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Level of S-100 protein in different rat brain areas after short-term protein restriction
Brain Research, 129 0977) 187-191 ~.~ Elsevier/North-Holland Biomedical Press
187
Level of S-100 protein in different rat brain areas after short-term protein restriction
ANDRZEJ WRONSKI, ALEXANDRA VON DER DECKEN and KENNETH G. HAGLID The Wenner-Gren Institute for Experimental Biology, Stockholm, and Department of Neurobiology, University o/ GOteborg, GSteborg (Sweden)
(Accepted March 3rd, 1977)
It has been shown that nutritional alterations, such as protein restriction or change in protein composition, decreased the in vitro incorporation of radioactive amino acids into proteins of liver and skeletal muscle 1°-15. Reduced protein synthesis in vivo has been observed in mice brain 5 subjected to neonatal infection and undernutrition. A decrease in brain protein metabolism in vivo has been observed as a result of restricted protein supply s. in comparison with other organs, such as skeletal muscle and liver, brain was less affected by partial protein starvation when protein synthesis was measured in a cell-free system 16. In a previous work no significant differences were observed when the incorporation of radioactive amino acids into total rat brain protein was measured in tissue slices after short-term protein restriction is. But the incorporation into the brain specific S- 100 protein was markedly reduced. In the present work, using similar dietary conditions, the amount of S-100 protein was determined in different areas of the brain and the results were compared with those obtained earlier on the in vitro synthesis of the protein in the whole tissue is. Male Sprague-Dawley rats, 25 days old, weighing 80 g, were caged individually as described previously iv,is. Groups of 8 rats received a diet containing either 20 ~o or 3 % of the high-quality protein casein supplemented with DL-methionine (3 g/kg). Details of the diets are given elsewhere is. After 6 days on the experimental diet, the rats were killed by decapitation, without previous starvation. The brains were removed and dissected on ice into 200 mg wet weight portions of frontal, middle and dorsal cerebral cortex, hypothalamus, brain stem and cerebellum. The tissue pieces were pooled from 2 animals per dietary group and 4 such series were prepared. The brain portions were immediately homogenized in a tightly fitting teflon-glass homogenizer, in 2 ml of 0.1 M barbital buffer (pH 8.6, ionic strength 0.02) containing 2.5 m M EDTA and 0.1 m M 2-mercaptoethanol. The homogenates were sonicated 17 and centrifuged at 105,000 7~ g for 60 rain and the supernatants were stored at - - 2 0 °C. Similarly dissected brain areas obtained from another group of 8 animals per diet were sliced and incubated in Hank's medium as described previously iv,is, but in a final volume of 4 ml. After incubation at 37 °C for 240 rain the slices were
188 homogenized, sonicated and centrifuged for 60 min at 105,000 x g. The supernatants were dialyzed against 10 vols. of immunoetectrophoresis buffer and stored at -20 '~C. Quantitative immunoelectrophoresis was carried out according to LauretH modified for S-100 protein 9. The immunoelectrophoresis was run for 6 h at a: current of 1 mA/cm at 15 °C in 1 ~ Litex agarose (Glostrup, Denmark) and 1 o;, polyethyleneglycol (mol. wt. 6,000) in 0.1 M barbital buffer (pH 8.6, ionic strength 0.02), containing 2.5 m M EDTA, 0.1 m M 2-mercaptoethanol and 4~, rabbit anti-(beef-S-100)serum ~. Purified beef S-100 was used as standard. It gave linearity of the precipitation rockets in the range of 25-200 ng. Rat S-100 values were expressed in terms of beef-S-100 equivalents. The amount of S-100 protein was calculated per g wet weight and per mg protein of the 105,000 × g supernatant (soluble proteins).
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Fig. 1. Quantitative immunoelectrophoretic analysis of S-100 protein in various parts of the brain. The results are presented as g S-100 protein per g wet weight. F, frontal; M, middle; D, dorsal cerebral cortex; T, hypothalamus; BS, brain stem; and C, cerebellum. Rats fed 20 ~o protein (black bars); 3 ~ protein (cross-hatched bars): The results are expressed as % of dorsal cerebral cortex of rats fed 20 ~ protein. The 1 0 0 ~ value corresponds to 82/~g of S-100 protein/g wet weight. The results are the mean, per dietary group, of 4 independent tissue preparations pooled from 2 rats per preparation. Each preparation was analysed immunoelectrophoreticalty in duplicates. Thus, the statistical evaluation according to Fiscber's permutation test 7 was based on 8 values per dietary group and brain area. ** P ~< 0.01.
189
Statistical analysis was carried out with Fisher's permutation test as used by Od6n and WedeF. This non-parametric test, which avoids assumption of normality, takes the numerical values of the results into account. Protein was determined by the method of Lowry et al. 6. Bovine serum albumin was used as a reference standard. The content of S-100 protein was quantitatively determined by means of immunoelectrophoresis in 6 different areas of the brain. As compared with S-100 protein obtained from protein-fed rats, there was a significant increase of 10-20 ~ per g wet weight of tissue after protein restriction (Fig. 1). Similar results were obtained when the dissected brain areas were incubated prior to the quantitative analysis. The similarity in results before and after incubation allowed for a comparison between the previously obtained results on the synthesis of S-100 protein]7, is and those presented here on the content of the protein. The dietary conditions and protein supply were identical in the two series. Rats fed a 3 ~ protein-containing diet increased less in body and brain wet weight than rats fed 20 ~ protein 18. Thus, the amount of S-100 protein per total brain wet weight was not significantly changed by the dietary conditions used.
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Fig. 2. Quantitative immunoelectrophoretic analysis of S-100 protein calculated per mg protein of the 105,000 :z g supernatant (total soluble proteins). For further details, see Fig. I. The results are expressed as ~ of dorsal cerebral cortex of rats fed 20~ protein. The 100~ value corresponds to 6.65 Mg S-100 protein/rag soluble protein. * P < 0.05.
190 The ratio of dry to wet weight was constant between the 6 brain areas :,tudied within a dietary g r o u p a n d between the 2 dietary groups. A change in wet weight with diet seemed to be parallelled by a simultaneous change in dry weight. The results are ir~ line with the data expressed as a m o u n t of S-100 protein per mg total soluble proteh~ (Fig. 2). Except in the case of frontal cerebral cortex, the differences between the dietary groups were non-significant, indicating the small effect dietary protein restriction had on the ratio of S-100 to soluble proteins. The distribution of S-100 protein between various parts of the brain differed when calculated per g wet weight (see Fig. 1). Brain stem contained the highest a m o u n t of the protein followed by cerebellum a n d h y p o t h a l a m u s , while frontal cerebral cortex showed the lowest content. As the glial cells are the major localization of the proteine~ the n u m b e r of these cells may vary between the areas of the brain. The diminished synthesis of S-100 protein at)or protein restriction t~ was not followed by a s i m u l t a n e o u s decrease in c o n t e n t of the protein. The data presented here extend the previous results a n d d e m o n s t r a t e that the reduced synthesis was c o m p e n sated for by a decrease of catabolic events in brain. Thus, after 6 days o f protein restriction the a m o u n t of S-100 protein in brain remained unchanged. The work was supported by a grant from the Swedish Medical Research Council to A.v.d.D. (project No. 4266) a n d Wilhelm and M a r t i n a L u n d g r e n ' s F o u n d a t i o n to K.H. The a u t h o r s wish to t h a n k Mrs. A g n e t a Nilsson for her valuable technical assistance. 1 Haglid, K. G., Stavrou, D., R6nnb~ick, L.. Carlsson. C. A. and Weidenbach. W.. f he S-100 protein in water-soluble and pentanol-extractable form in normal human brain and turnouts of the human nervous system. A quantitative study, J. neurol. Sci.. 20 [1973) 103-11 I. 2 Haglid, K. G., Hamberger, A., Hansson, H.-A., Hyd6n, H.. Persson, L. and R6nnback, I... Cellular and subcellular distribution of the S-100 protein in rabbit and rat CNS. J. Neurosc'i. Res.. 2(1976J 175-191. 3 Herschman. J. R.. Levine, L. and DeVellis, 1.. Appearance of a brain specific tlnr~gen ~S-100 protein) in the developing rat brain, J. Neurorhem., 18 (1971) 629-633. 4 Laurell, C.-B., Quantitative estimation of proteins by electrophoresis in agarose gel contaimng antibodies, Analyt. Bioehern., 15 (1966) 45-52. 5 Lee, C.-J., Biosynthesis and characteristics of brain protein and ribonucleic acid in m~ce subjected to neonatal infection and undernutrition, J. biol. Chem., 245 (1970) 1998-2004. 6 Lowry, O. H.. Rosebrough, N. I.. Farr, A. L. and Randall, R. I.. Protein measurement with the Folin phenol reagent, J. bioL Chem.. 193 (195t) 265-275. 7 0 d e n , A and Wedel, H., Arguments for Fischer's permutation tesl. Ann. Star., 3 ~1975) 518-520. 8 0 ' N e a l , R. M., Pla. G. W., Fox, S., Gibbon, F. and Fry, B. E.. Effect of zinc deficiency and restricted feeding on protein and ribonucleic acid metabolism of rat brain, J. Nutr., 100 ~1970/491 497. 9 Stavrou, D., Lubbe, I. and Haglid, K. G.. lmmunelektrophoretische Quantifizierung des hirnspezifischen S-t00 proteins, Acta neuropath, rBerl.), 29 (1974) 275-280. 10 Von Der Decken. A., Evidence for regulation of protein synthesis at the translation level in response to dietary alterations. J. Cell bioL. 33 (1967) 657-663. 11 Von Der Decken, A., Modification of the in vitro amino acid incorporation capacity of rat liver after in vivo and in vitro treatments, Europ. J. Biochem., 4 (1968) 87-94. 12 Von Der Decken, A., Amino acid incorporation by rat liver microsomal and ribosomal preparation. Modification of activity by in vivo and in vitro treatments, Abh. dtsch. Akad, Wiss. Berl.. I ( 1968} 541-551.
191 13 Von Der Decken, A., Activation in vitro of rat liver polyribosomes, J. Cell. Biol., 43 (1969) 138-147. 14 Von Der Decken, A., Cytoplasmic factors affecting the in vitro amino acid incorporating activity of rat liver preparation after dietary alterations or stress induction. In Szafranski, P., Klita, S. and Mastowski, P. (Eds.), Protein Biosynthesis, Pol. Biochem. Soc., Warszawa, 1969, pp. 33-46. 15 Von Der Decken, A. and Omstedt, P., Protein feeding of rats after protein starvation : incorporation of amino acid into polypeptide by skeletal muscle polyribosomes, J. Nutr., 10 (1970) 623-630. 16 Von Der Decken, A. and Wr6nski, A., Protein synthesis in vitro in rat brain after short-term protein starvation and refeeding, J. Neurochem., 18 (1971) 2383-2388. 17 Wr6nski, A. and Von Der Decken, A., Synthesis of brain-specific acidic proteins in rat and mouse cerebral slices, Actaphysiol. scand., 95 (1975) 482-493. 18 Wr6nski, A. and Von Der Decken, A., Protein synthesis with special reference to S-100 protein in brain slices from rats receiving a restricted protein supply, Acta physiol, scand., 97 (1976) 20-30.
187
Level of S-100 protein in different rat brain areas after short-term protein restriction
ANDRZEJ WRONSKI, ALEXANDRA VON DER DECKEN and KENNETH G. HAGLID The Wenner-Gren Institute for Experimental Biology, Stockholm, and Department of Neurobiology, University o/ GOteborg, GSteborg (Sweden)
(Accepted March 3rd, 1977)
It has been shown that nutritional alterations, such as protein restriction or change in protein composition, decreased the in vitro incorporation of radioactive amino acids into proteins of liver and skeletal muscle 1°-15. Reduced protein synthesis in vivo has been observed in mice brain 5 subjected to neonatal infection and undernutrition. A decrease in brain protein metabolism in vivo has been observed as a result of restricted protein supply s. in comparison with other organs, such as skeletal muscle and liver, brain was less affected by partial protein starvation when protein synthesis was measured in a cell-free system 16. In a previous work no significant differences were observed when the incorporation of radioactive amino acids into total rat brain protein was measured in tissue slices after short-term protein restriction is. But the incorporation into the brain specific S- 100 protein was markedly reduced. In the present work, using similar dietary conditions, the amount of S-100 protein was determined in different areas of the brain and the results were compared with those obtained earlier on the in vitro synthesis of the protein in the whole tissue is. Male Sprague-Dawley rats, 25 days old, weighing 80 g, were caged individually as described previously iv,is. Groups of 8 rats received a diet containing either 20 ~o or 3 % of the high-quality protein casein supplemented with DL-methionine (3 g/kg). Details of the diets are given elsewhere is. After 6 days on the experimental diet, the rats were killed by decapitation, without previous starvation. The brains were removed and dissected on ice into 200 mg wet weight portions of frontal, middle and dorsal cerebral cortex, hypothalamus, brain stem and cerebellum. The tissue pieces were pooled from 2 animals per dietary group and 4 such series were prepared. The brain portions were immediately homogenized in a tightly fitting teflon-glass homogenizer, in 2 ml of 0.1 M barbital buffer (pH 8.6, ionic strength 0.02) containing 2.5 m M EDTA and 0.1 m M 2-mercaptoethanol. The homogenates were sonicated 17 and centrifuged at 105,000 7~ g for 60 rain and the supernatants were stored at - - 2 0 °C. Similarly dissected brain areas obtained from another group of 8 animals per diet were sliced and incubated in Hank's medium as described previously iv,is, but in a final volume of 4 ml. After incubation at 37 °C for 240 rain the slices were
188 homogenized, sonicated and centrifuged for 60 min at 105,000 x g. The supernatants were dialyzed against 10 vols. of immunoetectrophoresis buffer and stored at -20 '~C. Quantitative immunoelectrophoresis was carried out according to LauretH modified for S-100 protein 9. The immunoelectrophoresis was run for 6 h at a: current of 1 mA/cm at 15 °C in 1 ~ Litex agarose (Glostrup, Denmark) and 1 o;, polyethyleneglycol (mol. wt. 6,000) in 0.1 M barbital buffer (pH 8.6, ionic strength 0.02), containing 2.5 m M EDTA, 0.1 m M 2-mercaptoethanol and 4~, rabbit anti-(beef-S-100)serum ~. Purified beef S-100 was used as standard. It gave linearity of the precipitation rockets in the range of 25-200 ng. Rat S-100 values were expressed in terms of beef-S-100 equivalents. The amount of S-100 protein was calculated per g wet weight and per mg protein of the 105,000 × g supernatant (soluble proteins).
%D
t.
.v,.
::iii
. m
1000 0
!i~::i:i::
c~
F
M
D
T
BS
C
Fig. 1. Quantitative immunoelectrophoretic analysis of S-100 protein in various parts of the brain. The results are presented as g S-100 protein per g wet weight. F, frontal; M, middle; D, dorsal cerebral cortex; T, hypothalamus; BS, brain stem; and C, cerebellum. Rats fed 20 ~o protein (black bars); 3 ~ protein (cross-hatched bars): The results are expressed as % of dorsal cerebral cortex of rats fed 20 ~ protein. The 1 0 0 ~ value corresponds to 82/~g of S-100 protein/g wet weight. The results are the mean, per dietary group, of 4 independent tissue preparations pooled from 2 rats per preparation. Each preparation was analysed immunoelectrophoreticalty in duplicates. Thus, the statistical evaluation according to Fiscber's permutation test 7 was based on 8 values per dietary group and brain area. ** P ~< 0.01.
189
Statistical analysis was carried out with Fisher's permutation test as used by Od6n and WedeF. This non-parametric test, which avoids assumption of normality, takes the numerical values of the results into account. Protein was determined by the method of Lowry et al. 6. Bovine serum albumin was used as a reference standard. The content of S-100 protein was quantitatively determined by means of immunoelectrophoresis in 6 different areas of the brain. As compared with S-100 protein obtained from protein-fed rats, there was a significant increase of 10-20 ~ per g wet weight of tissue after protein restriction (Fig. 1). Similar results were obtained when the dissected brain areas were incubated prior to the quantitative analysis. The similarity in results before and after incubation allowed for a comparison between the previously obtained results on the synthesis of S-100 protein]7, is and those presented here on the content of the protein. The dietary conditions and protein supply were identical in the two series. Rats fed a 3 ~ protein-containing diet increased less in body and brain wet weight than rats fed 20 ~ protein 18. Thus, the amount of S-100 protein per total brain wet weight was not significantly changed by the dietary conditions used.
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Fig. 2. Quantitative immunoelectrophoretic analysis of S-100 protein calculated per mg protein of the 105,000 :z g supernatant (total soluble proteins). For further details, see Fig. I. The results are expressed as ~ of dorsal cerebral cortex of rats fed 20~ protein. The 100~ value corresponds to 6.65 Mg S-100 protein/rag soluble protein. * P < 0.05.
190 The ratio of dry to wet weight was constant between the 6 brain areas :,tudied within a dietary g r o u p a n d between the 2 dietary groups. A change in wet weight with diet seemed to be parallelled by a simultaneous change in dry weight. The results are ir~ line with the data expressed as a m o u n t of S-100 protein per mg total soluble proteh~ (Fig. 2). Except in the case of frontal cerebral cortex, the differences between the dietary groups were non-significant, indicating the small effect dietary protein restriction had on the ratio of S-100 to soluble proteins. The distribution of S-100 protein between various parts of the brain differed when calculated per g wet weight (see Fig. 1). Brain stem contained the highest a m o u n t of the protein followed by cerebellum a n d h y p o t h a l a m u s , while frontal cerebral cortex showed the lowest content. As the glial cells are the major localization of the proteine~ the n u m b e r of these cells may vary between the areas of the brain. The diminished synthesis of S-100 protein at)or protein restriction t~ was not followed by a s i m u l t a n e o u s decrease in c o n t e n t of the protein. The data presented here extend the previous results a n d d e m o n s t r a t e that the reduced synthesis was c o m p e n sated for by a decrease of catabolic events in brain. Thus, after 6 days o f protein restriction the a m o u n t of S-100 protein in brain remained unchanged. The work was supported by a grant from the Swedish Medical Research Council to A.v.d.D. (project No. 4266) a n d Wilhelm and M a r t i n a L u n d g r e n ' s F o u n d a t i o n to K.H. The a u t h o r s wish to t h a n k Mrs. A g n e t a Nilsson for her valuable technical assistance. 1 Haglid, K. G., Stavrou, D., R6nnb~ick, L.. Carlsson. C. A. and Weidenbach. W.. f he S-100 protein in water-soluble and pentanol-extractable form in normal human brain and turnouts of the human nervous system. A quantitative study, J. neurol. Sci.. 20 [1973) 103-11 I. 2 Haglid, K. G., Hamberger, A., Hansson, H.-A., Hyd6n, H.. Persson, L. and R6nnback, I... Cellular and subcellular distribution of the S-100 protein in rabbit and rat CNS. J. Neurosc'i. Res.. 2(1976J 175-191. 3 Herschman. J. R.. Levine, L. and DeVellis, 1.. Appearance of a brain specific tlnr~gen ~S-100 protein) in the developing rat brain, J. Neurorhem., 18 (1971) 629-633. 4 Laurell, C.-B., Quantitative estimation of proteins by electrophoresis in agarose gel contaimng antibodies, Analyt. Bioehern., 15 (1966) 45-52. 5 Lee, C.-J., Biosynthesis and characteristics of brain protein and ribonucleic acid in m~ce subjected to neonatal infection and undernutrition, J. biol. Chem., 245 (1970) 1998-2004. 6 Lowry, O. H.. Rosebrough, N. I.. Farr, A. L. and Randall, R. I.. Protein measurement with the Folin phenol reagent, J. bioL Chem.. 193 (195t) 265-275. 7 0 d e n , A and Wedel, H., Arguments for Fischer's permutation tesl. Ann. Star., 3 ~1975) 518-520. 8 0 ' N e a l , R. M., Pla. G. W., Fox, S., Gibbon, F. and Fry, B. E.. Effect of zinc deficiency and restricted feeding on protein and ribonucleic acid metabolism of rat brain, J. Nutr., 100 ~1970/491 497. 9 Stavrou, D., Lubbe, I. and Haglid, K. G.. lmmunelektrophoretische Quantifizierung des hirnspezifischen S-t00 proteins, Acta neuropath, rBerl.), 29 (1974) 275-280. 10 Von Der Decken. A., Evidence for regulation of protein synthesis at the translation level in response to dietary alterations. J. Cell bioL. 33 (1967) 657-663. 11 Von Der Decken, A., Modification of the in vitro amino acid incorporation capacity of rat liver after in vivo and in vitro treatments, Europ. J. Biochem., 4 (1968) 87-94. 12 Von Der Decken, A., Amino acid incorporation by rat liver microsomal and ribosomal preparation. Modification of activity by in vivo and in vitro treatments, Abh. dtsch. Akad, Wiss. Berl.. I ( 1968} 541-551.
191 13 Von Der Decken, A., Activation in vitro of rat liver polyribosomes, J. Cell. Biol., 43 (1969) 138-147. 14 Von Der Decken, A., Cytoplasmic factors affecting the in vitro amino acid incorporating activity of rat liver preparation after dietary alterations or stress induction. In Szafranski, P., Klita, S. and Mastowski, P. (Eds.), Protein Biosynthesis, Pol. Biochem. Soc., Warszawa, 1969, pp. 33-46. 15 Von Der Decken, A. and Omstedt, P., Protein feeding of rats after protein starvation : incorporation of amino acid into polypeptide by skeletal muscle polyribosomes, J. Nutr., 10 (1970) 623-630. 16 Von Der Decken, A. and Wr6nski, A., Protein synthesis in vitro in rat brain after short-term protein starvation and refeeding, J. Neurochem., 18 (1971) 2383-2388. 17 Wr6nski, A. and Von Der Decken, A., Synthesis of brain-specific acidic proteins in rat and mouse cerebral slices, Actaphysiol. scand., 95 (1975) 482-493. 18 Wr6nski, A. and Von Der Decken, A., Protein synthesis with special reference to S-100 protein in brain slices from rats receiving a restricted protein supply, Acta physiol, scand., 97 (1976) 20-30.