Multihormonal control of proliferation and cytosolic glycerol phosphate dehydrogenase, lactate dehydrogenase and malic enzyme in glial cells in culture

Neurochem. Int. Vol. 9, No. 2, pp. 247 253, 1986 Printed in Great Britain

0197-0186/86 $3.00+ 0.00 PergamonJournals Ltd

MULTIHORMONAL CONTROL OF PROLIFERATION AND CYTOSOLIC GLYCEROL PHOSPHATE DEHYDROGENASE, LACTATE DEHYDROGENASE A N D MALIC ENZYME IN GLIAL CELLS IN CULTURE FATIMA MONTIEL,* LOUIS SARLII~VE,'I"ANGEL PASCUAL* and ANA ARANDA*~ *Departamento Endocrinologia Experimental, Instituto Invest.Biomed. C.S.I.C., Facultad de Medicina, Universidad Autonoma de Madrid, 28029 Madrid, Spain and tUnit6 44 de L'INSERM and Centre de Neurochimie da CNRS, 67084 Strasbourg Cedex, France (Received 21 November 1985; accepted 10 February 1986) Abstract--We have examined the effect of a physiological concentration of L-triiodothyronine on the

activity of cytosolic enzymes in the C6 rat glioma cell line. L-Triiodothyronine decreased glycerol phosphate dehydrogenase activity. This effect seems to be rather specific, since L-triiodothyronine did not change malic enzyme or lactate dehydrogenase activity and did not alter the amount of either cytosolic or total cell protein. Dexamethasone greatly increased glycerol phosphate dehydrogenase and L-triiodothyronine also decreased the response to the glucocorticoid. Noradrenaline or dibutyryl cyclic AMP potentiated the dexamethasone-induced specific activity of this enzyme, and L-triiodothyronine lowered the response to the combined effects of these agents. The effect of L-triiodothyronine is not restricted to the C6 cells, since it also decreased basal glycerol phosphate dehydrogenase activity in primary cultures of cells dissociated from brains of embryonic mice. The results indicate that thyroid hormones have a direct effect on the modulation of cytosolic glycerol phosphate dehydrogenase in cultured cells of glial origin.

It is well known that thyroid hormones play an important role in the growth, development and maturation of the brain. Thyroid hormones actions are not restricted to neurons, since thyroid status affects myelination in vivo (Dalal et al., 1971; Valcana et al., 1975; Sarli6ve et al., 1983), and thyroid hormone addition increases the formation of myelin in brain explant cultures (Hamburgh, 1969) and in brain primary cultures (Bath et al., 1979; 1981a; 1981b) indicating that these hormones are important for a normal maturation and differentiation of glial cells. C6 cells, a rat glioma cell line, has been widely used for the study of hormonal regulation of enzyme activities in glial cells, since the responsiveness of these cells to several hormones is very similar to that found in normal astrocytes and oligodendrocytes (de Vellis et aL, 1977). C6 cells possess nuclear thyroid hormone receptors (Ortiz-Caro et al., 1986) and

:~Address correspondence to: Dr Ana Aranda, Departamento de Endocrinologia Experimental, Instituto de Investigaciones Biom6dicasdel C.S.I.C., Facultad de Medicina, Universidad Aut6noma, C/Arzobispo Morcillo 4, 28029 Madrid, Spain. 247

respond to the addition of high concentrations of thyroxine in the culture medium by increasing the activity of arylsulphatases A and B (Farooqui et al., 1977). The glucocorticoid induction of glycerol phosphate dehydrogenase (EC 1.1.1.8; GPDH) is a very wellcharacterized hormonal effect in oligodendrocytes and C6 cultures and it has been demonstrated in vivo to be specific for the nervous system (de Vellis et al., 1977). The development of GPDH in the rat brain coincides with myelination and its expression and maintenance requires the presence of glucocorticoids. These steroids control GPDH activity in C6 cells, by increasing the amount of specific mRNA for the enzyme (Kumar et al., 1984). Lactate dehydrogenase (EC 1.1.1.27; LDH) is specifically regulated by catecholamines in C6 cells. These hormones, acting through cyclic AMP, increase its rate of synthesis (Kumar et al., 1980) and the rate of transcription of the enzyme specific mRNA (Jungmann et al., 1983). Malic enzyme (EC 1.1.1.40) activity is under thyroid hormone control in liver (Tarentino et al., 1966). Other hormones and factors (glucocorticoids, insulin

248

FATIMA MONTIEL et al.

a n d c a r b o h y d r a t e s ) act synergistically with t h y r o i d h o r m o n e s in h e p a t o c y t e s to increase malic e n z y m e synthesis ( W i l s o n a n d M c M u r r a y , 1981; G o o d r i d g e , 1983; M a r i a s h a n d O p p e n h e i m e r , 1983). A l t h o u g h it is c o n s i d e r e d that this e n z y m e fails to r e s p o n d to t h y r o i d h o r m o n e s in the a d u l t or the n e o n a t a l rat brain ( H e m o n , 1968), m o r e recent w o r k indicates that t h y r o i d h o r m o n e s p r o m o t e d the d e v e l o p m e n t o f malic e n z y m e in the brain o f n e o n a t a l h y p o t h y r o i d rats ( D i e z - G u e r r a et al., 1981). In the light o f the a b o v e o b s e r v a t i o n s we have e x a m i n e d the effect o f a physiological c o n c e n t r a t i o n o f L - t r i i o d o t h y r o n i n e (T3) o n b o t h the u n i n d u c e d a n d i n d u c e d levels o f G P D H , L D H a n d malic e n z y m e in C6 cells. O u r results indicate that T3 decreases the basal activity as well as the G P D H r e s p o n s e to d e x a m e t h a s o n e , or the c o m b i n a t i o n o f d e x a m e t h a s o n e a n d n o r e p i n e p h r i n e or dexa m e t h a s o n e a n d dibutyryl cyclic A M P . In c o n t r a s t , the L D H o r malic e n z y m e activities are n o t affected by T3. Finally, the effect o f T 3 o n G P D H activity was also seen in p r i m a r y cultures o f dissociated brain cells f r o m e m b r y o n i c mice. EXPERIMENTAL PROCEDURES

Hormones and chemicals Culture media and sera were obtained from Gibco. Resin AG I-XI0 was from Bio-Rad. [3H]leucine (sp.act.= 142Ci/mmol) was obtained from Amersham. Hormones, dibutyryl cyclic AMP and all substrates and cofactors used in enzymatic determinations were from Sigma. Cells and cell cultures C6 cells were grown in monolayer culture in 75 cm 2 Falcon tissue culture flasks in RPMI 1640 medium supplemented with 10% horse serum, 2.5% fetal calf serum and 57 units Penicillin/ml. The cultures were incubated at 37'C in a humidified atmosphere with 5% CO 2 and 95% air. For the experiments the cells were plated at an initial density of 8000-12000 cells/cm2 in either 25 cm 2 flasks or 24 well plates. After two to three days the medium was replaced by a medium containing 10% newborn calf serum treated with resin AG l-X10 and charcoal as previously described (Samuels et al., 1979a, 1979b) to eliminate thyroid hormones and glucocorticoids. The cells were incubated in this medium without hormones for an additional 24 h period before the beginning of the experiments to ensure hormonal depletion. Cerebral hemispheres of 14- to 15-day old mouse embryos were dissociated mechanically into single cells and cultured as described (Sarlieve et al., 1981). The cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal calf serum and 0.6% glucose. The growth medium was replaced by D M E M medium without serum 24 h before the addition of the hormones. Dexamethasone was dissolved in ethanol at a 0.0I M concentration and diluted in culture medium before added to the cells. Unless otherwise stated both C6 cells and primary cultures were incubated with 5 nM T3 for 72 h and with 50 nM dexamethasone during the last 48 h, and the medium was changed every 24 h.

All data presented are the mean + SD ot three to six separated cultures. An asterisk indicates the presence of statistically significant differences (P < 0.05) between the groups incubated with and without T3 and/or dexamethasone. Preparation o]' samples [or enzyme assays All operations were performed at 4 C . The cells were washed three times with saline and scraped from the flasks with a rubber policeman. They were homogenized with 0.64).8ml of 0.1 M sodium phosphate buffer, pH 7.4, containing 1 mM EDTA and 5 mM 2-mercaptoethanol, and centrifuged at 800g to remove nuclei and cellular debris. The supernatant was then centrifiuged at 17,500rpm for 60 rain in a SM-24 rotor in a Sorvall RC-5 centrifuge. The supernatants were used for protein determination (Lowry et al., 1951) and enzyme assays. GPDH and LDH were assayed as previously described (McGinnis and de Vellis, 1974), with the exception that the assays were carried out at room temperature instead of 30C. Malic enzyme was determined by the method of Hsu and Lardy (1969). One unit of enzyme is defined as that amount which causes the oxidation of 1 nmol of NADH/min in the case of GPDH and LDH, and the formation of 1 nmol NADPH/min in the case of malic enzyme. Specific activity is expressed as units of enzyme activity/mg of cytosolic protein. Effect q[' hormones on total protein and DNA content C6 cells were grown as described above in Falcon 24-well plates. Total DNA (Burton, 1956) and protein (Lowry et aL, 1951) were determined after incubation for 48 h or 120 h with T3 and/or dexamethasone. Incorporation q['[ ~H]leueine into total prolein The incorporation of [~H]leucine into trichloroacetic acidinsoluble material was used as a measure of total protein synthesis. C6 cells were incubated for 2 h at 37'C with 5/~Ci/ml of [3H]leucine in leucine-depleted medium supplemented with 6 # M cold leucine. After incubation the cultures were chilled and washed three times with phosphatebuffered saline, the cells then received l ml of 18% trichloroacetic acid. After 30rain trichloroacetic acidsoluble material was removed and the cellular material was lysed with 0.2 M sodium hydro×yde. An aliquot was used for protein determination and another aliquot was counted in a liquid scintillation counter. Data are expressed as cpm incorporated into protein/100 ~tg of total cell protein. RESULTS Effect o f T3 and dexamethasone on cell growth I n c u b a t i o n o f C6 cells with 5 n M T3 for 48 or 120 h d o e s n o t c h a n g e total p r o t e i n o r D N A / w e l l (Table 1). T h e effect o f the g l u c o c o r t i c o i d d e p e n d s on the cell density: 50 n M d e x a m e t h a s o n e significantly dec r e a s e d total p r o t e i n a n d D N A by a p p r o x i m a t e l y 5 0 % , w i t h o u t c h a n g i n g p r o t e i n / D N A ratio, w h e n the t r e a t m e n t with the h o r m o n e was initiated in rapidly g r o w i n g cultures. H o w e v e r , d e x a m e t h a s o n e did n o t alter these p a r a m e t e r s w h e n a d d e d to d e n s e cultures. T3 did n o t m o d i f y the effect o f the steroid o n cell p r o l i f e r a t i o n in any o f the c o n d i t i o n s studied, since

H o r m o n a l control of enzyme activities in glial cells

249

Table 1. Effect of T3 and dexamethasone on the growth of cultures of C6 cells 48 h of treatment Low density Prot. T3 Dx T3 + Dx

120 h of treatment High density

DNA Prot. (% of control)

98+6 54 + 3* 56 + 8*

100+3 60 + 4* 55_+ 2*

95+ 13 93 + 10 84 +4"

Low density

DNA

Prot.

110+6 100 + 5 96 + 3

101 + 4 48 + 6* 50 + 4*

High density

DNA Prot. (% of control) 100+6 53 + 4* 45 + 3*

116+7 87 + 7 88 + 9

DNA 93+2 83 + 4* 89 + 8

c6 cells were grown in RPMI 1640 medium containing 10% horse serum and 2.5% fetal calf serum. After three days the growth medium was replaced by a medium containing 10% newborn calf serum depleted of thyroid hormones and glucocorticoids by treatment with resin and charcoal. Twenty-four hours later the cultures were incubated with 5 nM T3, 50 nM dexamethasone (Dx), or the combination of both for 48 or 120 h. The treatment was initiated either in log phase cultures or in nearly confluent cultures. In our culture conditions, a confluent 2 cm2 plate will contain about 17 pg of DNA and 190 #g protein, which is equivalent to 1.5-2 million cells approximately. All values represent % of control cultures which did not receive any treatment. At the end of the experimental period, the DNA content in the control cultures was 18 19/~g DNA in the high density and 11 12 #g DNA in the low density cultures. The data represent the mean _+SD of three to six cultures. *: P < 0.05 vs control cultures. There were no statistically significant differences among control and T3, or Dx and Dx + T3 groups.

incubation with T3 plus dexamethasone had the same effect as i n c u b a t i o n w i t h d e x a m e t h a s o n e a l o n e . T 3 , dexamethasone or the combination of both did not affect p r o t e i n s y n t h e s i s e v e n w h e n d e x a m e t h a s o n e d e c r e a s e d t o t a l p r o t e i n c o n t e n t in t h e c u l t u r e s . [3H]leucine incorporation into proteins was 74610 + 9166 c p m / 1 0 0 # g cell p r o t e i n in c o n t r o l cells, 7 7 6 0 7 + 3386 in cells i n c u b a t e d w i t h 5 n M T 3 , 86702 + 5437 in cells i n c u b a t e d w i t h 5 0 n M d e x a m e t h a s o n e a n d 83676 + 2625 in t h e cells r e c e i v i n g t h e c o m b i n a t i o n o f b o t h f o r 72 h,

Hormonal effects on G P D H and L D H activities T a b l e 2 s h o w s t h e effect o f T 3 a n d d e x a m e t h a s o n e o n G P D H levels in C 6 cells a n d in p r i m a r y c u l t u r e s o f fetal m i c e b r a i n cells. T r e a t m e n t o f C 6 cells w i t h 50riM dexamethasone increased GPDH activity n e a r l y 5-fold o v e r a 48 h p e r i o d . F i v e n M T3 in t h e medium decreased basal activity by 70% and prod u c e d a s l i g h t r e d u c t i o n in t h e r e s p o n s e to dexa m e t h a s o n e . I n this a n d in t h e f o l l o w i n g e x p e r i m e n t s the treatment with the hormones was initiated before

the cultures reached confluency and accordingly with t h e d a t a in T a b l e 1, T 3 d i d n o t a l t e r t o t a l o r c y t o s o l i c p r o t e i n , w h e r e a s in this p a r t i c u l a r e x p e r i m e n t d e x a m e t h a s o n e d e c r e a s e d b o t h by a p p r o x i m a t e l y 3 5 % . A p a r t i a l b l o c k o f t h e g l u c o c o r t i c o i d i n d u c t i o n by T 3 w a s also o b s e r v e d in C 6 cells i n c u b a t e d in s e r u m - f r e e m e d i u m ( d a t a n o t s h o w n ) . T h e effect o f T3 o n b a s a l a n d i n d u c e d G P D H levels w a s q u a n t i t a t i v e l y v a r i a b l e a m o n g different e x p e r i m e n t s , r a n g i n g b e t w e e n 15 a n d 8 0 % . I n T a b l e 2 t h e r e s u l t s o b t a i n e d in p r i m a r y c u l t u r e s o f fetal m i c e b r a i n cells a r e also r e p r e s e n t e d . E n z y m e activity w a s d e t e r m i n e d at 14 a n d 60 d a y s in c u l t u r e . W i t h t h e c o n d i t i o n s e m p l o y e d (Sarli6ve et al., 1981) t h e c u l t u r e s are m a i n l y a s t r o g l i a l a n d olig o d e n d r o g l i a l b e t w e e n 10 a n d 25 d a y s in c u l t u r e , a n d p o s s i b l y m i c r o g l i a l f r o m 25 to 60 d a y s ( a l t h o u g h o l i g o d e n d r o c y t e s a r e still o b s e r v e d d u r i n g this period). B a s a l G P D H levels a r e l o w e r in t h e s e c u l t u r e s t h a n in C 6 cells, at least at t h e p e r i o d s s t u d i e d , a n d this d o e s n o t a p p e a r to be d u e to t h e u s e o f s e r u m free m e d i u m d u r i n g t h e e x p e r i m e n t , since b a s a l levels were t h e s a m e w h e n a n a l y z e d in cells at 14 d a y s in

Table 2. Effect of T3 and dexamethasone (Dx) on GPDH levels in C6 cells and in primary cultures of cells from embryonic brain GPDH activity C6 cells

Control

Dx

T3

T3 + Dx

53 + 10

273 + 28*

20 + 10"

223 + 16"

2.6±0.6 9.9+1.8 0.8+0.1

6.5+0.1" 16.8+3.3" ND

1.3+0.1" ND 0.3+0.03*

5.7+0.6* ND ND

Primary cultures (DICt) 14

60 tDays in culture; ND: not determined; *P < 0.05 vs controls. C6 cells or cells dissociated from embryonic mice were incubated with 5 nM T3 for 72 h or 50 nM dexamethasone (Dx) for 48 h. Values represent GPDH specific activity expressed as units/mg cytosolic protein and are the mean + SD of three to six different cultures.

250

FATIMA MONTIEL el al.

culture grown in the presence o f 15% fetal calf serum (8.7_+0.8 vs 9.3 _+ 1,9 U / m g protein in serum-free and serum-containing medium, respectively), G P D H induction by dexamethasone was also found in the primary cultures, though to a lesser extent than in C6 cells. The addition o f T3 decreased enzyme levels by more than 50% at both 14 and 60 days in culture, whereas the response to dexamethasone was only slightly decreased. Noradrenaline has been reported to potentiate the glucocorticoids-induced specific activity o f G P D H (Breen et al., 1978). Therefore we tested the effect in C6 cells o f T3 on this induction. Figure 1 shows that 3 p M noradrenaline for 18 h did not alter the uninduced level, but potentiated the response to dexamethasone and that T3 decreased both basal and stimulated G P D H levels. Figure 1 also illustrates L D H specific activity obtained in the same cells. As expected, noradrenaline increased L D H levels and in contrast with the results obtained with G P D H , T3 did not alter either basal activity nor the catecholamine induction, thus demonstrating that the effect o f T3 on the G P D H response is rather specific. The effects o f noradrenaline appear to be mediated by a transient rise in the intracellular cyclic A M P levels (Breen et al., 1978). Figure 2 shows the influence o f T3 on the G P D H response to dexamethasone and dibutyryl c A M P (1 m M for 48 h) in C6 cells. Dibutyryl cyclic A M P greatly potentiated dexamethasone induction, and T3 very significantly diminished G P D H specific activity both under basal conditions and in conditions o f very high stimulation. Dibutyryl cyclic A M P increased L D H activity and, as already described with norepinephrine (Fig. 1), T3 did not change basal or induced LDH levels (data not shown). H o r m o n a l £~kcts on malic e n z y m e Table 3 shows that malic enzyme, a good parameter of thyroid h o r m o n e action in other cell types, is

[ ] T 3 5nM

30

~

~-~8

O O.

u~

E 15 I n 0

c

~m T 3 5nM

o

? o x u~

C) -A

C

Dx

N"

D;c+ NA

Fig. 1. Differential effect ofT3 on GPDH (upper panel) and LDH (lower panel) levels in C6 cells. The cells were first incubated with T3 or with medium alone for 24 h. Dexamethasone was then added, followed by 3,uM noradrenaline 30 h later. The experiment was carried out 18 h after the addition of noradrenaline. Therefore, the overall time of incubation with T3 was 72 h, with dexamethasone 48 h and with noradrenaline 18 h. C represents uninduced controls. Dx represents dexamethasone-treated cells, NA cells incubated with noradrenaline and Dx + NA cells incubated with dexamethasone and noradrenaline. In all groups, hatched bars represent T3-treated cells and open bars represent cells which did not receive T3. Data are expressed as mean + SD of three or four cultures. *: P < 0.05 or less between T3 treated and untreated cells. not induced by T3 either alone or in combination with dexamethasone in C6 cells. It can also be observed in Table 3 that T3 did not affect cytosolic protein, whereas dexamethasone decreased it by 3040°/,,. It has been reported that a full rnalic

"fable 3. Malic enzyme activity in hormone-treatedC6 cells Experiment ll Experiment [

Control Dx T3 T3+Dx

ME activity

Cytosolic protein

3.8 + 0.4 2.8±0.5* 4.1 ±0.2 2.9k0.7

44 + 3 30±4* 43+ 3 28+ 1"

Insulin ME activity {}.96± 0.11 0.80±0.21 0.91 _~0.18 1.19±0.12

~ Insulin

Cytosolic protein

ME activil~

Cytosolic protein

36 + 3 2693* 36+5 26±2"

0.97 ~ {1.20 0.88 ~ 0.09 1.00+0.03 (1.91 + 0.02

32 f 26 ~- 2" 31 ! 27! I*

C6 cells were incubated with T3 and/or dexamethasone (Dx) for 48 h in the absence (Experiment I) or presence (Experiment II) of 5#g/ml bovine insulin. Malic Enzyme (MEI activity is expressed as D.O x 102/min/mg cytosolicprotein. Protein data representug protein/50itl cytosolicextract. Data are mean ± SD of three or four separated cultures. *: P < 0.05 vs controls.

Hormonal control of enzyme activities in glial cells

c ]

T3 5riM

o

400 u~ c I 2OO C~ 0_ (.9

¢,

1"1,*, Dx

Dx ÷

J

db cAMP

Fig. 2. Effect of T3 on GPDH induction by dexamethasone and dibutyryl cyclic AMP. C6 cells were incubated with or without 5 nM T3 for 24 h. The cells were then treated with medium alone (C), dexamethasone (Dx), or dexamethasone + 1 mM dibutyryl cyclic AMP (Dx + dbcAMP) for 48 h in the absence (open bars) or presence (hatched bars) of T3. Data presented as mean + SD of three or four cultures. *: P < 0.05 or less between T3 treated and untreated groups.

enzyme induction in hepatocytes only occurs in the presence of thyroid hormones, insulin and glucocorticoids (Wilson and McMurray, 1981). However, as shown in Table 3, 5/~g/ml of insulin for 48 h did not induce malic enzyme activity, even in the presence of T3 and dexamethasone in C6 cell cultures. DISCUSSION

Thyroid hormones have been long recognized as very important regulators of brain development and function. However, the variety of effects induced by thyroid hormones in vivo, make it difficult to decide which observations represent direct effects of thyroid hormones, and which are due to changes in other hormones and/or generalized metabolic effects. This prompted us to investigate the possible effects of thyroid hormones alone, as well than in combination with other hormones, on proliferation and enzyme activities of cultured cells of glial origin. We could not demonstrate any effect of T3 on the growth of C6 cell cultures, as measured by total DNA and protein content, nor any influence on total cellular protein synthesis. This is not restricted to C6 cells, since the same is also observed in the present primary cultures and in other cell lines (RN2, G26-20 and G26-24) of glial origin (Montiel and Aranda, unpublished observations).

251

In contrast, dexamethasone inhibited cell proliferation in rapidly growing C6 cells, in agreement with the results obtained by Grasso (1976) using cortisol. However, when treatment with dexamethasone was initiated in dense cultures, the steroid had little if any effect on protein and D N A content. These data, together with the finding that the incorporation of [3H]leucine into proteins is not altered in the presence of dexamethasone, supports the idea that neither cytotoxicity nor lethality are responsible for the reduction of cell numbers in glucocorticoid-treated cultures. We find that basal GPDH levels are much lower in primary cultures of cells from embryonic mice (Sarli6ve et al., 1981) than in C6 cells, probably because of the heterogeneity of cell types in the primary cultures. In the central nervous system GPDH is exclusively localized in oligodendrocytes (McCarthy and de Vellis, 1980), and in agreement with our results GPDH activity is low in primary cultures from rat brain (de Vellis et al., 1977) and it is high in pure rat oligodendrocytes (McCarty and de Vellis, 1980). As expected (de Vellis et al., 1977), dexamethasone treatment caused an increase in GPDH activity in C6 cells. We also observed that the steroid induced enzyme activity in the mouse brain cells, although the increase was less marked than that described for rat cells (McCarthy and de Vellis, 1980), and that this increase occurs even in the absence of other factor/s and/or hormones present in serum, since it is observed in cells cultured in serum-free conditions. T3 decreases basal GPDH activity in C6 cells. The fact that this inhibition is also seen in cultures of non-tumor cells, indicates that it is not merely a reflection of malignant transformation and opens the possibility that thyroid hormones could modulate this activity in the brain in vivo. In addition, this inhibition does not represent a generalized effect of T3 on cytosolic enzyme activities, since LDH basal level and its induction by noradrenaline (Kumar et al., 1980) remained unaffected. T3 not only influences GPDH basal activity, but it also causes a significant inhibition of the dexamethasone-mediated response. This phenomenom cannot merely be explained by a decreased cell permeability or hormone availability leading to a diminished biological action of the glucocorticoid, since T3 did not affect the inhibition of cell proliferation produced by the steroid. In agreement with results obtained by others (Breen et al., 1978), we found that noradrenaline or dibutyryl cyclic AMP enhances the dexamethasone-induced response. T3 reduced the response to the combination of glu-

252

FATIMA MONTIEL et al.

cocorticoids and cathecolamines, and this reduction was observed even in conditions of very high stimulation which were obtained when d e x a m e t h a s o n e a n d cyclic A M P were administered together. The inhibitory effect o f T3 on G P D H is rather surprising, since this enzyme is believed to play a role in the synthesis of lipids which could be used as myelin precursors and f u r t h e r m o r e it has been described t h a t the n o r m a l developmental increase in rat brain G P D H is impaired in h y p o t h y r o i d rats (Schwark et al., 1971). However, we have yet to clarify the underlaying mechanism, most notably whether the h o r m o n e is altering the synthesis of the enzyme or affecting other(s) protein(s) which could shorten its half-life. In this respect, it should be pointed out that thyroid h o r m o n e s not only increases myelin synthesis, but also myelin disappearance in organ explants ( H a m b u r g h , 1969). Interestingly, a n o n - h o r m o n a l c o m p o u n d , sodium butyrate, which has a p r o f o u n d influence on the c h r o m a t i n a n d greatly increases T3 receptor levels in C6 cells (Ortiz-Caro et al., 1986), has an effect similar to T3 blocking the G P D H induction by glucocorticoids (Weingarten et al., 1981). Therefore it will be of interest to determine whether T3 and butyrate m o d u l a t e enzyme activity t h r o u g h similar mechanism/s. Finally, a widely used biochemical index of thyroid function in non-neural tissues, cytosolic malic enzyme, failed to respond to T3 in C6 cells even in the presence of glucocortieoids and insulin. This lack of response to T3 agrees with the results obtained by H e m o n (1968) in the rat brain in vivo, and d e m o n strates the cell a n d tissue specificity in the response to thyroid hormones. In conclusion our data d e m o n s t r a t e direct effects of thyroid h o r m o n e s on glial cells, and indicate t h a t the use of glial cells in culture can contribute to our u n d e r s t a n d i n g of the mechanisms by which thyroid h o r m o n e s regulate brain function. Acknowledgements The authors are grateful to Dr. G. Rebel for the C6 cells and to Dra. G. Morreale de Escobar for the critical revision of this manuscript. The technical assistance of Ms. A. Jimenez is also acknowledged. This work was supported by research grants from the Comisi6n Asesora de Investigaci6n Cientifica y Tecnica and Fondo de Investigaciones Sanitarias de la Seguridad Social (Spain) and from grants from the CNRS and I'INSERM (France).

REFERENCES Bhat N. R., Shanker G. and Pieringer R. A. (1981) Investigations on myelination in vitro: Regulation of 2'3'-cyclic nucleotide 3'-phosphohydrolase by thyroid

hormone in cultures of dissociated brain cells from embryonic mice. J. Neurochem. 37, 695 701. Bhat N. R., Sarli6ve L. L., Subba Rao G. and Pieringer R. A. (1979) Investigations on myelination in vitro. Regulation by thyroid hormone in cultures of dissociated brain cells from embryonic mice. J. biol. Chem. 254, 9342 9344. Bhat N. R., Subba Rao G. and Pieringer R. A. (1981) Investigations on myelination in vitro. Regulation of sulpholipid synthesis by thyroid hormone in cultures of dissociated brain cells from embryonic mice. J. biol. Chem. 256, 1167 1171. Breen G. A. M., McGinnis J. F. and de Vellis J. (1978) Modulation of the hydrocortisone induction of glycerol phosphate dehydrogenase by N6,0Z-dibutyryl cyclic AMP, norepinephrine and isobutylmethylxanthine in rat brain cell cultures. J. biol. Chem. 253, 2554-2562. Burton K. (1956) A study of the conditions and mechanisms of the diphenylamine reactions for the colorimetric estimation of deoxyribonucleic acids. Biochem. J. 62, 315323. Dalai K. B., Valcana T., Timiras P. S. and Einstein E. R. (1971) Regulatory role of thyroxine on myelogenesis in the developing rat. Neurobiology I, 211 224. De Vellis J. McGuinnis J. F., Breen G. A. M., keveille P., Bennet K. and McCarthy K. (1977) Hormonal effects on differentiation in neural cultures. In: Cell, Tissue and Organ Cultures in Neurohiologl', (Fedoroff S. and Hertz g., eds), pp. 485 511. Academic Press, New York. Diez-Guerra J., Aragon M. C.. Gimenez C. and Valdivieso F. ( 198 I) Effect of thyroid hormones on the malic enzyme activity in rat brain during development. Devl Neurosci. 4, 130 133. Farooqui A. A., Elkoubi A. and Mandel P. (1977) Effect o1" hydrocortisone and thyroxine on arylsulphatases A and B of cultured cells of neuronal and glial origin. J. Neurochem. 29, 365 369. Goodridge A. G. (1983) Regulation of malic enzyme in hepatocytes in culture. A model system for analyzing the mechanism of action of thyroid hormones. In: Molecular Basis ~/' Thyroid Hormone Action, (Oppenheimer J. H. and Samuels H. H., eds), pp. 245 263. Academic Press, New York. Grasso R. J. (1976) Transient inhibition of cell proliferation in rat glioma monolayer cultures by cortisol. Cancer Res. 36, 2408 2414. Hamburgh M. (1969) The role of thyroid and growth hormones in neurogenesis. In: Current Topics in Developmental Biology, (Moscona A. and Mouroy A., eds), Vol. 4, 109 148. Academic Press, New York. Hemon P. (1968) Malate dehydrogenase (decarboxylating) (NADPH) and :~-glycerophosphate oxidase in the developing rat. Biochim. biophys. Acta 151, 681-683. Hsu R. J. and Lardy H. A. (1969)Malic enzyme. In: Meth. En:ym. 13, 230 235. Jungmann R. A., Kelley D. C., Miles M. F. and Milkowski D. M. (1983) Cyclic AMP regulation of lactate dehydrogenase. J. biol. ('hem, 258, 5312 5318. Kumar S., McGinnis J. F, and de Vellis J. (1980) Catecholamine regulation of lactate dehydrogenase in rat brain cell culture. J. biol. Chem. 255, 2315 2321. Kumar S., Weingarten D. P., Callahan J. W., Sachar K. and de Vellis J. (1984) Regulation of mRNAs for three enzymes in the glial cell model C6 cell line. J. Neurochem. 43, 1455 1463.

Hormonal control of enzyme activities in glial cells Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mariash C. N. and Oppenheimer J. H. (1983) Thyroid hormone-carbohydrate interaction. In: Molecular Basis o f Thyroid Hormone Action, (Oppenheimer J. H. and Samuels H. H., eds), pp. 265-292. Academic Press, New York. McCarthy K. D. and de Vellis J. (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890-902. McGinnis J. F. and de Vellis J. (1974) Purification and characterization of rat brain glycerol phosphate dehydrogenase. Biochim. biophys. Acta 364, 17-27. Ortiz-Caro J., Montiel F., Pascual A. and Aranda A. (1986) Regulation by n-butyrate of thyroid hormone receptor levels in cultured cells of glial origin. In: Proceedings of the I X International Thyroid Congress. In press. Plenum Press, New York. Samuels H. H., Stanley F. and Casanova J. (1979) Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105, 80-85. Samuels H. H., Stanley F. and Shapiro L. E. (1979) Control of growth hormone synthesis in cultured GH1 cells by 3,5,Y-triiodo-L-thyronine and glucocorticoid agonists and antagonists: studies on the independent and synergistic regulation of the growth hormone response. Biochemistry 18, 715 721. Sarlieve L. L., Delaunoy J. P., Dierich A., Ebela., Fabre M., Mandel P., Rebel G., Vincendon G., Winthzerith M. and Yusufi A. N. K. (1981) Investigations on myelination in

253

vitro. III. Ultrastructural, biochemical, and immunohistochemical studies in cultures of dissociated brain cells from embryonic mice. J. Neurosci. Res. 6, 659~83. Sarlieve L. L., Bouchon R., Koehl C. and Neskovic N. M. (1983) Cerebroside and sulfatide biosynthesis in the brain of Snell dwarf mouse: effects of thyroxine and growth hormone in the early postnatal period. J. Neurochem. 40, 1058-1062. Schwark W. S., Singhal R. L. and Ling G. M. (1971) Metabolic control mechanisms in mammalian systems: thyroid hormone control of alpha glycerol phosphate dehydrogenase activity in rat cerebral cortex and cerebellum. Can. J. Physiol. Pharmac. 49, 598607. Tarentino A. L., Richert D. A. and Westerfeld W. W. (1966) The concurrent induction of hepatic x-glycerophosphate dehydrogenase and malate dehydrogenase by thyroid hormone. Biochim. biophys. Acta. 124, 295-309. Valcana T., Einstein E. R., Csejtey J., Dalai K. B. and Timiras P. S. (1975) Influence of thyroid hormones on myelin proteins in the developing rat brain. J. Neurol. Sci. 25, 19-27. Weingarten D. and de Vellis J. (1980) Selective inhibition by sodium butyrate of the glucocorticoid induction of glycerol phosphate dehydrogenase in glial cultures. Biochem. biophys. Res. Commun. 93, 1297-1304. Wilson E. J. and McMurray W. C. (1981) Regulation of malic enzyme and mitochondrial c¢-glycerophosphate dehydrogenase by thyroid hormones, insulin, and glucocorticoids in cultured hepatocytes J. biol. Chem. 256, 11657-11662.