ENDOCRINE RESEARCH COMMUNICATIONS, 3(5),
273-279
(1976)
EFFECTS OF HUMAN CHORIONIC GONADOTROPIN PREPARATIONS ON AMINO A C I D UPTAKE AND INCORPORATION INTO PROTEIN I N VITRO
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
M. S. GOLBUS and P. K. SIITERI Department of O b s t e t r i c s and Gynecology and t h e Reproductive Endocrinology C e n t e r , U n i v e r s i t y of C a l i f o r n i a San F r a n c i s c o , C a l i f o r n i a 94143
ABSTRACT Human c h o r i o n i c gonadotropin p r e p a r a t i o n s were t e s t e d € o r t h e i r e f f e c t on p r o t e i n metabolism by c u l t u r e d f i b r o b l a s t s . Commercial p r e p a r a t i o n s o f HCG s t r o n g l y inhi’uit amino a c i d u p t a k e and i n c o r p o r a t i o n i n t o p r o t e i n by a v a r i e t y of human a d u l t and f e t a l f i b r o b l a s t l i n e s , whereas, h i g h l y p u r i f i e d HCG i s w i t h o u t such a n e f f e c t .
INTRODUCTION Human c h o r i o n i c gonadotropin h a s been r e p o r t e d t o have a number o f m e t a b o l i c e f f e c t s i n a v a r i e t y of s y s t e m s .
These i n c l u d e
enhancement of amino a c i d i n c o r p o r a t i o n i n t o p r o t e i n by homogenates of p l a c e n t a ( l ) , i n d u c t i o n of enzymes ( 2 ) , i n c r e a s i n g c h o l e s t e r o l s i d e c h a i n c l e a v a g e ( 3 ) , and s u p p r e s s i o n of m a t e r n a l lymphocyte r e s p o n s e t o phytohemagglutinin and i n mixed-lymphocyte r e a c t i o n s (4-8).
culture
I n t h e c o u r s e of s t u d i e s on t h e e f f e c t of human
c h o r i o n i c g o n a d o t r o p i n on amino a c i d u p t a k e and i n c o r p o r a t i o n i n t o p r o t e i n by c u l t u r e d c e l l s t h e q u e s t i o n w a s r a i s e d as t o whether t h e e f f e c t noted w a s due t o t h e g o n a d o t r o p i n , p e r se.
Our r e s u l t s
i n d i c a t e t h a t commercial p r e p a r a t i o n s of HCG s t r o n g l y i n h i b i t amino
273 Copyright 0 1976 by Marcel Dekker, Inc All Rights Reserved. Neither this work noi any p d r t may be reproduced or transmitted in a n y f o r m or by any means, electronic or mechanical. Including photocopying, microfilming, a n d recording, or by a n y information storage a n d retrievdl s y s t e m without permission in writing from t h e publisher.
274
GOLBUS AND SIITERI
a c i d uptake and i n c o r p o r a t i o n by a v a r i e t y of human f i b r o b l a s t l i n e s , whereas, h i g h l y p u r i f i e d HCG i s without e f f e c t .
METHODS AND MATERIALS F i b r o b l a s t c u l t u r e s w e r e maintained i n MEM (IS1 Corp.) sup-
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
plemented w i t h 20% f e t a l c a l f serum (IS1 Corp.), glutamine, penic i l l i n and streptomycin.
A l l experiments w e r e done when c u l t u r e s
w e r e c o n f l u e n t i n T-30 f l a s k s (Falcon P l a s t i c s ) .
Incubations w i t h
t r i t i a t e d amino a c i d s were f o r two hours a f t e r which c e l l s were removed from t h e f l a s k w i t h a "rubber-ducky".
The c e l l s were put
i n t o suspension and a l i q u o t e d i n t o d u p l i c a t e samples f o r amino a c i d uptake, p r o t e i n s y n t h e s i s , and t o t a l p r o t e i n determinations. Amino a c i d uptake and i n c o r p o r a t i o n i n t o p r o t e i n were measured as p r e v i o u s l y described ( 9 ) .
T o t a l p r o t e i n determination w a s by t h e
method of Lowry (10). The commercial c h o r i o n i c gonadotropin p r e p a r a t i o n used w a s APL (Ayerst).
P u r i f i e d HCG (Lot CR-119) w a s k i n d l y provided by D r s . R.
Canfield and G. B i a l y and had an estimated potency of 11,600 IU/mg w i t h 95% confidence l i m i t s a t 8,060 and 16,900 IU/mg. used w e r e t r i t i a t e d t y r o s i n e
Amino a c i d s
s p e c a c t i v i t y = 5 4 C i / m o l ) and l y s i n e
(spec a c t i v i t y = 1 8 Ci/mmol) obtained from Amersham.
Rabbit anti-HCG
(Miles Lab.) was used t o p r e c i p i t a t e t h e gonadotropin i n APL d u r i n g
a 30 minute i n c u b a t i o n a t 37'
C.
275
HCG EFFECTS ON FIBROBLAST AMINO ACID UPTAKE RESULTS
Amino acid uptake and incorporation into protein as a function of the concentration of APL in the culture medium is shown in Fig. 1A-1C.
Fetal lung, fetal skin, and adult skin fibroblasts
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
A 0--0 0-0
UPTAKE INCORPORATION
80 60
-
40
-
20
-
0
-
I
I
*
_
I
B
80 60
I
Fetal Skin
-
40 20
-
0 -
I
I
I
J
C
6ot
401
Adult S k i n ' f G I
20
I
O'l
I
I
10 100 1,000 CONCENTRATION APL ( IU /cc medium)
I
10.000
FIGURE 1 Amino acid uptake and incorporation into protein as a function fetal skin (B), and adult skin (C) fibroblasts. Conditions as described in "Methods".
of APL concentration by fetal lung ( A ) ,
GOLBUS AND SIITERI
276
all demonstrated a marked inhibition of both amino acid uptake and protein synthesis at HCG concentrations between 100 and 1000 IU/cc medium.
The degree of inhibition was directly related to hormone
concentration. Trypan blue exclusion tests demonstrated that these metabolic effects were not secondary to cell death.
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
To investigate whether the metabolic inhibitor was chorionic gonadotropin or some other component of the AF'L, the gonadotropin was precipitated with antibody and the remaining AF'L components tested with adult skin fibroblasts. The quantity used was equivalent to 1000 IU/cc of HCG medium.
In a series of four experiments
the amino acid uptake was 15.6 (t 2.6) % and the incorporation into protein was 9.9 ( 2 1.9) % of control values.
The inhibiting
activity of immunoglobulins, per se, was studied using human gamma globulin (Sigma Chem. Co.) at concentrations up to 33 mg/cc medium. No significant effect on adult skin fibroblast amino acid uptake or protein synthesis was found. The metabolic inhibitor properties of chorionic gonadotropin were further investigated using HCG CR-119 having an estimated potency of 11,600 IU/mg.
Fig. 2 indicates that this relatively
pure HCG had no effect on amino acid uptake or protein synthesis by adult skin fibroblasts. DISCUSSION Although a number of metabolic properties have been attributed to human chorionic gonadotropin, virtually all of these studies have been done employing crude gonadotropin preparations (1-4,8).
277
HCG EFFECTS ON FIBROBLAST AMINO ACID UPTAKE
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
T
c
6o
t
0--0
UPTAKE
0-0
INCORPORATION
20 -
0,
I
I
I
Amino a c i d u p t a k e and i n c o r p o r a t i o n i n t o p r o t e i n by a d u l t s k i n Conditions f i b r o b l a s t s a s a f u n c t i o n of HCG CR-119 c o n c e n t r a t i o n . a s d e s c r i b e d i n "Methods".
S i m i l a r l y , w e found a marked i n h i b i t i o n of amino a c i d u p t a k e and p r o t e i n s y n t h e s i s i n b o t h f e t a l and a d u l t f i b r o b l a s t s u s i n g a commercially a v a i l a b l e HCG p r e p a r a t i o n .
However, when t h e e x p e r i -
ments w e r e r e p e a t e d w i t h a p u r i f i e d HCG t h e hormone had no e f f e c t
on p r o t e i n s y n t h e s i s .
Furthermore, t h e c r u d e p r e p a r a t i o n r e t a i n e d
t h e metabolic i n h i b i t o r a c t i v i t y a f t e r t h e c h o r i o n i c gonadotropin had been removed from t h e commercial p r e p a r a t i o n by immunologic precipitation.
1
GOLBUS AND SIITERI
278
The anti-complement a c t i o n of HCG p r e p a r a t i o n s h a s been shown t o b e due t o t h e p r e s e n c e o f immunoglobulins i n t h e p r e p a r a t i o n s and n o t t o t h e g o n a d o t r o p i n ( 1 1 ) .
W e , t h e r e f o r e , examined t h e
a b i l i t y o f human gamma g l o b u l i n , a t c o n c e n t r a t i o n s e q u i v a l e n t t o t h o s e found i n u r i n a r y g o n a d o t r o p i n p r e p a r a t i o n s (12, 1 3 ) , t o
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
i n h i b i t amino a c i d u p t a k e and p r o t e i n s y n t h e s i s .
No i n h i b i t i n g
( o r s t i m u l a t i n g ) e f f e c t w a s found. The p r e s e n t s t u d y i n d i c a t e s t h a t many of t h e r e p o r t e d metab o l i c e f f e c t s o f HCG p r e p a r a t i o n s are l i k e l y n o t t o b e due t o t h e g o n a d o t r o p i n , p e r se.
It may b e n e c e s s a r y t o r e c o n s i d e r p r e v i o u s
p u b l i s h e d works o n t h e m e t a b o l i c e f f e c t s of human c h o r i o n i c gonadotropin. ACKNOWLEDGMENTS
Supported i n p a r t by t h e N a t i o n a l Foundation-March of D i m e s (CRBS-321) and t h e R o c k e f e l l e r Foundation. The a u t h o r s w i s h t o acknowledge t h e e x p e r t t e c h n i c a l a s s i s t a n c e of M r . Leonard B e r r y .
REFERENCES
1.
O r l a n d i , C . , S e g a t a , L. and Becca, B . , 50: 233, 1966.
2.
Maudsley, D. V. and Kobayashi, Y . , 1974.
3.
Menon, K. M. J . , Dorfman, R. I. and F o r c h i e l l i , E . , Biochim. Biophys. Acta, 148,486, 1967.
4.
Kaye, M. D. and J o n e s , W . R . , 1029, 1971.
Riv.
I t a l . Ginecol.,
Biochem. Pharm,,
2: 2697,
Am. J. O b s t e t . Gynecol.,
109:
279
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
HCG EFFECTS ON FIBROBLAST AMINO ACID UPTAKE
5.
J e n k i n s , D. M . , Acres, M. G . , P e t e r s , J. and R i l e y , J , , , Am. J . O b s t e t . Gynecol., 114:1 3 , 1972.
6.
Adcock, E. W . , T e a s d a l e , F . , August, C. S . , Cox, S . , Meschia, G . , B a t t a g l i a , F . C. and Naughton, M. A., S c i e n c e 845, 1973.
7.
C o n t r a c t o r , S . F. and Davies, H . , N a t u r e New B i o l . , 1973.
a.
T e a s d a l e , F . , Adcock, E. W . , August, C. S . , Cox, S . , B a t t a g l i a , F. C. and Naughton, M. D . , Gynecol. I n v e s t . , 4: 263, 1.973.
9.
E p s t e i n , C. J . and Smith, S . A . ,
z:
Devel. B i o l . ,
33:
43:
284,
1 7 1 , 1973.
10.
Lowry, 0. H . , Rosebrough, M. J . , F a r r , A. L . and R a n d a l l , R . J . , J. B i o l . Chem., 193: 265, 1951.
11.
Loke, Y . W. and Pepys, M. B . , 3 7 , 1975.
12.
Boyce, W. H . , Garvey, F. K. and N o r f l e e t , C. M. J r . , J . C l i n . I n v e s t . , 3: 1 2 8 7 , 1954.
13.
McGarry, E . , 832, 1955.
Am.
J . O b s t e t . Gynecol.,
Sehon, A. H. and Rose, B . ,
121:
J. C l i n . I n v e s t .
Received:
3:
A p r i l 1976
273-279
(1976)
EFFECTS OF HUMAN CHORIONIC GONADOTROPIN PREPARATIONS ON AMINO A C I D UPTAKE AND INCORPORATION INTO PROTEIN I N VITRO
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
M. S. GOLBUS and P. K. SIITERI Department of O b s t e t r i c s and Gynecology and t h e Reproductive Endocrinology C e n t e r , U n i v e r s i t y of C a l i f o r n i a San F r a n c i s c o , C a l i f o r n i a 94143
ABSTRACT Human c h o r i o n i c gonadotropin p r e p a r a t i o n s were t e s t e d € o r t h e i r e f f e c t on p r o t e i n metabolism by c u l t u r e d f i b r o b l a s t s . Commercial p r e p a r a t i o n s o f HCG s t r o n g l y inhi’uit amino a c i d u p t a k e and i n c o r p o r a t i o n i n t o p r o t e i n by a v a r i e t y of human a d u l t and f e t a l f i b r o b l a s t l i n e s , whereas, h i g h l y p u r i f i e d HCG i s w i t h o u t such a n e f f e c t .
INTRODUCTION Human c h o r i o n i c gonadotropin h a s been r e p o r t e d t o have a number o f m e t a b o l i c e f f e c t s i n a v a r i e t y of s y s t e m s .
These i n c l u d e
enhancement of amino a c i d i n c o r p o r a t i o n i n t o p r o t e i n by homogenates of p l a c e n t a ( l ) , i n d u c t i o n of enzymes ( 2 ) , i n c r e a s i n g c h o l e s t e r o l s i d e c h a i n c l e a v a g e ( 3 ) , and s u p p r e s s i o n of m a t e r n a l lymphocyte r e s p o n s e t o phytohemagglutinin and i n mixed-lymphocyte r e a c t i o n s (4-8).
culture
I n t h e c o u r s e of s t u d i e s on t h e e f f e c t of human
c h o r i o n i c g o n a d o t r o p i n on amino a c i d u p t a k e and i n c o r p o r a t i o n i n t o p r o t e i n by c u l t u r e d c e l l s t h e q u e s t i o n w a s r a i s e d as t o whether t h e e f f e c t noted w a s due t o t h e g o n a d o t r o p i n , p e r se.
Our r e s u l t s
i n d i c a t e t h a t commercial p r e p a r a t i o n s of HCG s t r o n g l y i n h i b i t amino
273 Copyright 0 1976 by Marcel Dekker, Inc All Rights Reserved. Neither this work noi any p d r t may be reproduced or transmitted in a n y f o r m or by any means, electronic or mechanical. Including photocopying, microfilming, a n d recording, or by a n y information storage a n d retrievdl s y s t e m without permission in writing from t h e publisher.
274
GOLBUS AND SIITERI
a c i d uptake and i n c o r p o r a t i o n by a v a r i e t y of human f i b r o b l a s t l i n e s , whereas, h i g h l y p u r i f i e d HCG i s without e f f e c t .
METHODS AND MATERIALS F i b r o b l a s t c u l t u r e s w e r e maintained i n MEM (IS1 Corp.) sup-
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
plemented w i t h 20% f e t a l c a l f serum (IS1 Corp.), glutamine, penic i l l i n and streptomycin.
A l l experiments w e r e done when c u l t u r e s
w e r e c o n f l u e n t i n T-30 f l a s k s (Falcon P l a s t i c s ) .
Incubations w i t h
t r i t i a t e d amino a c i d s were f o r two hours a f t e r which c e l l s were removed from t h e f l a s k w i t h a "rubber-ducky".
The c e l l s were put
i n t o suspension and a l i q u o t e d i n t o d u p l i c a t e samples f o r amino a c i d uptake, p r o t e i n s y n t h e s i s , and t o t a l p r o t e i n determinations. Amino a c i d uptake and i n c o r p o r a t i o n i n t o p r o t e i n were measured as p r e v i o u s l y described ( 9 ) .
T o t a l p r o t e i n determination w a s by t h e
method of Lowry (10). The commercial c h o r i o n i c gonadotropin p r e p a r a t i o n used w a s APL (Ayerst).
P u r i f i e d HCG (Lot CR-119) w a s k i n d l y provided by D r s . R.
Canfield and G. B i a l y and had an estimated potency of 11,600 IU/mg w i t h 95% confidence l i m i t s a t 8,060 and 16,900 IU/mg. used w e r e t r i t i a t e d t y r o s i n e
Amino a c i d s
s p e c a c t i v i t y = 5 4 C i / m o l ) and l y s i n e
(spec a c t i v i t y = 1 8 Ci/mmol) obtained from Amersham.
Rabbit anti-HCG
(Miles Lab.) was used t o p r e c i p i t a t e t h e gonadotropin i n APL d u r i n g
a 30 minute i n c u b a t i o n a t 37'
C.
275
HCG EFFECTS ON FIBROBLAST AMINO ACID UPTAKE RESULTS
Amino acid uptake and incorporation into protein as a function of the concentration of APL in the culture medium is shown in Fig. 1A-1C.
Fetal lung, fetal skin, and adult skin fibroblasts
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
A 0--0 0-0
UPTAKE INCORPORATION
80 60
-
40
-
20
-
0
-
I
I
*
_
I
B
80 60
I
Fetal Skin
-
40 20
-
0 -
I
I
I
J
C
6ot
401
Adult S k i n ' f G I
20
I
O'l
I
I
10 100 1,000 CONCENTRATION APL ( IU /cc medium)
I
10.000
FIGURE 1 Amino acid uptake and incorporation into protein as a function fetal skin (B), and adult skin (C) fibroblasts. Conditions as described in "Methods".
of APL concentration by fetal lung ( A ) ,
GOLBUS AND SIITERI
276
all demonstrated a marked inhibition of both amino acid uptake and protein synthesis at HCG concentrations between 100 and 1000 IU/cc medium.
The degree of inhibition was directly related to hormone
concentration. Trypan blue exclusion tests demonstrated that these metabolic effects were not secondary to cell death.
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
To investigate whether the metabolic inhibitor was chorionic gonadotropin or some other component of the AF'L, the gonadotropin was precipitated with antibody and the remaining AF'L components tested with adult skin fibroblasts. The quantity used was equivalent to 1000 IU/cc of HCG medium.
In a series of four experiments
the amino acid uptake was 15.6 (t 2.6) % and the incorporation into protein was 9.9 ( 2 1.9) % of control values.
The inhibiting
activity of immunoglobulins, per se, was studied using human gamma globulin (Sigma Chem. Co.) at concentrations up to 33 mg/cc medium. No significant effect on adult skin fibroblast amino acid uptake or protein synthesis was found. The metabolic inhibitor properties of chorionic gonadotropin were further investigated using HCG CR-119 having an estimated potency of 11,600 IU/mg.
Fig. 2 indicates that this relatively
pure HCG had no effect on amino acid uptake or protein synthesis by adult skin fibroblasts. DISCUSSION Although a number of metabolic properties have been attributed to human chorionic gonadotropin, virtually all of these studies have been done employing crude gonadotropin preparations (1-4,8).
277
HCG EFFECTS ON FIBROBLAST AMINO ACID UPTAKE
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
T
c
6o
t
0--0
UPTAKE
0-0
INCORPORATION
20 -
0,
I
I
I
Amino a c i d u p t a k e and i n c o r p o r a t i o n i n t o p r o t e i n by a d u l t s k i n Conditions f i b r o b l a s t s a s a f u n c t i o n of HCG CR-119 c o n c e n t r a t i o n . a s d e s c r i b e d i n "Methods".
S i m i l a r l y , w e found a marked i n h i b i t i o n of amino a c i d u p t a k e and p r o t e i n s y n t h e s i s i n b o t h f e t a l and a d u l t f i b r o b l a s t s u s i n g a commercially a v a i l a b l e HCG p r e p a r a t i o n .
However, when t h e e x p e r i -
ments w e r e r e p e a t e d w i t h a p u r i f i e d HCG t h e hormone had no e f f e c t
on p r o t e i n s y n t h e s i s .
Furthermore, t h e c r u d e p r e p a r a t i o n r e t a i n e d
t h e metabolic i n h i b i t o r a c t i v i t y a f t e r t h e c h o r i o n i c gonadotropin had been removed from t h e commercial p r e p a r a t i o n by immunologic precipitation.
1
GOLBUS AND SIITERI
278
The anti-complement a c t i o n of HCG p r e p a r a t i o n s h a s been shown t o b e due t o t h e p r e s e n c e o f immunoglobulins i n t h e p r e p a r a t i o n s and n o t t o t h e g o n a d o t r o p i n ( 1 1 ) .
W e , t h e r e f o r e , examined t h e
a b i l i t y o f human gamma g l o b u l i n , a t c o n c e n t r a t i o n s e q u i v a l e n t t o t h o s e found i n u r i n a r y g o n a d o t r o p i n p r e p a r a t i o n s (12, 1 3 ) , t o
Endocr Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/19/14 For personal use only.
i n h i b i t amino a c i d u p t a k e and p r o t e i n s y n t h e s i s .
No i n h i b i t i n g
( o r s t i m u l a t i n g ) e f f e c t w a s found. The p r e s e n t s t u d y i n d i c a t e s t h a t many of t h e r e p o r t e d metab o l i c e f f e c t s o f HCG p r e p a r a t i o n s are l i k e l y n o t t o b e due t o t h e g o n a d o t r o p i n , p e r se.
It may b e n e c e s s a r y t o r e c o n s i d e r p r e v i o u s
p u b l i s h e d works o n t h e m e t a b o l i c e f f e c t s of human c h o r i o n i c gonadotropin. ACKNOWLEDGMENTS
Supported i n p a r t by t h e N a t i o n a l Foundation-March of D i m e s (CRBS-321) and t h e R o c k e f e l l e r Foundation. The a u t h o r s w i s h t o acknowledge t h e e x p e r t t e c h n i c a l a s s i s t a n c e of M r . Leonard B e r r y .
REFERENCES
1.
O r l a n d i , C . , S e g a t a , L. and Becca, B . , 50: 233, 1966.
2.
Maudsley, D. V. and Kobayashi, Y . , 1974.
3.
Menon, K. M. J . , Dorfman, R. I. and F o r c h i e l l i , E . , Biochim. Biophys. Acta, 148,486, 1967.
4.
Kaye, M. D. and J o n e s , W . R . , 1029, 1971.
Riv.
I t a l . Ginecol.,
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