J. Nutr.-1992-Yamamoto-871-7
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Transcript of J. Nutr.-1992-Yamamoto-871-7
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Biochemical and M olecular Roles of Nutrients
Collagen Synthesis in Human Skin Fibroblasts is
Stimulated by a Stable Form of Ascorbate,
2 - O-oc-D-Glucopyranosy l-L-Ascorb ic Acid1
ITA R YA MA MO TO 2 N OR IO M OT O K O K I M U RA KA MI
ANDJN ICHIAKIYAMA
D ep artm en t o f Im m un oc he mis try F ac ulty o f P ha rm ac eu tic al S cie nc es
O kayam a U niversity Tsushim a naka 1 1 1 O kayam a 700 Japan
ABSTRACT We evaluated the effect of 2-O-a-D-
glucopyranosyl-L -ascorbic acid (A A-2G ) on collagen
synthesis in cultured hum an skin fibroblasts and on pro
liferation of fibroblasts. A t concentrations of 0.1-0.5
m mol/L , AA-2G effectively stim ulated collagen syn
thesis with an effectiveness comparable to that of L-
ascorbic acid. On the other hand, 6-O-ct-D-
glucopyranosyl-L -ascorbic acid show ed a w eak effect.
The stimulation of collagen synthesis by AA-2G was
attenuated by the addition of a collagen synthesis inhib
ito r, L -a ze tid in e 2 -c arb ox ylic a cid , in a d ose -d ep en de nt
m an ner. In ad dition , AA-2G -ind uced stim ulatio n o f co l
lagen synthesis could be completely inhibited by the
addition of castanosperm ine, an inhibitor of neutral
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COLLA GEN SY N THES IS A ND A SCOR BIC A CID 2-GLUCOS IDE
87 3
AA -2G , and the cells w ere cultured f or v arious tim e s
until 24 d. T he m edium w as changed tw ice w eek ly .
A fter rem oval of the m edium , the cell layer w as
rinsed tw ice w ith 2 m L of PB S (pH 7.2) and processed
f or the m easurem ent of D NA content as described by
G iles and M y er (24).
D eter min ation of en zym e a ctivity of cell lysa te.
T he cell suspension (4 x IO9 cells/L ) in 0.1 m ol/L
b arb itu rate b uf fe r (pH 7 .2 ) w as so nicated an d ce ntri-
fuged at 1700 x g for 10 m in. T he supernatant w as
used f or the determ ination of v arious enz ym e activ
ities at 25 Cand at optim al pH . a-G lucosidase (EC
3.2.1.20) activ ity w as determ ined by m easuring the
rate o f f orma tio n o f g lu co se f rom ma lto se as d esc rib ed
p re v io usly (1 7). A lk alin e pho sphatase (EC 3 .1 .3 .1 ) ac
tiv ity w as m e asu red w ith 7 .0 5 mmo l/L p -n itro ph en yl
phosphate in 0.72 m o l/L diethanolam ine-HCl buf fer
(pH 9 .8 ). A ry lsu lf atase (EC 3 .1 .6.1 ) activ ity w as m e a
sured w ith 1.81 m m ol/L p-nitropheny l sulfate in 87
mm o l/L ac etate b uf f er (pH 6 .2 ). p -N i tro ph enol f ormed
in these reactions w as m easured at 405 nm . L -
A s corbic acid-liberating activ ities of the cell ly sate
w ere also determ ined at pH 7.2 w ith A A -2G , A A -2P
or A A -2S . T he reaction m ix ture consisting of 30 uL of
enz ym e solution and 10 uL of 50 m m ol/L L -ascorbic
acid deriv ativ e w as incubated at 37 Cfor 20 m in,
follow ed by addition of 2 v olum es of 0.61 m ol/L
trichloroacetic acid. T o the reaction m ix ture w as
added an equal v olum e of 52 mmo l/L dithiothreitol in
1 m ol/L potassium phosphate buf fer (pH 7.0). T he
samp le obtain ed was analy z ed f or to tal L -asc orb ic ac id
am ount by H PL C (16) equipped w ith an Eicom elec
tro ch em ic al d etec to r. Pro te in co nce ntratio n w as d e
term ined by the m ethod of L ow ry et al. (25) using
b ov in e se rum alb um i n as a stan dard . S pec if ic ac tiv ity
of each enzy m e w as ex pressed as nm ol of substrate
hydro lyzed /(min-mg pro te in ).
S ta tistic al a n a lysis. Me an s a nd sta nd ar d d evia tio n s
are presented and w ere com pared by S tudent's t test
w ith sig nif ican t p ro bab ility lev els o f
o p
u
O
O C
4
Cu lt iv at ion h
60
F IGURE 2 Ra te of disa ppea r a nce of L-a scorbic a cid AA
and 2-O -a-D -glucopy ranosy l-L -ascorbic acid (A A -2G ) in
culture m edium . A A or A A -2G w as added to M EM -10
(m inim um essential m edium w ith 10 fetal bov ine serum )
at a f inal concentration of 0.25 m mol/L in the presence of
f ibroblasts and then incubated at 37 C for 60 h in an at
m osphere of 5 CU2-air. T he rem aining am ounts of A A (
and A A -2G ()n the m edium w ere m easured by HPLC.
Points are the m eans of quadruplicate cultures.
T he ef f ects of A A -2G and other L -ascorbic acid
deriv ativ es on collagen sy nthesis by cultured f ibro
blasts are sum mariz ed in T able 1. A A -2G signif i
cantly stim ulated collagen sy nthesis as w ell as L -
ascorbic acid and A A -2P did, w hereas A A -6G and A A -
2S show ed w eak or no enhancing ef f ects. A m ong
TABLE 1
E ffect of con cen tr ation of L -a scor bic a cid AA der iva tives on
colla gen syn th esis a s a percen tage of tota l protein syn th esis
in h um an skin fibr obla sts*
ConcentrationStimulatorNoneAAAA-2G2AA-6GAA-2PAA-2S0.1
mmol/LC3.3
.311.0
0.4*
8.6 .9*6.3
.4*9.1
.4*3.4
0.20.2 5
mmol/L3.3
.310.1
0.7 *
9.0 .6*6.2
.3*8.0
.5*4.8
0.4*
lCel\s were incubated in 2.0 mL of fresh MEM 10 minimum
essential m edium w ith 10 fetal bov ine serum ) containing
[3H ]proline (74 M B q/L ) and A A deriv ativ es (0.1 and 0.25 mm ol/L )
for 24 h. T he relative rate of collagen sy nthesis to total protein
sy nthesis w as determ ined by the bacterial collagenase m ethod.
V alues are the m eans so of triplicate cultures. 'S ignif icantly
dif ferent com pared w ith corresponding control (none) (P < 0.05).
Abb re v iat io ns u sed: AA -2G , 2 -O -a-D -g lu copy ranos y l-L -as co rb ic
ac id ; A A -6 G, 6 -O -o t-D -g lu co py rano sy l-L -asco rb ic acid; A A -2 P, L -
as co rb ic acid 2-p ho sp hate; A A -2 S, L -as co rb ic acid 2 -su lf ate .
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874 Y A M A MOTO ET A L .
en'ut0>1C(DS1(3no>.Cj^C'ssCL
1 2 3
m M
4 5
F IGURE 3 Ef fect of increasing concentrations of L -
as co rb ic ac id ( AA ) and 2 -O -c e-D- glu copy ranos y l- L -as co rb ic
acid (A A -2G) on collagen sy nthesis in hum an sk in f ibro-
blasts. T he cells w ere incubated in 2.0 m L of fresh M EM -10
(m inim um essential m edium w ith 10 fetal bov ine serum )
containing [3H ]proline (74 M B q/L ) and A A ()r A A - 2G ()
(0.03-0.5 m mol/L ) for 24 h. T he relativ e rate of collagen
sy nthesis to total protein sy nthesis w as determ ined by the
bacterial collagenase m ethod. Points are the m eans S D of
trip lic ate cu ltures. 'S ig nif ican tly dif feren t than v alue d eter
m ined in the absence of A A or A A -2G (P < 0.05).
these L -ascorbic acid-related com pounds, L -ascorbic
acid and A A -6G show ed a reducing activ ity tow ard
cy tochrom e c and a redox dye, w hereas A A -2G, A A -
2P, and A A -2S had no direct reducing activ ity . Conse
quently , there w as no relationship betw een their
stim ulating ef fect on collagen synthesis and their
direct reducibility . A s show n in Figure 3, the stim u
lation of collagen sy nthesis by A A -2G was dose-
dependent at concentrations ranging from 0.03 to 0.5
m mol/L . How ever, L -ascorbic acid strongly stim u
lated collagen synthesis at a concentration as low as
0.06 m mol/L and reached a m ax im um at 0.125 m mol/
L . T he reason for this relativ ely w eak ef fect of L -
ascorbic acid at concentrations m ore than 0.25 m m ol/
L on collagen synthesis is not clear, but this m ay be
due to cy totox icity of hydrogen perox ide produced by
ox idativ e reaction of L -ascorbic acid under culture
conditions (26). O n the contrary , a high concentration
of A A -2G (2.0 mm ol/L ) w as as ef f ectiv e as 0.25
m mol/L in stim ulating collagen synthesis (data not
shown).
Figure 4 illustrates the effect of AzC on AA
2G-induced collagen synthesis. L -A zetidine 2-
carboxy lic acid, an inhibitor of collagen sy nthesis
induced by L -ascorbic acid (27), attenuated the
stim ulatory ef fect of A A -2G on collagen synthesis in
a dose-dependent m anner. W e further determ ined the
ef fect of castanosperm ine, a neutral cc-glucosidase in
hibitor (28), on A A -2G-induced collagen synthesis.
A s show n in T able 2, this com pound com pletely in
hibited A A -2G-induced collagen synthesis, but not
that induced by L -ascorbic acid. T able 3 show s the
I
I
A A-2 G m m o l/L
Az c m m ol/L
0
0
0 . 25
0
0 . 25
0 .5
0 . 25
1 .0
F I GU RE 4 E ff ect of azeti di ne 2-car boxyl ic aci d A zC on
2-O-a-D-g lucopyranosy l -L -ascorb ic ac id (AA -2G) -s timu la ted
collagen sy nthesis in hum an sk in f ibroblasts. T he cells w ere
incubated in 2.0 m L of f resh M EM -10 (m inim um essential
m edium w ith 10 fetal bov ine serum ) containing
[3H]proline (74 M B q/L ), A A -2G (0.25 m m ol/L ) and A z C for
24 h. T he relativ e rate of collagen sy nthesis to total protein
sy nthesis w as determ ined by the bacterial collagenase
m ethod. Points are the m eans S D of triplicate cultures.
*S ignif icantly dif f erent than v alue f rom cells stim ulated by
A A -2G only (P < 0.05).
three enz ym e activ ities in the ly sate of cultured f ibro
blasts. T he cell ly sate ex hib ited a-glucosidase activ ity
and had the ability to release L -ascorbic acid from A A -
2G. T hese values w ere relativ ely higher than the
other tw o enzym e activ ities exam ined. A mong the
three L -ascorbic acid deriv ativ es, A A -2S w as the m ost
TAB LE 2
Abo li shmen t of 2 -O -a-D -g lucopyranosyl -L -asco rb ic acid
A A-2G -i ndu ced col lagen syn th esi s i n h uman sk in
f ibrob lasts by castanospermine1
StimulatorNoneNoneAA-2GAA-2GL-Ascorbic
acidL-Ascorbic
acidCastanospermine
Collagenynthesis
to ta l proteinynthesis5.2
.7+
5.7 .58.6
.3*+
5.1 .313.0
.8*+
13. 1 1 .2*
'Cells w ere incubated in 2.0 m L of f resh M EM -10 (m inim um
essential m edium w ith 10 fetal bov ine serum ] containing
[3 H]p roline (7 4 MBq /L ) an d L -asc orbic acid o r AA -2 G (0 .1 mm o l/L )
in the presence or absence of castanosperm ine (0.1 m m ol/L ) for 24
h. T he relativ e rate of collagen sy nthesis to total protein sy nthesis
w as determ ined by the bacterial collagenase m ethod. V alues are the
m e an s Do f trip licate cu ltu res. S ig nif icantly d if fe ren t c om p ared
w ith control value (none) in the absence of castanosperm ine (P