 versus Ad-siRNA-control without HT treatment.
9J. Hao et al. / Journal of Nutritional Biochemistry xx (2009) xxx–xxx
ARTICLE IN PRESS
subsequent activation of calcium-sensitive signaling of calcium/
calmodulin-dependent protein kinase  and nitric oxide  and
cAMP-responsive element binding protein [29–31]. We investigated
the possible involvement of the AMPK signaling pathway by detecting
the phosphorylation of AMPK and ACC (Fig. 10). Whether HT also
affects other signaling pathways of PPARGC1α activation needs to be
In addition to stimulating mitochondrial biogenesis, AMPK
was also shown to increase muscle fatty acid oxidation and
insulin sensitivity. The antidiabetic drug metformin activates
AMPK . gACRP30 or globular adiponectin, the globular subunit
of ADIPOQ, improves insulin sensitivity and increases fatty acid
oxidation. The mechanism by which gACRP30 exerts these effects is
possibly due to activation of AMPK and inactivation of ACC .
The potent effect of HT on phosphorylation of AMPK and ACC is
consistent with the decrease in FFA and the increases in Cpt1 and
Ppara as indexes of increased fatty acid oxidation. The potent effect
of HT on phosphorylation of AMPK and ACC in our 3T3-L1
adipocytes suggests that HT might be potentially effective in
increasing fatty acid oxidation and improving insulin sensitivity in
diabetes and obesity.
In addition to the effects on increasing the mRNA expression of
Ppara and Cpt1a for stimulation of fatty acid oxidation, HT also
induced gene mRNA expression, but not transactivation, of Pparg. Oil
Red O staining indicates smaller oil droplets in adipocytes. At the
same time, the cellular triglyceride level did not increase (Fig. 9), and
the FFA levels in the cell culture supernatant were reduced, suggesting
that HT promoted fat burning. This reasoning is in accordance with
Wilson-Fritch et al. , who describe that PPARγ plays an important
role not only in adipogenesis but also in regulating lipid metabolism
in mature adipocytes. PPARγ activity can be modulated by direct
binding of low-molecular-weight ligands. For example, the clinically
effective antidiabetic drugs such as TZD are high-affinity agonist
ligands for PPARγ, leading to a net flux of fatty acids from the
circulation and other tissues into adipocytes . Interestingly, HT did
not increase the levels of cellular triglycerides and FFAs but rather led
to decreased level of FFAs in the cell culture supernatant and to
smaller lipid droplets in adipocytes. Since HT only up-regulates
PPARα/PPARγ expression but has no PPAR agonistic activity, these
findings could be interpreted by the presence of sufficient PPAR
agonistic molecules (e.g., fatty acids) in the cellular system, to favor
Our findings indicate that HT in adipocytes may support adipocyte
differentiation but does not result in increased fat storage. This is in
accordance with observations that a 2-week supplementation of HT
did not cause any change in body weight in normal rats [Albino:
HanWistar (SPF), doses up to 450 mg/kg]. Also, HT did not influence
body weight or body composition in mice [C57BL/6NCrl: (SPF), doses
up to 300 mg/kg]. The net effect of HT on adipogenesis can be
compared with the action of the novel non-TZD selective PPARγ
modulator (nTZDpa). Berger et al.  found that, in cell-based assays
for transcriptional activation, nTZDpa served as a selective, potent
PPARγ partial agonist and was able to antagonize the activity of PPARg
full agonists; nTZDpa also displayed partial agonist effects when its
ability to promote adipogenesis in 3T3-L1 cells was evaluated.
Interestingly, nTZDpa, unlike the TZD, caused reductions in weight
gain and adipose depot size when it was administered to fat-fed
In conclusion, we showed that HT is a nutrient that effectively
stimulates mitochondrial biogenesis and function. This mitochon-
drial targeting property may provide a possible mechanism for the
efficacy of the Mediterranean diet for lowering the risk of
cardiovascular disease and also suggests that HT may be used as a
therapeutic intervention for preventing and treating type 2 diabetes
We thank Ji Zhang for technical assistance in performing the
Western blotting assays. This study was supported by a UC Davis
Center for Human and Nutrition Pilot Award (CHNR08-318) and by
DSM Nutritional Products Ltd.
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