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The effect of insulin on the formation of protein and ribonucleic acid by fetal rat thyroid glands in organ culture
Lüe Sciences Vol. Great Britain.
4, pp . 1603-1809, 1985 . Pergamon Press Ltd. Printed in
THE EFFECT OF INSULIN ON THE FORMATION OF PROTEIN AND RTnp~iUCT.RT C ACID BY FETAL RAT THYROID GLANDS IN ORGAN CULTURE*
V . N . Singh, B . M . Nataf and I . L . Chaikoff Departriment of Physiology, University of California Berkeley
(Received 1 June
1985)
introduction The ability of insulin to enhance 1311 uptake and incorpora_ tion of the isotope into protein bound monoiodotyrosine, duodo_ tyrosine and thyroxine by fetal rat thyroid glands in organ cul_ ture was recently demonstrated (1) .
Since these iodoamino acids
are components of thyroglobulin, we were led to study the action of insulin on the synthesis of protein and ribonucleic acid by the thyroid gland explants . Materials and Methods Fetuses were removed by Caesarean operation from 21-day_ pregnant rats of the Long_Evans strain and their thyroid glands were excised under sterile conditions
(1) .
The thyroid explants
were cultured by the watch-glass method of Chen (2) as modified by Elias and Rivers (3), the details of which have been described by Nataf et al . (4) . Determination of Protein 14C - At the end of the experiment, the tissue in each dish was removed, washed once with fresh * This investigation was supported by grants from the U . S . Public Health Service . 1603
Vol. 4, No . 18
EFFECT OF INSULIN
1804
culture medüun, and transferred to a centrifuge tube containing One ml of an aqueous solo_
one ml of 1096 trichloroacetic acid .
tion containing 2 mg of bovine serum albwnin was added as carrier, The homogenate was centrifuged
and the tissue was homogenized .
and the protein precipitate was prepared for 14 C counting as de_ scribed by Singh et al . (5) . Determination of Ribonucleic Acid 14C _ The glands were re_ moved, washed once with fresh medium, and immersed in one ml of chilled 0 .05 M tris_HCl buffer the mixture was frozen .
(p~H 7 .6) .
Immediately thereafter
Ribonucleic acid was isolated by the
procedure of Schemer and Darnell (6) with the following modifi_ cations :
(a) polyvinyl sulfate was not used ; (b) after adding
sodium dodecyl sulfate, the homogenate was kept at 60° for 5 minutes before the addition of phenol ; and (c) two mg of yeast ribonucleic acid were added as carrier before adding alcohol to precipitate the ribonucleic acid .
The precipitate was dissolved
in 0 .05 M tris_HC1 buffer of pH 7 .6, and an aliquot was taken for determination of its 14C content (5) . Results The 14 C of each amino acid added to the medium was readily incorporated into protein (Table 1) .
The presence of insulin in
the medium enhanced the incorporation in each case .
Insulin in_
creased the 14C incorporation into protein regardless of the age of the culture (Table 2) .
The 2~hour incorporation of leucine
carbon into thyroidal protein was about the same in one, two, end three_day_old cultures (Table 2) .
Vol . 4, No. 18
EFFECT OF INSULIN
180 5
TAHLB 1 $ffect of Insulin on Incorporation of 14C of 14 C-labeled L-Amino Acids into Protein by Fetal Rat Thyroid Glands in Organ Culture for Two Days Thyroid glands excised from 21_day_old fetuses were cu~~ured for Twenty_ two days in the basal medium* with or without insulin . four hours before harvesting the culture, the medium was replaced with fresh medium (with or without insulin) to which a 14 Clabeled amino acid had been added (0 .5 ~c/ml) . Incorporation of all four amino acids was studied in each experiment which was tarried out with the glands excised from the fetuses of a single pregnant rat . Each value is the mean ± 5 .8 . of the results of four separate experiments . 14~p~ino acid#
nou in a to medium hg
`~ recover n protein of one whole gland cpm
L-Leucine L-Leucine
None 5
4,200 ± 380 6,800 ± 550
L-Phenylalanine L-Phenylalanine
None 5
9,300 ± 210 5,300 ± 840
L-Tyrosine L-Tyrosine
None 5
1,600 f 170 2,600 ± 250
L-Valine L-Valine
None 5
4,700 ± 240 5,900 ± 350
* Medium 199 (7) to which penicillin G had been added (50 units per ml) . +~* Amorphous insulin, 20 unite per mg and practically devoid of glucagon (less than 0 .0003%), was furnished by the Lilly Laboratories, $li Lilly and Co ., Indianapolis, Ind. The amino acids were uniformly labeled with 14C and their specific activities ranged from 200 to 360 me per amole .
Puromycin, an inhibitor of protein biosynthesis (8,9), and 2,4_dinitrophenol and arsenate, both of which uncouple oxidative phoaphorylation (10), appreciably inhibited incorporation of leucine carbon into protein by the fetal thyroid explants 3) .
(Table
The inhibition by 2,4_dinitropihenol and arsenate indicates
1808
EFFECT OF INSULIN
Vol. 4, No. 18
TABLE 2 Effect of Insulin on Incorporation of 14C of L-Leucine-U-14 C into Protein by One, TWO and ThreeDay_Old Cultures of Fetal Rat Thyroid Glande The experimental details are given in Table 1 . The labeled leu_ cine waä added 24 hours before harvesting the culture . Fetuses from a single pregnant rat were used for each experiment . Each value is the mean ± S .E . of the results of four experiments . e o cu ure when harvested days
nau in a to medium Fig
in `tC rernver protein of one whole gland cpn
1 1
None 5
4,300 ± 360 6,600 ± 460
2 2
None 5
4, 500 f 260 7,800 ± 790
3 3
None 5
4,900 ± 670 7,800 + 740
TABLE 3 Effects of Puromycin, Arsenate and 2,4_Dinitrophenol on Incorporation of 14C of L-Leucine_U_ 14 C into Protein by Fetal Rat Thyroid Glanda in Organ Culture fór Two Days Each experiment made use of all inhibitors and was carried out With the thyroid glands excised from the fetuses of a single pregnant rat . Each value is the mean ~ S .E . of the results of four separate experiments . Addition to medium None
i~ rernvered in protein of one whole gland cpm 6, 000 f 470
Puromycin (100 Wg)
150 ± 30
Arsenate (5 Eunolea)
240 ± 30
2,4_Dinitrophenol (1 Funole)
400 ± 70
EFFECT OF INSULIN
Vol . 4, No. 16
1607
that the incorporation is energy dependent . Table 4 shows that the addition of insulin to the medium increased considerably the incorporation of 14 C of adenine_8_14C into ribonucleic acid by the fetal rat thyroid glands . TABLE 4 Effect of Insulin on Incorporation of 14C of Adenine_8_ 14 C into RNA by Fetal Rat Thyroid Glands in Organ Culture for Two Days Thyroid glands excised from 21_day_old fetuses were cultured for 2 days in the basal medium with or without insulin . Twenty four hours before termination of culture, the medium was replaced with a fresh medium (with or without insulin) to which was added one we/ml of adenine_8_14C (specific ac_ tivity, 5 mc/mmole) . Thyroid glands from two fetuses were used for each analysis . Each value is the mean ± S .S . of five analyses . Insulin added to medium
4 recover as RDiA of one whole gland cpm .
None
1,380 ± 70
5 wg
2,490 ~ 140
Discussion The ability of insulin to enhance the incorporation of amino acids and amino acid precursors into protein has been demonstrated in several tissues (11_13) .
Although the precise
mechanism of this action of insulin has not yet been established, there is evidence suggesting that the hormone enhances protein formation by increasing the synthesis of ribonucleic acid (13) . It is of interest to recall here that the role of ribonucleic acid, possibly messenger ribonucleic acid, in the enhancement of
1808
EFFECT OF INSULIN
Vol. 4, No. 18
protein synthesis bY other hormones concenred with protein anabo_ liam has also been pointed out (14,15) . Our findings show that insulin enhanced the formation of both protein and ribonucleic acid by fetal rat thyroid glands in organ çulture .
The increased rate of ribonucleic acid formation
could have stimulated protein synthesis in the gland by increas_ ing the availability of messenger or ribosomal ribonucleic acid . It is conceivable, therefore, that insulin enhanced protein for_ oration in the embryonic thyroid explante solely or partly via its influence on the metabolism of ribonucleic acid .
1Pe cannot, how_
ever, exclude other mechanisms, for example an enhanced transport of nutrients across the cell membrane . Ackna rled c~ment The technical assistance of Mr . 1larner E . Freeman is grate_ fully acknowledged . References 1.
H . M . Nataf and i . L.. Chaikoff, Life Sci . 3, 895 (1964) .
a.
J . M. Cher, Exp. Cell Res . 7, 518 (1954) .
3.
J . J . Elias,and E . M . Rivera, Cancer Res . 19, 505 (1959) .
4.
B . M. Nataf, E . M . Rivera and I . L. Chaikoff, Endocrinology _76, 35 (1965) . V. N. Singh, E . Raghupathy and I . L . Chaikoff, Biochim . Biophys . Acta , in pxeas (1965) .
6.
R . Schemer and J . E. Darnell, Biochem . Bio
e . Res . Comet.
7, 486 (1962) . 7.
J . F. Morgan, H . J. Morton and R. C . Parker, Proc . Soc . Exp . Biol . Med . 73, 1 (1950) .
EFFECT OF INSULIN
Vol. 4, No. 18 8.
M . B . Yarmolinsky and G . L . de la Haba, Proc . Nat . Acad .
9.
Sci . U .S . 45, 1721 (1959) . a D . Nathans, Federation Proc . _23, 984 (1964) .
1809
10 .
E . Racker, Adv . Enzymol . 23,
11 .
K . L. Manchester and F . G . Young, Vitamins and Hormones _19, 95
3 (1961) .
(1961) ,
12 .
F . D . W . Lukens, Diabetes 13, 451 (1964) .
13 .
I . G . Wool, Actions of Hormones on Molecular Processes p . 422 .
John Wiley and Sons, Inc., New York (1964) .
14 .
A . Korner, Biochem. J . 92, 449 (1964) .
15 .
T . M. Tomkins and E . S . Ma3ave11, Ann . Rev. Biochem. _32, 677 (1963) .
4, pp . 1603-1809, 1985 . Pergamon Press Ltd. Printed in
THE EFFECT OF INSULIN ON THE FORMATION OF PROTEIN AND RTnp~iUCT.RT C ACID BY FETAL RAT THYROID GLANDS IN ORGAN CULTURE*
V . N . Singh, B . M . Nataf and I . L . Chaikoff Departriment of Physiology, University of California Berkeley
(Received 1 June
1985)
introduction The ability of insulin to enhance 1311 uptake and incorpora_ tion of the isotope into protein bound monoiodotyrosine, duodo_ tyrosine and thyroxine by fetal rat thyroid glands in organ cul_ ture was recently demonstrated (1) .
Since these iodoamino acids
are components of thyroglobulin, we were led to study the action of insulin on the synthesis of protein and ribonucleic acid by the thyroid gland explants . Materials and Methods Fetuses were removed by Caesarean operation from 21-day_ pregnant rats of the Long_Evans strain and their thyroid glands were excised under sterile conditions
(1) .
The thyroid explants
were cultured by the watch-glass method of Chen (2) as modified by Elias and Rivers (3), the details of which have been described by Nataf et al . (4) . Determination of Protein 14C - At the end of the experiment, the tissue in each dish was removed, washed once with fresh * This investigation was supported by grants from the U . S . Public Health Service . 1603
Vol. 4, No . 18
EFFECT OF INSULIN
1804
culture medüun, and transferred to a centrifuge tube containing One ml of an aqueous solo_
one ml of 1096 trichloroacetic acid .
tion containing 2 mg of bovine serum albwnin was added as carrier, The homogenate was centrifuged
and the tissue was homogenized .
and the protein precipitate was prepared for 14 C counting as de_ scribed by Singh et al . (5) . Determination of Ribonucleic Acid 14C _ The glands were re_ moved, washed once with fresh medium, and immersed in one ml of chilled 0 .05 M tris_HCl buffer the mixture was frozen .
(p~H 7 .6) .
Immediately thereafter
Ribonucleic acid was isolated by the
procedure of Schemer and Darnell (6) with the following modifi_ cations :
(a) polyvinyl sulfate was not used ; (b) after adding
sodium dodecyl sulfate, the homogenate was kept at 60° for 5 minutes before the addition of phenol ; and (c) two mg of yeast ribonucleic acid were added as carrier before adding alcohol to precipitate the ribonucleic acid .
The precipitate was dissolved
in 0 .05 M tris_HC1 buffer of pH 7 .6, and an aliquot was taken for determination of its 14C content (5) . Results The 14 C of each amino acid added to the medium was readily incorporated into protein (Table 1) .
The presence of insulin in
the medium enhanced the incorporation in each case .
Insulin in_
creased the 14C incorporation into protein regardless of the age of the culture (Table 2) .
The 2~hour incorporation of leucine
carbon into thyroidal protein was about the same in one, two, end three_day_old cultures (Table 2) .
Vol . 4, No. 18
EFFECT OF INSULIN
180 5
TAHLB 1 $ffect of Insulin on Incorporation of 14C of 14 C-labeled L-Amino Acids into Protein by Fetal Rat Thyroid Glands in Organ Culture for Two Days Thyroid glands excised from 21_day_old fetuses were cu~~ured for Twenty_ two days in the basal medium* with or without insulin . four hours before harvesting the culture, the medium was replaced with fresh medium (with or without insulin) to which a 14 Clabeled amino acid had been added (0 .5 ~c/ml) . Incorporation of all four amino acids was studied in each experiment which was tarried out with the glands excised from the fetuses of a single pregnant rat . Each value is the mean ± 5 .8 . of the results of four separate experiments . 14~p~ino acid#
nou in a to medium hg
`~ recover n protein of one whole gland cpm
L-Leucine L-Leucine
None 5
4,200 ± 380 6,800 ± 550
L-Phenylalanine L-Phenylalanine
None 5
9,300 ± 210 5,300 ± 840
L-Tyrosine L-Tyrosine
None 5
1,600 f 170 2,600 ± 250
L-Valine L-Valine
None 5
4,700 ± 240 5,900 ± 350
* Medium 199 (7) to which penicillin G had been added (50 units per ml) . +~* Amorphous insulin, 20 unite per mg and practically devoid of glucagon (less than 0 .0003%), was furnished by the Lilly Laboratories, $li Lilly and Co ., Indianapolis, Ind. The amino acids were uniformly labeled with 14C and their specific activities ranged from 200 to 360 me per amole .
Puromycin, an inhibitor of protein biosynthesis (8,9), and 2,4_dinitrophenol and arsenate, both of which uncouple oxidative phoaphorylation (10), appreciably inhibited incorporation of leucine carbon into protein by the fetal thyroid explants 3) .
(Table
The inhibition by 2,4_dinitropihenol and arsenate indicates
1808
EFFECT OF INSULIN
Vol. 4, No. 18
TABLE 2 Effect of Insulin on Incorporation of 14C of L-Leucine-U-14 C into Protein by One, TWO and ThreeDay_Old Cultures of Fetal Rat Thyroid Glande The experimental details are given in Table 1 . The labeled leu_ cine waä added 24 hours before harvesting the culture . Fetuses from a single pregnant rat were used for each experiment . Each value is the mean ± S .E . of the results of four experiments . e o cu ure when harvested days
nau in a to medium Fig
in `tC rernver protein of one whole gland cpn
1 1
None 5
4,300 ± 360 6,600 ± 460
2 2
None 5
4, 500 f 260 7,800 ± 790
3 3
None 5
4,900 ± 670 7,800 + 740
TABLE 3 Effects of Puromycin, Arsenate and 2,4_Dinitrophenol on Incorporation of 14C of L-Leucine_U_ 14 C into Protein by Fetal Rat Thyroid Glanda in Organ Culture fór Two Days Each experiment made use of all inhibitors and was carried out With the thyroid glands excised from the fetuses of a single pregnant rat . Each value is the mean ~ S .E . of the results of four separate experiments . Addition to medium None
i~ rernvered in protein of one whole gland cpm 6, 000 f 470
Puromycin (100 Wg)
150 ± 30
Arsenate (5 Eunolea)
240 ± 30
2,4_Dinitrophenol (1 Funole)
400 ± 70
EFFECT OF INSULIN
Vol . 4, No. 16
1607
that the incorporation is energy dependent . Table 4 shows that the addition of insulin to the medium increased considerably the incorporation of 14 C of adenine_8_14C into ribonucleic acid by the fetal rat thyroid glands . TABLE 4 Effect of Insulin on Incorporation of 14C of Adenine_8_ 14 C into RNA by Fetal Rat Thyroid Glands in Organ Culture for Two Days Thyroid glands excised from 21_day_old fetuses were cultured for 2 days in the basal medium with or without insulin . Twenty four hours before termination of culture, the medium was replaced with a fresh medium (with or without insulin) to which was added one we/ml of adenine_8_14C (specific ac_ tivity, 5 mc/mmole) . Thyroid glands from two fetuses were used for each analysis . Each value is the mean ± S .S . of five analyses . Insulin added to medium
4 recover as RDiA of one whole gland cpm .
None
1,380 ± 70
5 wg
2,490 ~ 140
Discussion The ability of insulin to enhance the incorporation of amino acids and amino acid precursors into protein has been demonstrated in several tissues (11_13) .
Although the precise
mechanism of this action of insulin has not yet been established, there is evidence suggesting that the hormone enhances protein formation by increasing the synthesis of ribonucleic acid (13) . It is of interest to recall here that the role of ribonucleic acid, possibly messenger ribonucleic acid, in the enhancement of
1808
EFFECT OF INSULIN
Vol. 4, No. 18
protein synthesis bY other hormones concenred with protein anabo_ liam has also been pointed out (14,15) . Our findings show that insulin enhanced the formation of both protein and ribonucleic acid by fetal rat thyroid glands in organ çulture .
The increased rate of ribonucleic acid formation
could have stimulated protein synthesis in the gland by increas_ ing the availability of messenger or ribosomal ribonucleic acid . It is conceivable, therefore, that insulin enhanced protein for_ oration in the embryonic thyroid explante solely or partly via its influence on the metabolism of ribonucleic acid .
1Pe cannot, how_
ever, exclude other mechanisms, for example an enhanced transport of nutrients across the cell membrane . Ackna rled c~ment The technical assistance of Mr . 1larner E . Freeman is grate_ fully acknowledged . References 1.
H . M . Nataf and i . L.. Chaikoff, Life Sci . 3, 895 (1964) .
a.
J . M. Cher, Exp. Cell Res . 7, 518 (1954) .
3.
J . J . Elias,and E . M . Rivera, Cancer Res . 19, 505 (1959) .
4.
B . M. Nataf, E . M . Rivera and I . L. Chaikoff, Endocrinology _76, 35 (1965) . V. N. Singh, E . Raghupathy and I . L . Chaikoff, Biochim . Biophys . Acta , in pxeas (1965) .
6.
R . Schemer and J . E. Darnell, Biochem . Bio
e . Res . Comet.
7, 486 (1962) . 7.
J . F. Morgan, H . J. Morton and R. C . Parker, Proc . Soc . Exp . Biol . Med . 73, 1 (1950) .
EFFECT OF INSULIN
Vol. 4, No. 18 8.
M . B . Yarmolinsky and G . L . de la Haba, Proc . Nat . Acad .
9.
Sci . U .S . 45, 1721 (1959) . a D . Nathans, Federation Proc . _23, 984 (1964) .
1809
10 .
E . Racker, Adv . Enzymol . 23,
11 .
K . L. Manchester and F . G . Young, Vitamins and Hormones _19, 95
3 (1961) .
(1961) ,
12 .
F . D . W . Lukens, Diabetes 13, 451 (1964) .
13 .
I . G . Wool, Actions of Hormones on Molecular Processes p . 422 .
John Wiley and Sons, Inc., New York (1964) .
14 .
A . Korner, Biochem. J . 92, 449 (1964) .
15 .
T . M. Tomkins and E . S . Ma3ave11, Ann . Rev. Biochem. _32, 677 (1963) .