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Liver glycogen in mice. Effect of a single triiodothyronine injection
Camp. Blochem Ph\uol. Vol. 70A. pp. 615 lo 617. PrInted I” Great Brltaln All rights reserved
LIVER
1981
0300-9629 XI I20615-03102.CQjO Copyright 0 1981 Pergamon Press Ltd
GLYCOGEN IN MICE. TRIIODOTHYRONINE
EFFECT OF A SINGLE INJECTION
C. PAINO, L. LABEAGA,J. L. R. ARRONDD,M. J. SANCHO and J. M. MACARULLA Departamento de Bioquimica, Facultad de Ciencias. Apartado 644, Bilbao, Spain (Receiced
23 March 1981)
Abstract-l. Administration of a single triiodothyronine injection to mice produces a decrease in hepatic glycogen levels. 2. The decrease is about 50% and has a lag time of 12 hr and a maximum effect 15 hr after injection. 3. The minimum dose to produce this event is 32 pg of hormone/l00 g of body weight and the process seems to be saturable because a higher dose does not change the pattern in the depletion, whereas a lower dose has less effect. 4. These results are compared with those obtained in chicken previously.
INTRODUCTION Thyroid hormones influence the metabolism of almost every class of foodstuff, but it is not yet fully clear their mechanisms of action. It is well known that most of the effects are mediated by an increase in protein synthesis following an interaction of the hormones with the nucleus (Oppenheimer, 1979) but it has been shown that there is also a specific binding of the hormones to mitochondria (Sterling, 1979) and plasma membrane (Pliam & Goldfine, 1977). Early studies in our laboratory have shown that glycogen levels can be depleted in a newborn chicken after a single injection of triiodothyronine (T3) within the lag time for protein synthesis enhancement, and this effect cannot be supressed by using antibiotics inhibiting protein synthesis such as cycloheximide or actinomycin D (Arrondo et al., 1978). Thyroid hormones act in a different way in birds than in mammals because the lack of a transport protein in bird serum (Tata & Shellabarger, 1971) so the effect obtained previously could not correlate in mammals. In the present paper we have studied the effect of a single T, injection on glycogen levels in mice and discussed the differences in lag time and time for maximum action between chicken and mice.
10 hr with the maximum effect 6 hr after injection. Figure 1 shows the effect of a similar dose (16 pg TX/100 g body wt) when it is injected to mice. It can be seen that there is almost no decrease produced by the hormone in the time considered, but only a small difference at 15 hr. Increasing the dose to 32 fig T,/lOO g body wt, the decrease in glycogen levels produced by the hormone is similar to that observed previously in chicken as can be seen in Fig. 2. The lag time now is 12 hr whereas in chickens it was 4 hr, and the maximum effect is 15 hr after injection. Figure 3 shows that using a dose of 8Opg T,/lOOg body wt the action of the hormone on glycogen levels is close to that of 32 pg T3/100 g body wt, with a same lag time and time for maximum effect. Higher hormone doses were employed, even as high as 8OOpg TJ/lOOg body wt but the results were not different to those of Fig. 2. DISCUSSION It is well known that thyroid hormones interact with the nucleus and stimulate protein synthesis, but some other mechanisms of action have been recently reported. Sterling (1979) have found receptors in mitochondria and Bouhnick et al. (1979) have shown that thyroid hormones have an early and preferential action on the mitochondrial protein synthesizing system. Segal et al. (1977) have found that 2-deoxy-Dglucose uptake is affected by thyroid hormones and is independent from the protein synthesis enhancement produced by them, showing that some of the effects produced by thyroid hormone do not require new synthesis of either RNA or proteins. In a previous work (Arrondo et al., 1978) we have shown that after a single injection of T, to chicken a decrease in glycogen levels was produced before protein synthesis enhancement produced by the hormones and this effect was not affected by neither cicloheximide nor actinomicin D. We also found that glycogen depletion was mediated by an increase in phosphorylase a activity and higher CAMP levels were found after hormone injection so we suggested that there could be some interaction between thyroid
MATERIAL AND METHODS “Swiss” mice weighing 2c2.5 g were used in all experiments. The animals were injected intraperitoneally with 0.1 ml of 3,3’,5-triiodo-L-thyronine (Sigma, London) or 0.9oi;, NaCI. After injection the animals were kept in the same conditions and then killed by a blow in the head. They were exsanguinated and their liver was excised and homogenized in 20~01 of cold 0.3 N perchloric acid. The homogenate was centrifuged in a bench centrifuge at top speed and glycogen extracted and measured as previously described (Sancho et al., 1977).
RESULTS Previous work in chickens had shown that a single injection of T3 (12 yg/lOO g body wt) was able to produce a decrease in glycogen levels between 4 and 615
c. PAIN0
616
J----
et ul.
I
1
6
24
16
12
Time, hr
Fig. 1. Liver glycogen levels in mice after a single injection of 16 pg T,/lOO g body wt (---) or saline (--). Each point corresponds to the mean f SEM of eight mice. Student’s t-test parameters *P < 0.05.
I
18
12
24
Time, hr
Fig. 2. Glycogen levels after 32 pg TJlOOg body wt_(---) or saline (---). SEM of ten mice. Student’s test as in Fig. 1.
Each point is the mean j,
50 t 40 gki 5s
30-
=f
1 if 0, 20-
10
0
i
- _!
Fig. 3. Milligrams of glycogen per gram of wet liver in lots of eight mice injected with a single dose of either 80 fig T,/lOO g body wt (---) or saline (--). Student’s t-test as in Fig. 1.
Liver glycogen
hormone transport across the membrane and glycogen depletion (Arrondo et al., 1981). This suggestion is in agreement with recent findings that there is a protein mediated transport system for thyroid hormones and that receptors can be found in plasma membrane (Rao et al., 1976; Kremning et al., 1978; Helm 8~ Jacquemin, 1979; Gharby & Torresani, 1979; Cheng et (I/., 1980). Differences in response to thyroid hormones have been found between birds and mammals by Tata & Shellabarger (1971), so we have now studied the effect of a single T, injection on glycogen levels in mice. It can be seen that glycogen levels depletion after T, injection seems to be a saturable process because usmg even as much as 800 pg T3/100 g body wt there is no difference in percentage of glycogen depletion as compared with a dose of 32 pg T3/100 g body wt. One difference between the effect of the hormone in mice or chicken is the time lag, which is 12 hr for mice and only 4 hr for chicken. This could be explained by the fact that mammals have a protein in serum which has not been found in chicken (Shellabarger & Tata, 1961), so in chicken thyroid hormone can be exchanged more easily between serum and liver cells. This difference in time applies too to the increase in 14C-Leu uptake which is 12 hr in chickens (Arrondo et al., 1978) and 21 hr in mice (data not shown). Another difference between chicken and mice is that higher hormone concentrations must be used in mice to obtain a similar effect. This could be due to age difference as has been recently reported by Schwartz et al. (1979) or a different adsorption equilibrium. These results suggest that in mammals there is an effect prior to protein synthesis enhancement produced by thyroid hormones and comparable to that observed previously in chicken, which could be related to hormone transport across the plasma membrane, either by a direct effect of the hormone on the mechanisms regulating CAMP levels in the cell or by mean of an interaction with P-adrenergic receptors.
REFERENCES ARRONW J. L. R., SANCHO M. J. & MACARULLA J.
M.
(1978) Effect of acute administration of triiodothyronine in chicken. Liver glycogen depletion and aminoacid incorporation to proteins. Experientia 34, 1099-l 100.
c B.P.70/4* K
in mice
617
ARROND~ J. L. R., ARTETXEJ., SANCHO M. J. & MACAR-
J. M. (1981) Liver Glycogen metabolism in chicken. Activation prior to triiodothyronine-induced protein synthesis enhancement. Horm. Mrtuh. Rrs. 13, ULLA
92-95.
BOUHNIK J., CLOT J. P., BAUDRY M. & MICHEL R. (1979) Early effects of thyroidectomy and triiodothyronine administration on rat liver mitochondria M&c,. Cell. Endocr. 15, l-12. CHENG S. Y.. MAXFIELD F. R., BOBBINSJ.. WILLINGHAM M. C. & PASTAN I. H. (1980) Receptor-mediated uptake of 3,3’,5-triiodo-L-thyronine by cultured fibroblasts. Proc. nutn. Acud. Sci. ti.S.A. 71, 3425-3429. GHARBY J. & TORRESANI J. (1979) High affinity thyroxine binding to purified rat liver plasma membranes. Biothem. hiophys. Res. Commun. 88, 17C-177. HOLM A. C. & JACQUEMIN C. (1979) Membrane transport of L-triiodothyronine by human red cell ghosts Bkhem. hiophys. Res. Commun. 89, 1006-1017. KRENNING E. P., DOCTER R., BERNARD H. F., VISSER T. J. & HENNEMANNG. (1978) Active transport of triiodothyronine (T,) into isolated rat liver cells FEBS Lett. 91, 113-116. OPPENHEIMER J. H. (1979) Thyroid hormone action at the cellular level Science 203, 971-979. PLIAM N. B. & GOLDFINE 1. D. (1977) High affinity thyroid hormone binding sites on purified rat liver plasma membranes. Biochemr biophys. kes. Commun. 79.166172. RAO G. S.. ECKEL J.. RAO M. L. & BREUER H. (1976) Uptake of thyroid hormone by isolated rat liver cells. Biochem. biophys. Res. Commun. 13, 98-104. SANCHO M. J., MACARULLA J. M. & SEGOVIA J. L. (1977) Efecto de la triyodotironina sobre el glucbgeno hephtico en Gal/w domesticus. RecTu. esp. Fisiol. 33, 159-162. SCHWARTZ H. L., FORCIEA M. A., MARIASCH C. N. & OPPENHEIMER J. H. (1979) Age related reduction in response of hepatic enzymes to 3,5,3’-triiodothyronine administration. Endocrinology 105, 41-48. SEGAL J. & GORDON A. (1977) The effects of actinomycin cycloheximide and hidroxiurea on D, puromicin, 3’,5,3-triiodo-L-thyronine stimulated 2-deoxi-D-glucose uptake in chick embryo heart cells in ritro. Endocrinology 101, 15&156. SHELLABARGERC. J. & TATA J. R. (1961) Etrects of administration of human serum thyroxine-binding globulin on the disappearance rates of thyroid hormones in the chicken. Endocrinology 68, 105f%1058. STERLING K. (1979) Thyroid hormone action at the cell level. N. Engl. J. Med. 300, 117-123. TATA J. R. & SHELLABARGER C. J. (1963) An explanation for the difference between the response of mammals and birds to thyroxine and triiodothyronine. Endocrinology 72, 608-613.
LIVER
1981
0300-9629 XI I20615-03102.CQjO Copyright 0 1981 Pergamon Press Ltd
GLYCOGEN IN MICE. TRIIODOTHYRONINE
EFFECT OF A SINGLE INJECTION
C. PAINO, L. LABEAGA,J. L. R. ARRONDD,M. J. SANCHO and J. M. MACARULLA Departamento de Bioquimica, Facultad de Ciencias. Apartado 644, Bilbao, Spain (Receiced
23 March 1981)
Abstract-l. Administration of a single triiodothyronine injection to mice produces a decrease in hepatic glycogen levels. 2. The decrease is about 50% and has a lag time of 12 hr and a maximum effect 15 hr after injection. 3. The minimum dose to produce this event is 32 pg of hormone/l00 g of body weight and the process seems to be saturable because a higher dose does not change the pattern in the depletion, whereas a lower dose has less effect. 4. These results are compared with those obtained in chicken previously.
INTRODUCTION Thyroid hormones influence the metabolism of almost every class of foodstuff, but it is not yet fully clear their mechanisms of action. It is well known that most of the effects are mediated by an increase in protein synthesis following an interaction of the hormones with the nucleus (Oppenheimer, 1979) but it has been shown that there is also a specific binding of the hormones to mitochondria (Sterling, 1979) and plasma membrane (Pliam & Goldfine, 1977). Early studies in our laboratory have shown that glycogen levels can be depleted in a newborn chicken after a single injection of triiodothyronine (T3) within the lag time for protein synthesis enhancement, and this effect cannot be supressed by using antibiotics inhibiting protein synthesis such as cycloheximide or actinomycin D (Arrondo et al., 1978). Thyroid hormones act in a different way in birds than in mammals because the lack of a transport protein in bird serum (Tata & Shellabarger, 1971) so the effect obtained previously could not correlate in mammals. In the present paper we have studied the effect of a single T, injection on glycogen levels in mice and discussed the differences in lag time and time for maximum action between chicken and mice.
10 hr with the maximum effect 6 hr after injection. Figure 1 shows the effect of a similar dose (16 pg TX/100 g body wt) when it is injected to mice. It can be seen that there is almost no decrease produced by the hormone in the time considered, but only a small difference at 15 hr. Increasing the dose to 32 fig T,/lOO g body wt, the decrease in glycogen levels produced by the hormone is similar to that observed previously in chicken as can be seen in Fig. 2. The lag time now is 12 hr whereas in chickens it was 4 hr, and the maximum effect is 15 hr after injection. Figure 3 shows that using a dose of 8Opg T,/lOOg body wt the action of the hormone on glycogen levels is close to that of 32 pg T3/100 g body wt, with a same lag time and time for maximum effect. Higher hormone doses were employed, even as high as 8OOpg TJ/lOOg body wt but the results were not different to those of Fig. 2. DISCUSSION It is well known that thyroid hormones interact with the nucleus and stimulate protein synthesis, but some other mechanisms of action have been recently reported. Sterling (1979) have found receptors in mitochondria and Bouhnick et al. (1979) have shown that thyroid hormones have an early and preferential action on the mitochondrial protein synthesizing system. Segal et al. (1977) have found that 2-deoxy-Dglucose uptake is affected by thyroid hormones and is independent from the protein synthesis enhancement produced by them, showing that some of the effects produced by thyroid hormone do not require new synthesis of either RNA or proteins. In a previous work (Arrondo et al., 1978) we have shown that after a single injection of T, to chicken a decrease in glycogen levels was produced before protein synthesis enhancement produced by the hormones and this effect was not affected by neither cicloheximide nor actinomicin D. We also found that glycogen depletion was mediated by an increase in phosphorylase a activity and higher CAMP levels were found after hormone injection so we suggested that there could be some interaction between thyroid
MATERIAL AND METHODS “Swiss” mice weighing 2c2.5 g were used in all experiments. The animals were injected intraperitoneally with 0.1 ml of 3,3’,5-triiodo-L-thyronine (Sigma, London) or 0.9oi;, NaCI. After injection the animals were kept in the same conditions and then killed by a blow in the head. They were exsanguinated and their liver was excised and homogenized in 20~01 of cold 0.3 N perchloric acid. The homogenate was centrifuged in a bench centrifuge at top speed and glycogen extracted and measured as previously described (Sancho et al., 1977).
RESULTS Previous work in chickens had shown that a single injection of T3 (12 yg/lOO g body wt) was able to produce a decrease in glycogen levels between 4 and 615
c. PAIN0
616
J----
et ul.
I
1
6
24
16
12
Time, hr
Fig. 1. Liver glycogen levels in mice after a single injection of 16 pg T,/lOO g body wt (---) or saline (--). Each point corresponds to the mean f SEM of eight mice. Student’s t-test parameters *P < 0.05.
I
18
12
24
Time, hr
Fig. 2. Glycogen levels after 32 pg TJlOOg body wt_(---) or saline (---). SEM of ten mice. Student’s test as in Fig. 1.
Each point is the mean j,
50 t 40 gki 5s
30-
=f
1 if 0, 20-
10
0
i
- _!
Fig. 3. Milligrams of glycogen per gram of wet liver in lots of eight mice injected with a single dose of either 80 fig T,/lOO g body wt (---) or saline (--). Student’s t-test as in Fig. 1.
Liver glycogen
hormone transport across the membrane and glycogen depletion (Arrondo et al., 1981). This suggestion is in agreement with recent findings that there is a protein mediated transport system for thyroid hormones and that receptors can be found in plasma membrane (Rao et al., 1976; Kremning et al., 1978; Helm 8~ Jacquemin, 1979; Gharby & Torresani, 1979; Cheng et (I/., 1980). Differences in response to thyroid hormones have been found between birds and mammals by Tata & Shellabarger (1971), so we have now studied the effect of a single T, injection on glycogen levels in mice. It can be seen that glycogen levels depletion after T, injection seems to be a saturable process because usmg even as much as 800 pg T3/100 g body wt there is no difference in percentage of glycogen depletion as compared with a dose of 32 pg T3/100 g body wt. One difference between the effect of the hormone in mice or chicken is the time lag, which is 12 hr for mice and only 4 hr for chicken. This could be explained by the fact that mammals have a protein in serum which has not been found in chicken (Shellabarger & Tata, 1961), so in chicken thyroid hormone can be exchanged more easily between serum and liver cells. This difference in time applies too to the increase in 14C-Leu uptake which is 12 hr in chickens (Arrondo et al., 1978) and 21 hr in mice (data not shown). Another difference between chicken and mice is that higher hormone concentrations must be used in mice to obtain a similar effect. This could be due to age difference as has been recently reported by Schwartz et al. (1979) or a different adsorption equilibrium. These results suggest that in mammals there is an effect prior to protein synthesis enhancement produced by thyroid hormones and comparable to that observed previously in chicken, which could be related to hormone transport across the plasma membrane, either by a direct effect of the hormone on the mechanisms regulating CAMP levels in the cell or by mean of an interaction with P-adrenergic receptors.
REFERENCES ARRONW J. L. R., SANCHO M. J. & MACARULLA J.
M.
(1978) Effect of acute administration of triiodothyronine in chicken. Liver glycogen depletion and aminoacid incorporation to proteins. Experientia 34, 1099-l 100.
c B.P.70/4* K
in mice
617
ARROND~ J. L. R., ARTETXEJ., SANCHO M. J. & MACAR-
J. M. (1981) Liver Glycogen metabolism in chicken. Activation prior to triiodothyronine-induced protein synthesis enhancement. Horm. Mrtuh. Rrs. 13, ULLA
92-95.
BOUHNIK J., CLOT J. P., BAUDRY M. & MICHEL R. (1979) Early effects of thyroidectomy and triiodothyronine administration on rat liver mitochondria M&c,. Cell. Endocr. 15, l-12. CHENG S. Y.. MAXFIELD F. R., BOBBINSJ.. WILLINGHAM M. C. & PASTAN I. H. (1980) Receptor-mediated uptake of 3,3’,5-triiodo-L-thyronine by cultured fibroblasts. Proc. nutn. Acud. Sci. ti.S.A. 71, 3425-3429. GHARBY J. & TORRESANI J. (1979) High affinity thyroxine binding to purified rat liver plasma membranes. Biothem. hiophys. Res. Commun. 88, 17C-177. HOLM A. C. & JACQUEMIN C. (1979) Membrane transport of L-triiodothyronine by human red cell ghosts Bkhem. hiophys. Res. Commun. 89, 1006-1017. KRENNING E. P., DOCTER R., BERNARD H. F., VISSER T. J. & HENNEMANNG. (1978) Active transport of triiodothyronine (T,) into isolated rat liver cells FEBS Lett. 91, 113-116. OPPENHEIMER J. H. (1979) Thyroid hormone action at the cellular level Science 203, 971-979. PLIAM N. B. & GOLDFINE 1. D. (1977) High affinity thyroid hormone binding sites on purified rat liver plasma membranes. Biochemr biophys. kes. Commun. 79.166172. RAO G. S.. ECKEL J.. RAO M. L. & BREUER H. (1976) Uptake of thyroid hormone by isolated rat liver cells. Biochem. biophys. Res. Commun. 13, 98-104. SANCHO M. J., MACARULLA J. M. & SEGOVIA J. L. (1977) Efecto de la triyodotironina sobre el glucbgeno hephtico en Gal/w domesticus. RecTu. esp. Fisiol. 33, 159-162. SCHWARTZ H. L., FORCIEA M. A., MARIASCH C. N. & OPPENHEIMER J. H. (1979) Age related reduction in response of hepatic enzymes to 3,5,3’-triiodothyronine administration. Endocrinology 105, 41-48. SEGAL J. & GORDON A. (1977) The effects of actinomycin cycloheximide and hidroxiurea on D, puromicin, 3’,5,3-triiodo-L-thyronine stimulated 2-deoxi-D-glucose uptake in chick embryo heart cells in ritro. Endocrinology 101, 15&156. SHELLABARGERC. J. & TATA J. R. (1961) Etrects of administration of human serum thyroxine-binding globulin on the disappearance rates of thyroid hormones in the chicken. Endocrinology 68, 105f%1058. STERLING K. (1979) Thyroid hormone action at the cell level. N. Engl. J. Med. 300, 117-123. TATA J. R. & SHELLABARGER C. J. (1963) An explanation for the difference between the response of mammals and birds to thyroxine and triiodothyronine. Endocrinology 72, 608-613.