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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