Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor Complete study available in Free Radical Biology and Medicine. 2017 Sep;110:176-187. doi: 10.1016/j.freeradbiomed.2017.06.006. Epub 2017 Jun 9.

1. C
ontrol
5nM
50nM
100nM
500nM M1
M
10
0
5
10
15
20
***
***
***
**
pmolesQ9/well
Quercetin
B
C
ontrol
5nM
50nM
100nM
500nM M1
M
10
0
5
10
15
20
pmolesQ9/well
C
ontrol
5nM
50nM
100nM
500nM M1
M
10
0.0
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1.0
1.5
2.0
2.5
pmolesQ10/well
Flavonols
Kaempferol
C
ontrol
5nM
50nM
100nM
500nM M1
M
10
0.0
0.5
1.0
1.5
2.0
2.5
*** *** ***
**
pmolesQ10/well
Q9
Q9
Q10
Q10
Sirt3
C
ontrol
M
K

10 C
ontrol
M
K

10
0
1.0107
2.0107
3.0107
*
Full-length Sirt3 (48kDa)
Mitochondrial Sirt3 (23kDa)
a.u.
 Cellular cultures and treatments.- Cellular lines of different origins were used along the study: Tkpts (derived
from mouse kidney proximal tube epithelium), Hepa 1.6 (derived from mouse liver hepatoma), mouse embryonic
fibroblast (MEFs), human promyelocytic leukemia cells (HL-60), human liver hepatoma cells (Hep G2) and human
embryonic kidney cells 293 (HEK 293). Cells were treated and incubate in all cases during 48h in 5% CO2 at 37ºC.
Lucía Fernández-del-Río1,2, Anish Nag2, Elena Gutiérrez Casado1, Julia Ariza1, Agape M. Awad2, Akil I. Joseph3, Ohyun Kwon2, Eric Verdin4, Rafael de Cabo5, Claus
Schneider3, Jorge Z. Torres2, María I. Burón1, Catherine F. Clarke2, and José M. Villalba1 (jmvillalba@uco.es)
1Department of Cell Biology, Physiology and Inmunology, University of Córdoba, Campus de Excelencia Internacional Agroalimentario, ceiA3, Cordoba, Spain
2Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, CA, USA
3Department of Pharmacology, and the Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical School, Nashville, TN, USA
4Buck Institute for Research on Aging, Novato, CA, USA
5Translational Gerontology Branch, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
INTRODUCTION
Coenzyme Q (Q) is the only soluble antioxidant
synthesized endogenously and present in all cellular
membranes. It has a crucial role in numerous important
cellular functions such as metabolism, antioxidant
protection and signal regulation (1). Given the essential
functions of Q, a deficit in this molecule leads to
mitochondrial disorders with heterogeneous clinical
symptoms (2). Oral Q supplementation is used as a
treatment but it low bioavailability makes it ineffective in
most cases (3). Research is focused on alternative
procedures able to activate endogenous Q biosynthesis.
Polyphenols, which are widely present in foods and
beverages of plant origin, as wine, have received great
interest during the last years due to their positive effects
on human health. Their beneficial properties have been
partially attributed to an antioxidant role, as well as to
their ability to modulate molecular targets and signaling
pathways (4). Phenolic acids such as 4HB, vanillic acid,
protocatechuic acid and p-coumarate, as well as the
stilbenoid resveratrol, function as Q ring precursors in
yeast and mammals (5, 6). Little is known about the
possible interaction between complex polyphenols and the
metabolism of Q.
The main objective of this work was to deepen into the
regulation of the Q system through dietary
interventions involving plant polyphenols.
 Lipid extraction and CoQ measurements.- Lipids were isolated using SDS,
ethanol:isopropanol and hexane. The correspondant Q isoforms were
measured by reverse-phase HPLC with a Coulochen II electrochemical detector.
 Biosynthesis measurements.- Two different approaches were used:
(1) A radiolabeled precursor, 4-hydroxy-(U-14C)benzoate (14C-4HB), synthesized from (U-14C) tyrosine
(Amersham) as described in (9). Tkpts cells were radiolabeled with 100,000 cpm of 14C-4HB, in presence or absence
of kaempferol, during 48h. After fixing cells with TCA, total radioactive was extracted with NaOH and quantified in a
scincillation counter.
(2) 13C or deuterated labelled precursors were added to the cells during 48h and their lipid isolated using
methanol and petroleum ether. Dry lipids were resuspended using 1mg/ml ubiquinone and measured using HPLC-
MS/MS.
METHODS
RESULTS
CONCLUSIONS
Figure 1. Kaempferol strongly increases Q levels in Tkpts cells. Q9
and Q10 levels were determined in Tkpts cells at concentrations that do not
affect cell viability. (A) Stilbenoids. Resveratrol, but not piceatannol, produced
a modest increase of Q9 and Q10. (B) Flavonols. Quercetin did not affect Q
levels, but kaempferol produced a dramatic increase of both Q9 and Q10. (C-D)
Flavones and a flavanone. Apigenin, but not luteolin or naringenin, slightly
increased Q9 and Q10 levels. Data are represented as mean ± SEM of 6
replicates. Differences are represented as * (p < 0.05), ** (p < 0,01) and *** (p <
0.001).
HYPOTHESIS
REFERENCES
Our results confirm that kaempferol can act as Q ring precursor in kidney cells. The way this polyphenol carries
out this function is still unknown. We propose two hypotheses: A) kaempferol could be processed by an unknown
enzyme system to yield 4HB, a confirmed precursor of Q or B) kaempferol could enter directly in Q biosynthesis
pathway and be processed during one of the multiple reactions involved in the generation of the final product.
4-hydroxybenzoate
9-10
Coenzyme Q
9-10
PolyprenylPP
Kaempferol
Kaempferol
A
B
?
? Coq
proteins
Coq
proteins
Coq 2
• Kaempferol strongly increases coenzyme Q levels in kidney cells, whereas other phenolics, such as
resveratrol or apigenin, are much less active.
• Increase of coenzyme Q by kaempferol is not related to the upregulation of sirtuin activity, but is
due to a novel role of this phenolic as a biosynthetic ring precursor that competes with endogenous
4-hydroxybenzoate.
• Limited availability of endogenous biosynthetic ring precursors is a feature of renal cells that
determines enhanced sensitivity to supplementation with exogenous precursor substances, in
comparison with cells of different origins.
• The yeast Saccharomyces cerevisiae is unable to use efficiently kaempferol in coenzyme Q
biosynthesis.
?
Coenzyme Q biosynthesis pathway.
The biosynthesis of Q is a not completely
characterized process that can be divided into three
steps: the synthesis of the polyisoprenoid tail
through the mevalonate pathway in eukaryotes, the
attachment of the tail to the benzoquinone ring
precursor and, finally, the subsequent modifications
of the ring to yield the final product (7, 8).
Radiolabeled Q
Control 10M K
0
5000
10000
15000
***
cmp/protein
+ 1M 14
C-4HB
14C-4HB
PABA
4HB
Q9
Control
M
K

10 M
13C-K

10
0
100
200
300
12
C-CoQ
13
C-CoQ
*** ***
pmolsQ9/mgprotein
Q10
C
ontrol
M
K

10
M
13C
-K

10
0
20
40
60
*** ***
pmolsQ10/mgprotein
13C-Kaempferol
A B C
1. Villalba, J. M., Parrado C, Santos-Gónzalez M, Alcaín F.J. (2010). "Therapeutic use of coenzyme Q10 and coenzyme Q10-related
compounds and formulations." Expert Opin. Invetig. Drugs 19(4): 1-20.
2. Laredj, L. N., et al. (2014). "The molecular genetics of coenzyme Q biosynthesis in health and disease." Biochimie 100: 78-87.
3. Ozaltin, F. (2014). "Primary coenzyme Q10 (CoQ10) deficiencies and related nephropathies." Pediatr Nephrol 29(6): 961-969.
4. Del Rio, D., et al. (2013). "Dietary (poly)phenolics in human health: structures, bioavailability, and evidence of protective
effects against chronic diseases." Antioxid Redox Signal 18(14): 1818-1892.
5. Nambudiri, A. M., et al. (1976). "Alternate routes for ubiquinone biosynthesis in rats." Biochem Biophys Res Commun 76(2):
282-288.
6. Xie, L. X., et al. (2015). "Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis." J Lipid Res
56(4): 909-919.
7. Bentinger, M., et al. (2010). "Coenzyme Q--biosynthesis and functions." Biochem Biophys Res Commun 396(1): 74-79.
8. Acosta, M. J., et al. (2016). "Coenzyme Q biosynthesis in health and disease." Biochim Biophys Acta 1857(8): 1079-1085.
9. Clarke, C. F., et al. (1991). "Ubiquinone Biosynthesis in Saccharomyces cerevisiae." J Biol Chem 266(25): 16636-16644.
Acknowledgements:
Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor
B C
C
ontrol
M
cur

1
-cur
12
C13
M1
-van
6
C13
M
10
0
5
10
15
20
25
**
pmolsQ10/mgprotein
C
ontrol
M
cur

1
-cur
12
C13
M1
-van
6
C13
M
10
0
1
2
3
4
5
50
100
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200
* *****
13
C6-Q12
C-Q
pmolsQ9/mgprotein
C
ontrol
M
Fe

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-Fe
3
M
D

10
0
10
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30
40
pmolsQ10/mgprotein
C
ontrol
M
Fe

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-Fe
3
M
D

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0
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D3-Q12
C-Q
pmolsQ9/mgprotein
A B
Q9
Q10
Q9
Q10
TKPTS
C
ontrol
5nM
50nM100nM500nM
M1
M
10
M
20
M
50
M
100
0
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800
*
***
**
***
*
*** ***
**
**
Q10(%ofcontrol)
TKPTS
C
ontrol
5nM
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M1
M
10
M
20
M
50
M
100
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800
***
*** ***
***
***
***
***
**
*
Q9(%ofcontrol)
HEK-293
C
ontrol
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M1
M
10
M
20
M
50
M
100
0
200
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800
***
*** *** *** ***
*** *** ******
Q10(%ofcontrol)
A B
Hepa 1.6
C
ontrol
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M1
M
10
M
20
M
50
M
100
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800
** * * *
Q9(%ofcontrol)
Hepa 1.6
C
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M1
M
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M
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M
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M
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*** ** ** ***
Q10(%ofcontrol)
MEFs
C
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M
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M
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*****
Q9(%ofcontrol)
MEFs
Control
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HL-60
C
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M
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Q10(%ofcontrol)
Hep G2
C
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M1
M
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M
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M
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*
Q10(%ofcontrol)
Tkpts cells Hepa 1.6 cells MEFs HEK 293 cells Hep G2 cells HL-60 cells
 Yeast cultures.- BY4741 yeast were grown at at 30 °C and 250 rpm until a cell
density of 0.5 A600. At that point, designated polyphenol compounds were added
to attain a final concentration of 10 µM and incubation resumed during 3 h.
A
ResveratrolPiceatannol
C
ontrol
5nM
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500nM M1
M
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pmolesQ9/well
C
ontrol
5nM
50nM
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500nM M1
M
10
0.0
0.5
1.0
1.5
2.0
2.5
******
****
pmolesQ10/well
C
ontrol
5nM
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500nM M1
M
10
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pmolesQ9/well
C
ontrol
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M
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Stilbenoids
Q9
Q9
Q10
Q10
ApigeninLuteolin
C
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M
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pmolesQ9/well
C
ontrol
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M
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pmolesQ10/well
C
ontrol
5nM
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M
10
0
5
10
15
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*********
*
pmolesQ9/well
C
ontrol
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500nM
M1
M
10
0.0
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FlavonesC
Q9
Q9
Q10
Q10
Naringenin
C
ontrol
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M
10
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C
ontrol
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500nM M1
0.0
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FlavanoneD
Q9 Q10
M
uscle
Liver
K
idney
B
rain
0
20
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80
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800
Wt Sirt3 KO
pmolsQ9/mgprotein
C
ontrol
M
K

10
10
m
M
N
A
M
+N
A
M
+K
0
10
20
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40
50
d
*** ***
CoenzymeQ10
pmols/mgprotein
C
ontrol
M
K

10
10
m
M
N
A
M+N
A
M
+K
0
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***
***
CoenzymeQ9
pmols/mgprotein
M
uscle
Liver
K
idney
B
rain
0
2
4
6
8
10
20
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60
80
100
pmolsQ10/mgprotein
Q9 Q10
Q9 Q10
 Sirt3 KO tissues.- Kindly
provided by Dr. Verdin (Buck
Institute, CA, USA)
Figure 2. The mitochondrial NAD-dependent deacetylase sirtuin Sirt3 does not participate in the
regulation of Q levels. (A) Mitochondrial form of Sirt3 is upregulated by kaempferol at 10 μM. (B) Acetylation levels
were significantly increased in presence of nicotinamide (NAM) confirming the inhibitory effect of this product over
sirtuins. Arbitrary units depicted in the graph relate directly to the immunoblots shown underneath, and the
immunoblots derive from the same film. (C) General inhibition of sirtuin deacetylases by nicotinamide (NAM) at 10mM
did not prevent the increase of Q levels in Tkpts cells treated kaempferol (K) at 10 µM. (D) Q levels are not altered in
tissues (skeletal muscle, liver, kidney and brain) obtained from Sirt3 knockout mice in comparison with age-matched
controls. Data are represented as mean ± SEM of at least 3 replicates. Differences are represented as * (p < 0.05), ** (p <
0,01) and *** (p < 0.001).
A
Control NAM
0.0
0.5
1.0
1.5
2.0
2.5
***
Acetyl-lysine
a.u.
Control NAM
Anti-acetyl lysine
Control 10µM K
48 kDa
23 kDa
Sirt3
 Protein levels.- Were
measured using western blot
specific inmunostaining.
D
Control
1m
M
PABA
M
K

10
PABA
+
K
0
100
200
300
400
***
***
***
***
pmolsQ9/mgprotein
10 µM K - - + +
1mM PABA - + - +
C
ontrol
1m
M
PA
B
A
M
K

10
+PA
B
A
+K
0
10
20
30
40
50
4
***
***
***
***
pmolsQ10/mgprotein
10 µM K - - + +
1mM PABA - + - +
Q9 Q10
Figure 3. Kaempferol increase Q levels acting as a novel ring precursor. (A) Inhibition of Coq2
polyprenyltransferase with PABA decreased Q levels in control Tkpts cells and abolished the increase of Q9 and Q10 in cells
treated with kaempferol (K). (B) Competition assay of 14C-4HB incorporation into Q. Treatment of cells with 10 µM
kaempferol (K) inhibited the incorporation of 14C-4HB into Q, which indicates competition of kaempferol and 4HB as Q ring
precursors. (C) Demonstration of the role played by kaempferol as a Q ring precursor in Tkpts cells. Cells were cultured for
48 h in the presence of unlabeled (K) or 13C-labeled kaempferol (13C-K) at 10 µM. Unlabeled (12C, open bars) and 13C6-
labeled (closed bars) Q were then measured with HPLC-MS/MS. The majority of Q measured in cells treated with 13C-
kaempferol was 13C6-Q, demonstrating a role for kaempferol as a novel Q ring precursor. Data are represented as mean ±
SEM of 6 replicates. Asterisks denote statistically significant differences (***p<0.001).
Figure 4. Nor curcumin neither ferulic
acid serve as Q ring precursors. The effect
of curcumin, ferulic acid and vanillin (as a
positive control) on Q biosynthesis was tested in
Tkpts cells. (A) Q levels were slightly decreased
in Tkpts cells treated with curcumin and
vanillin. A small amount of 13C6-Q was detected
when Tkpts cells were treated with 13C6-Q -
vanillin, but not when treated with 13C12-Q -
curcumin, indicating that curcumin does not act
as a Q ring precursor. (B) Ferulic acid did not
alter Q levels and no deuterated signal was
recovered after the treatment with D3-ferulic
acid, indicating that ferulic acid does not serve
as a Q ring precursor. Statistically significant
differences between control and treatments are
represented by *p<0.05, **p<0.01 and
***p<0.001.
13C6-vanillin
Figure 5. 4HB is a limiting step in the
biosynthesis of Q in kidney cells. (A)
Different types of murine cells were treated with
increasing concentrations of 4HB. Tpkts cells
exhibited a dramatic increase in Q9 and Q10
levels. However, only a slight increase at some
concentrations of 4HB was observed for Hepa
1.6 and MEFs. (B) Similar experiments were
performed in human cell lines. Human kidney-
derived cells (HEK 293) displayed a significant
increase of Q10 levels, but this effect was not
found in human lines from another origin, such
as Hep G2 or HL-60. The dramatic increase of Q
levels in Tkpts and HEK 293 supports that
availability of 4HB is a limiting step for the Q
biosynthetic pathway in kidney cells. Statistically
significant differences in total Q9 and Q10
between control and treatments are represented
by *p<0.05, **p<0.01 and ***p<0.001.
C
ontrol
K
aem
pferol
Ferulic
acidVanillin
C
urcum
in
0
10
20
30
40
pmolsQ6/od600nm
C
ontrolC
-4HB
13C
-K
aem
pferol
13
C
-Vanillin
13
C
-C
urcum
in
13
0
10
20
30
40
50
13
C6-Q6
12
C-Q6
*
*
pmolsQ6/od600nm
Q6
Figure 6. Kaempferol, ferulic acid, vanillin and curcumin are
not utilized as Q ring precursors as efficiently as 4HB in yeast.
(A) Effect of kaempferol, ferulic acid, vanillin and curcumin on Q6 levels.
BY4741 yeast cells were treated with these compounds at 10 μM
concentrations each, or with EtOH to establish the vehicle control. Yeast
were subsequently grown for 3 h in PABA-minus folate-minus medium. (B)
Effect of 13C-ring-labeled compounds on de novo Q6 biosynthesis. BY4741
yeast cells were treated with 13C6-4HB, 13C-kaempferol, 13C6-vanilin, 13C12-
curcumin, or with EtOH to establish the vehicle control. Significant
differences refer to the control condition and are represented by *p<0.05.
A B
Complete study available in Free Radical Biology and Medicine. 2017 Sep;110:176-187.
doi: 10.1016/j.freeradbiomed.2017.06.006. Epub 2017 Jun 9.
D3-ferulic acid