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Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 1
1
Human Nutrition Center for Research on Ageing
Tufts University, Boston
angelo.azzi@tufts.edu
Cell Signaling by Vitamin E
Angelo Azzi, MD, PhD
2
3Molecular aspects of α-tocotrienol antioxidant action and cell signaling,
Lester Packer, Stefan U. Weber and Gerald Rimbach, Journal of Nutrition, 2001, 131: 369S-373S
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 2
4
Other chemical antioxidants
with different biological function
� Vitamin A
� Estrogens
� Melatonin
5
Evans, H. M. and Bishop, K. S., (1922), On the existence of a hitherto
unrecognized dietary factor essential for reproduction, Science,
(Washington, DC), 56: 649-651
"Factor X" was v itamin E missing f rom the diet,
and present in lettuce
The discovery
6
Epstein, S. S., Forsyth, J., Saporoschetz, I. B., Mantel, N., (1966),
An exploratory investigation on the inhibition of selected photosensitizers
by agents of varying antioxidant activity, Radiat. Res., 28 (2): 322-35
The antioxidant function
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 3
7
αααα -Tocopherol
L-ascorbate
GSH, thioredoxin reductase, etc.
A) Lipid peroxidation
B) Antioxidant
mechanism
of αααα -tocopherol
C) αααα -tocopherol recycling by L-ascorbate
8
The f ollowing thesis has been v ery f requently embraced
1. α-tocopherol has chemical radical scav enging properties,
shown by test-tube chemistry
2. All ev ents mediated by α-tocopherol are due to this property
The problem
9Min, K. C., Kovall, R. A., Hendrickson, W. A., Proc. Natl. Acad. Sci. USA, 2003, 100: 14713-8
Alpha-tocopherol transf er protein
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 4
10
α-tocopherol selection, through α-TTP structural conserv ation,
has been maintained ov er time: this requires the existence
of an ev olutionary pressure f or the uptake of a unique molecule,
α-tocopherol, rather than of a generic antioxidant
11
Concentration (µM)
Cell
nu
mb
er
(%)
Trolox C
α-tocopherol acetate
dl-α-tocopherol
RRR-α-tocopherol
12
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 5
13
Yoshimi Takai, Akira Kishimoto,
Ushio Kikkawa, Terutoshi Mori
and Yasutomi Nishizuka
Biochemical and Biophysical Research Communications,
91, 1979, 1218-1224
Unsaturated diacy lgly cerol
as a possible messenger
f or the activ ation
of calcium-activ ated,
phospholipid-dependent
protein kinase sy stem
14
PKC inhibition by dif f erent tocopherols in smooth muscle cells
PP2A
PKC
PKC -P
α-tocopherol
Active
Inactive
+
Boscoboinik, D., Szewczyk, A., et al., (1991), Inhibition of cell proliferation by alpha-tocopherol. Role of protein
kinase C, J. Biol. Chem., 266: 6188-6194
Ricciarelli, R., Tasinato, A., et al., (1998), Alpha-tocopherol specifically inactivates
cellular protein kinase C alpha by changing its phosphorylation state, Biochem. J.,
334 (Pt 1): 243-9
15
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 6
16
Cell
pro
life
rati
on
, %
Pro
tein
kin
ase C
acti
vit
y
% o
f co
ntr
ol
Contr
ol
d-α
-tocophero
l
d-β
-tocophero
l
d-α
-tocophero
ld-β
-tocophero
l
Contr
ol
d-α
-tocophero
l
d-β
-tocophero
l
d-α
-tocophero
l+
d-β
-tocophero
l
17
Inhibition of NADPH-oxidase assembly
by ⟨⟨⟨⟨ -tocopherol
Diabetes, 1994, 43: 1372-7;Cachia, O., Benna, J. E., Pedruzzi, E., Descomps, B., Gougerot-Pocidalo, M. A., Leger, C. L.,
Alpha-tocopherol inhibits the respiratory burst in human monocytes. Attenuation of p47(phox) membrane translocation and phosphorylation, J. Biol. Chem., 1998, 273: 32801-5
18
Circulation, 2000, 102: 82-7
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 7
19
FACS analysis of oxLDL uptake (macrophage)
Log fluorescence
Co
un
ts
20
� Animal studies (Barella, et al.; Gohil, et al.)
� No genes expressing antioxidant enzy mes appear
to be up regulated in the absence of alpha-tocopherol
as expected by an obv ious compensatory mechanism
21
Kempna, P., Zingg, J. M., Ricciarelli, R., Hierl, M., Saxena, S., Azzi, A., Cloning of novel human SEC14p-like proteins: ligand binding
and functional properties, Free Radic. Biol. Med., 2003, 34: 1458-72
Stocker, A., Tomizaki, T., Schulze-Briese, C., Baumann, U., Structure (Camb.), 2002, 10: 1533-40, Crystal structure of the human supernatant protein factor
Zimmer, S., Stocker, A., Sarbolouki, M. N., Spycher, S. E., Sassoon, J., Azzi, A., A novel human tocopherol-associated protein: cloning,
in vitro expression, and characterization, J. Biol. Chem., 2000, Aug. 18, 275: 25672-80
A new family of tocopherol associated proteins
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 8
22
Specificity of αααα-tocopherol binding to TAP
Tocopherol-associated protein is a ligand-dependent transcriptional activator, Jun Yamauchi, Taku Iwamoto, Satoshi Kida, Shoichi Masushige, Kazuhiko Yamada, Takatoshi Esashi, Biochemical and Biophysical Research Communications, Vol. 285, No. 2, Jul. 2001, pp. 295-299
23
P 0 15 30 60 2h P 0 15 30 60 2h P 0 15 30 60 2hTime (min)
GDP -
GTP -
hTAP1 hTAP2 hTAP3
0
100
200
300
% G
TP
ase a
cti
vit
yo
f co
ntr
ol
GTPase activity of hTAPs
CGAGESGKSTIVKQMK α-common peptide of G proteinsNYGGDIPRKYYVRDQV hTAP1NYGGEIPKSMYVRDQV hTAP2NYGGEVPKSYYLCEQV hTAP3
24
Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol
on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B
and peroxisome proliferator-activated receptor-gamma,
Adelina Munteanu, Michele Taddei, Ilaria Tamburini, Ettore Bergamini, Angelo Azzi
and Jean-Marc Zingg, JBC, 2006, 281(10): 6489-97
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 9
25
The PKC-PI3 kinase regulation systems
26αααα -tocopherol
L-ascorbate
GSH, thioredoxin reductase, etc.
A) Lipid peroxidation
B) Signalling by αααα -tocopherol
C) αααα -tocopherol protection by L-ascorbate
27Menno, P. J. de Winther and Marten H. Hofker, (2000), J. Clin. Invest., 105 (8): 1039-41
Production and uptake of oxLDL by macrophages � Foam cells
� Atherosclerotic plaques � Smooth muscle cell
♫NADPH-OX♫
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 10
28Munteanu, A., Zingg, J. M. and Azzi, A., (2004), JCMM, 8: 59-76
29
Higher baseline serum concentrations of v itamin E are associated
with lower total and cause-specif ic mortality in the alpha-tocopherol,
beta-carotene cancer prev ention study
Margaret E. Wright, Karla A. Lawson, Stephanie J. Weinstein, Pirjo Pietinen, Philip R. Taylor,
Jarmo Virtamo, and Demetrius Albanes, Am. J. Clin. Nutr., 2006, 84: 1200–7
30
� AVED prev ention and some regression of ataxia sy mptoms
� Protection against Alzheimer's and Parkinson's diseases
with high intake of dietary antioxidants, including v itamin E (Jimenez-Jimenez et al., 1997; Sano et al., 1997)
� Supplemental intake of v itamin E has been shown to improv e
cell-mediated immunity and reduce the incidence of upper
respiratory tract inf ection in elderly subjects (Meydani et al., 2004)
Documented effects of α-tocopherol
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 11
31
� Weinberg, P. D., Analy sis of the v ariable ef f ect of dietary
v itamin E supplements on experimental atherosclerosis,
Plant Physiol., 2005, Jul., 162 (7): 823-33
� Almost all the mice trials showed a signif icant ef f ect of v itamin E
� One-third of the rabbit trials responded positiv ely
� The high doses of v itamin E required to af f ect experimental
atherosclerosis may , if applicable to other species, help explain
the absence of ef f ects in many human trials
In animal studies α-tocopherol is generally active
32Robert Gianello, Roksan Libinaki, Angelo Azzi, Paul D. Gavin, Yesim Negis, Jean-Marc Zingg, Phillip Holt, Hooi-Hong Keah, Annike Griffey, Andrew Smallridge, Simon M. West and Esra Ogru,
α-tocopheryl phosphate: a novel, natural form of vitamin E, Free Radic. Biol. Med., 2005
33Negis, Y., Zingg, J. M., Ogru, E., Gianello, R., Libinaki, R., Azzi, A., On the existence of cellular tocopheryl
phosphate, its synthesis, degradation and cellular roles: a hypothesis, IUBMB Life, 2005, 57: 23-5
[32P]ATP + α-tocopherol � [ 32P]-α-tocopherol
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 12
34
No cytotoxicity of tocopheryl phosphate
35Menno P.J. de Winther and Marten H. Hofker, (2000), J. Clin. Invest., 105 (8): 1039-41
Production and uptake of oxLDL by macrophages � Foam cells
� Atherosclerotic plaques � Smooth muscle cell
♫NADPH-OX
36
Inhibition of superoxide production by tocopherol and tocopheryl phosphate
in THP cells
Time, h
Supero
xid
e
pro
ductio
n
µm
ol/h/1
06
cells
Control αααα -tocopherol
αααα -tocopheryl phosphate
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 13
37
Inhibition of CD36 expression by α-tocopherylphosphate occurs at concentrations
lower than by α-tocopherol
Co
un
ts
38
0
0.1
0.2
0.3
0.4
0.5
0.6 Control
10 α-toc
25 α-toc
50 α-toc
10 TP
25 TP
50 TP
TP inhibits - tocopherol does not –
THP-1 cell proliferation
Cell p
rolife
rati
on
, arb
itra
ry u
nit
s
39
THP-1 cells treated with TP
pmol TP/µµµµg protein pmol αααα -tocopherol/µµµµg protein
(nmol αααα -toc/mg protein)
Control 0,00 0,00
10 µM TP 10,62 ± 1,93 1,89 ± 0,40
25 µM TP 16,99 ± 0,08 2,83 ± 0,00
50 µM TP 23,99 ± 1,62 3,59 ± 0,07
TP is only minimally converted to tocopherol
TP ⟨⟨⟨⟨ -tocopherol
pm
ol/
µg
pro
tein
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 14
40
2,31 ± 0,38
11,03 ± 3,1850 µµµµM αααα -toc
5,16 ± 2,3825 µµµµM αααα -toc
10 µµµµM αααα -toc
0,03Control
pmol αααα -tocopherol/µµµµg protein(nmol αααα -toc/mg protein)
THP-1 cells treated with αααα -tocopherol
Tocopherol is transported into cells
Control 10 25 50
pm
ol
α-t
oco
ph
ero
l/µg
pro
tein
41
The structure of glybenclamide
Gly benclamide abolishes ABCA1-mediated
cholesterol ef f lux, phospholipid ef f lux, apoA-I binding
42
Incubation of THP-1 cells with glybenclamide
and tocopheryl phosphate and measurement
of their tocopheryl phosphate content
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 15
43
Inhibition of cell proliferation by tocopheryl phosphate
and its prev ention by glybenclamide
10-200 µM TP + 10 µM glybenclamide
Me
an
ab
so
rba
nc
e (
49
0 n
m)
0 hour control
24 hour control
10 microM glybenclamide
10 microM TP
10 microM TP + 10 microM GC
25 microM TP
25 microM TP + 10 microM GC
50 microM TP
50 microM TP + 10 microM GC
100 microM TP
100 microM TP + 10 microM GC
200 microM TP
200 microM TP + 10 microM GC
44Menno P.J. de Winther and Marten H. Hofker, (2000), J. Clin. Invest., 105 (8): 1039-41
Production and uptake of oxLDL by macrophages � Foam cells
� Atherosclerotic plaques � Smooth muscle cell
♫NADPH-OX
45
Tocopheryl phosphate given with the diet
is not converted to tocopherol
� Plasma concentrations of tocopherol and tocophery l phosphate
� Group α-tocopherol (µg/mL) TP (µg/mL)
� TA (1 g/kg) 32 ± 11.79** 4.80 ± 6.39
� TP (1.33 g/kg) 11.95 ± 7.14* 15.02 ± 1.29*
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 16
46
A →→→→ Control diet (no cholesterol, no treatments)
B →→→→ 2% cholesterol diet alone
C →→→→ 2% cholesterol + TP 0.33 g/kg
in the diet (62% diminutionof plaque formation)
D →→→→ 2% cholesterol + TA 1 g/kg in the diet (17% diminutionof plaque formation)
Tocopheryl phosphate is more effective
than the acetate ester in vivo
47
Disease protection
48
Cell Signaling by Vitamin E
Prof. Angelo Azzi
The screen versions of these slides have full details of copyright and acknowledgements 17
49
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