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

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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