Effect of hibernation, thyroid hormones and dexamethasone on cytosolic and mitochondrial glycerol-3-phosphate
dehydrogenase from jerboa (Jaculus orientalis )
Widad Berrada a , Abdallah Naya b , L’houcine Ouafik c , Noureddine Bourhim a, *
a Laboratoire de Biochimie, Biologie Cellulaire et Mole´culaire ( Unite´ de Biochimie ) , De´partement de Biologie, Faculte´ des Sciences, Uni 6 ersite´ Hassan II-Aı¨n-Chock, Route d ’ Eljadida B.P. 5366 , Maaˆrif, Casablanca, Morocco
b Laboratoire de Biologie et d ’ Ecologie Animale, Faculte´ des Sciences, De´partement de Biologie, Uni 6 ersite´ Hassan II-Aı¨n-Chock, Route d ’ Eljadida B.P. 5366 , Maaˆrif, Casablanca, Morocco
c Laboratoire de Cance´rologie Expe´rimentale, Faculte´ de Me´decine Nord, IFR Jean Roche, Boule 6 ard Pierre Dramard, 13916 MarseilleCedex 20 , France
Received 24 September 1998; received in revised form 9 December 1999; accepted 20 December 1999
Abstract
Tissue distribution of the cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase (cGPDH and mGPDH) activities in jerboa (Jaculus orientalis), a hibernator, shows the highest level of enzyme activity in skeletal muscle and brown adipose tissue, respectively. The effect of hibernation on cGPDH indicates an increase of activity in all tissues examined. In contrast, hibernation decreases mGPDH activity in all tissues, except skeletal muscle. The effect of thyroid hormones on GPDH activity was tissue specific: in kidneys, cGPDH activity doubled in euthermic jerboas treated with T4. In contrast, 6-n-propyl-2-thiouracil treatment provokes an increase of enzyme activity in brown adipose tissue, liver and brain. T4 treatment leads to a 2.7-fold increase in liver mGPDH activity. 6-n-propyl-2-thiouracil treatment decreases mGPDH activity in the skeletal muscle whereas the opposite effect was observed in brain. Dexamethasone stimulates cGPDH in all tissues examined, except skeletal muscle and kidneys. In the case of mGPDH activity, this increase was observed only for brown adipose tissue and brain. Our results suggest that hibernation, thyroid hormones and dexamethasone probably play a role in the regulation of cGPDH and mGPDH activities in jerboa. Our findings confirm that these enzymes are involved in metabolic adaptation to thermal stress in Jaculus orientalis. © 2000 Elsevier Science Inc. All rights reserved.
Keywords : Jaculus orientalis; Tissue distribution; Hibernation; Cytosolic glycerol-3-phosphate dehydrogenase; Mitochondrial glycerol-3- phosphate dehydrogenase; Thyroid hormones; T3; T4; PTU; Dexamethasone
Abbre 6 iations : BAT, brown adipose tissue; cGPDH, cytosolic glycerol-3-phosphate dehydrogenase; DHAP, dihydroxyacetone phosphate; FFA, free fatty acids; G3P, glycerol-3-phosphate; INT, 2-p-iodophenyl-5-phenyl-5-phenyltetrazolium chloride; KCN, potassium cyanide; mGPDH, mitochondrial glycerol-3-phosphate dehydrogenase; PTU, 6-n-propyl-2-thiouracil; T3, 3, 3 % , 5-tri-iodothy- ronine; T4, 3, 5, 3 % , 5 % -tetra-iodothyronine (thyroxine).
* Corresponding author. Tel.: +212-2-230680; fax: +212-2-230674.
E-mail address : [email protected] (N. Bourhim)
0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 3 0 5 - 0 4 9 1 ( 0 0 ) 0 0 1 6 1 - 9
1. Introduction
Previous work from our laboratory has indi- cated that many neurotransmitters (Bourhim et al., 1993, 1997) and enzymes (Soukri et al., 1995, 1996; Kabine et al., 1998) were modified during the hibernating state of jerboa (Jaculus orientalis ), a subdesert rodent of Morocco, which is known as a natural hibernator in comparison with the other species of jerboa (J. jaculus) which is con- sidered a non hibernating species (El Hilali and Veillat, 1975). During the prehibernating state, J.
orientalis accumulates simple lipids to be used in the hibernation period. Earlier works have re- ported for many species, including J. orientalis, that during hibernation, plasma glycerol was low and no obvious difference in plasma free fatty acids (FFA) compared to the euthermic animal (Moreau-Hamsany et al., 1988; Yeh et al., 1995;
Flechtner-Mors et al., 1999). The decrease in the plasma glycerol concentration during hibernation suggests that the gluconeogenic precursors may be mainly from glycerol released from triacylglycerol (Moreau-Hamsany et al., 1988; Yeh et al., 1995).
Another observation has demonstrated that the level of serum glycerol was elevated by exposure to cold, fasting, and during the arousal period of ground squirrels (Spermophilus richardsonii ) (Lin, 1977), and during hibernation of little brown bats (Damassa et al., 1995). In this case, the high level of use of the lipids could explain the increased level of glycerol during the deep hibernation pe- riod. Indeed, hibernating animals degrade lipids accumulated during the prehibernating state (Wang, 1982). Lipolysis generates glycerol and FFA. Glycerol accumulates when it is not used in the gluconeogenesis pathway, while FFA are oxi- dized by the b-oxidation pathway. Glycerol is biochemically important as a precursor of sn-glyc- erol-3-phosphate (G3P), a pivotal metabolite with triple roles: (i) providing the carbon skeleton for gluconeogenesis; (ii) carrying reducing equivalents from the cytosol to mitochondria for oxidative phosphorylation; and (iii) acting as the backbone for triacylglycerol synthesis. These reactions are catalyzed by different enzymes among them glyc- erol kinase (EC 2.7.1.30), cytosolic NADH-depen- dent GPDH (EC 1.1.1.8), and mitochondrial FAD-Dependent GPDH (EC 1.1.99.5). Glycerol kinase catalyses the phosphorylation of free glyc- erol to G3P in the presence of ATP. cGPDH catalyzes the reversible reduction of dihydroxy-
acetone phosphate to G3P. mGPDH catalyzes an irreversible conversion of G3P to dihydroxyace- tone phosphate. Mitochondrial GPDH is also im- plicated in the glycerol phosphate shuttle system in conjunction with the cGPDH, and transfers reducing equivalents to the electron transport chain. cGPDH and mGPDH occupy a key posi- tion in metabolism linking glycolysis to phospho- lipid and triacylglycerol synthesis. Lipids rather than carbohydrates are the principal substrate used during hibernating bouts (Willis, 1982). This utilization of lipids produces glycerol that is im- mediately transformed to G3P by glycerol kinase.
This hypothesis suggests an induction of cGPDH enzyme activity by transforming the G3P to DHAP in the aim to maintain glucose homeosta- sis during hibernation. In contrast, the energy requirements are greatly reduced during hiberna- tion suggesting a decrease in mGPDH activity.
All these biochemical and physiological changes are in general controlled by hormones like thyroid and steroid hormones. Thyroid hormone has a profound effect on cellular respiration; high levels of this hormone accelerate respiration in conjunc- tion with a general increase in metabolism. While low amounts cause low levels of respiration with a general slowing of metabolic activity (Dummler et al., 1996). Concerning steroid hormones, bio- chemical evidence has suggested that glucocorti- coids induced GPDH activity (Kumar et al., 1985). Glucocorticoids are also important for thermoregulatory responses and carbohydrates homeostasis during hypothermia (Steffen and Musacchia, 1985).
This work presents data on: (i) the tissue distri- bution of cGPDH and mGPDH activities in the euthermic J. orientalis; (ii) the effect of hiberna- tion on the activity of these two enzymes; (iii) the effect of PTU and T4 on the activity of cGPDH and mGPDH; and (iv) the effect of one synthetic glucocorticoid (dexamethasone) on the activity of cGPDH and mGPDH in different tissues of jerboa.
2. Material and methods 2 . 1 . Animals
Male adult jerboas (120 – 150 g), 4 – 6 months,
were captured in the subdesert land of eastern
Morocco and transferred to the laboratory in a
preacclimated room (229 2°C). Food was avail- able ad libitum (barleycorn and sunflower seeds), occasionally complemented with fresh lettuce and carrot; and free access to water. The light cycle in the entire experiment was set to 14-h light and 10-h dark (but it cannot be excluded that the dark period may have been interrupted on occasions). Age identification was made on the first day the animals arrived to the labo- ratory according to the body mass, body length and appearance (Aulagnier and Thevenot, 1986).
2 . 2 . Induced and natural hibernation
Hibernation was induced as described (Bourhim et al., 1993; Bourhim et al., 1997).
Jerboas (n =8), were transferred to the cold room and kept at (4 9 1°C) for 3 weeks of accli- mation with food and free access to water. At the end of the three weeks, food was removed and hibernation was soon induced; this group of jerboas is defined as ‘induced hibernating jer- boas’.
Another group of jerboas (n = 4) was left in a room where the temperature varied between (10 – 15°C) with food and free access to water.
This group entered the hibernating state natu- rally (from November to January); they are defined as ‘natural hibernating jerboas’. Rectal temperature of natural and induced hibernating jerboas was 6 – 7°C; heart rate about 10 beats/
min and periods of apnea of about one hour were observed. Jerboas were killed while deeply hibernating.
Control jerboas (n =10) were kept at room temperature (22 9 2°C) with food and free access to water and they are defined as ‘euthermic jerboas’.
2 . 3 . Animal treatment
Hyperthyroidism was provoked by daily in- traperitonial (ip) injection of T4 to euthermic jerboas (n = 10) (15 m g/g of body weight (BW);
Sigma, Paris, France) for 21 days; this treatment led to a body weight loss. Similarly, euthermic jerboas (n =10) were rendered hypothyroid by daily ip injection of 6-n -propyl-2-thiouracil (PTU) (10 m g/g BW; Sigma); in this case, no change in body weight was observed. T4 and PTU were dissolved in 0.1 N NaOH and diluted
to the appropriate concentration in saline (Fraboulet et al., 1996). Another group of jer- boas (n =10) was injected by dexamethasone (10 m g/g BW) for 21 days (Grino et al., 1989); this treatment led to a body weight gain.
Control animals received ip injection of vehicle during the same experimental period. At the end of the treatment, jerboas were killed by decapita- tion and trunk blood was collected. Serum was separated promptly and frozen for subsequent determination of serum free T3 and free T4 lev- els. Tissues (brain, skeletal muscle, brown adipose tissue, liver and kidneys) were quickly removed, placed on ice and frozen until use.
Hypo- and hyperthyroidism were confirmed by serum free T3 and free T4 measurement.
2 . 4 . Serum free T 3 and free T 4
Serum free T3 and free T4 were determined by radioimmunoassay using a commercially avail- able kit (Immunotech, France). Values are ex- pressed in pmol/l.
2 . 5 . Preparation of crude extract
Tissues removed from jerboas were homoge- nized in a Potter – Elvehjem homogenizer with a motor-driven Teflon pestle (10 strokes, the highest speed) in an extract solution containing 0.25 M sucrose, 10 mM HEPES, (pH 7.5) using 6 ml buffer per gram of wet tissue, according to the procedure described by Fleisher et al. with slight modification (Fleisher et al., 1966). For skeletal muscle, we first used the Ultraturax (T25 Janke and Kunkel) followed by a Potter – Elve- hjem homogenizer under the same conditions as described above. The homogenate was cen- trifuged two times for 10 min at 350 g. The supernatant was then centrifuged two times for 5 min at 5590 g. This supernatant was the source of cytosolic GPDH. The pellet was resuspended in sucrose (0.25 M, pH 7) and it was the source of mitochondrial GPDH.
2 . 6 . Protein estimation
The protein concentration in each sample of
the supernatant and the pellet suspension was
determined by the Bradford method (Bradford,
1976) using bovine serum albumin (BSA) as
standard.
2 . 7 . Enzyme assays
2 . 7 . 1 . Cytosolic NADH -dependent GPDH acti6ity cGPDH activity was tested in 20 mM imida- zole buffer (pH 7.6) in a final vol. of 1 ml. The assay medium contained 0.1 mM NADH and 1 mM dihydroxyacetone phosphate (DHAP) (Fluka, Switzerland). Activity was determined with a spectrophotometer at 340 nm. Specific activity was calculated as unit per mg of protein (U/mg of protien). One unit of enzyme activity was defined as the conversion of one m mol sub- strate per minute at 25°C. All assays were done in triplicate.
2 . 7 . 2 . Mitochondrial FAD -dependent GPDH acti 6 ity
For the determination of mGPDH activity, we used the procedure of Garrib and McMurray
with slight modification (Garrib and McMurray, 1984). The mitochondrial pellets were suspended in 0.25 M sucrose, 0.1 M Tris – HCl, and 1 mM EDTA, (pH 7.5). To 10 ml of the suspension, a 0.5 ml aliquot of a 10% aqueous Triton X-100 solution was added. Assay cuvettes contained, in a final vol. of 1 ml: 100 ml a-glycerophosphate (0.3 M, pH 7.5); 100 ml KCN (0.01 M); 150 m l menadione (1 mg/ml stock solution); 300 m l 2-p-iodophenyl-5-phenyltetrazolium chloride (INT) (10 mM); and 350 ml of 0.25 M phosphate buffer (pH 7.5). A blank cuvette had the sub- strate omitted, but was otherwise identical. The reaction was started by the addition of the en- zyme solution (10 – 100 m l of mitochondrial prepa- ration) to both cuvettes. Absorbency at 25°C was determined in each cuvette at 500 nm. The molar absorption coefficient of reduced INT was taken as 11.5 × 10 6 M − 1 cm −1 at 500 nm. Specific activity was calculated as unit per mg of protein.
A unit of activity was the amount that catalyzed the reduction of 1 mmol INT/min under the assay conditions used. All assays were done in tripli- cate.
2 . 8 . Statistical analysis
All results are presented as the mean9 S.E.M.
Statistical significance of differences was deter- mined by one way analysis of variance followed by Fisher’s PLSD test (ANOVA, Statview 512).
3. Results
3 . 1 . Tissue distribution of GPDH acti 6 ity in euthermic jerboa
Fig. 1 shows the cGPDH and mGPDH activi- ties in all the tissues examined and the level of this activity was tissue specific. cGPDH activity far exceeded that of mGPDH. Maximal cGPDH and mGPDH activities were detected in skeletal muscle and brown adipose tissue, respectively. On the other hand, cGPDH activity in brain is lower than skeletal muscle by 21-fold. Concerning mG- PDH, the specific activity varies approximately by 28-fold from a low specific activity in the liver ((5.55 9 0.825)× 10 − 5 U/mg of protein) to a high activity in BAT ((157 925.2) ×10 − 5 U/mg of protein).
Fig. 1. Effect of natural hibernation on cGPDH and mGPDH activities in BAT, skeletal muscle, liver, kidney and brain.
Specific activities (U/mg of protein) in , euthermic jerboas (n=10) and , natural hibernating jerboas (n=4) are shown.
Results are means 9 S.E.M. of three separate experiments. The
asterisk indicates a significant difference from the control
value (P B 0.05).
Fig. 2. Determination of the level of serum free T4 and free T3 in, , euthyroid (n =10); , hyperthyroid (n =10); b , hy- pothyroid (n=10); , natural hibernating (n=4) and 3 , induced hibernating jerboas (n=8). Data are means 9 S.E.M.
Each sample was essayed in triplicate. The asterisk indicates a significant difference from the control value (P B 0.05).
The decrease was about 31-fold for BAT, three- fold to four-fold for kidneys, brain and liver.
3 . 3 . Serum le 6 els of free T 3 and free T 4
T4 injection during 21 days increased serum free T4 by approx. 100% and PTU injection decreased it by 23%. Furthermore, natural and induced hibernation led to a decrease in the con- centration of serum free T4 by 68 and 60%, respectively (Fig. 2). On the other hand, T4 injection increased serum free T3 by 13-fold and PTU injection decreased it by 30%. Natural and induced hibernation decreased the level of serum free T3 by 70 and 60%, respectively (Fig. 2).
3 . 4 . Effect of thyroid hormones on the GPDH acti 6 ity
T4 treatment increased the specific cGPDH activity only in kidneys (approx. 85%). In con- trast, the activity of cGPDH in BAT and liver were decreased by 33 and 38%, respectively. T4 treatment had no effect on skeletal muscle and brain cGPDH enzyme activity. PTU treatment provoked an increase in the activity of cGPDH in BAT by 35%, liver by 20%, and brain by 63%, but a decrease in skeletal muscle by 29%. No effect was detected in kidney (Fig. 3). T4 treat- ment led to an increase in the specific activity of mGPDH in liver by 173%, and a decrease in BAT by 72%. No effect was observed in skeletal mus- cle, brain or kidneys. PTU treatment increased the specific activity of mGPDH in brain, de- creased the specific activity in skeletal muscle by 54%, and had no effect on BAT, liver or kidneys (Fig. 4).
3 . 5 . Dexamethasone
Treatment with dexamethasone caused an in- crease in cGPDH activity in BAT by 35%, brain by 35%, and liver by 17%. Specific activity of mGPDH increased in BAT and brain by 52 and 30%, respectively. The liver mGPDH activity was insensible to dexamethasone treatment.
In contrast, we observed a decrease of cGPDH and mGPDH activities in skeletal muscle by 16 and 35%, respectively. Dexamethasone did not significantly alter mitochondrial and cytosolic GPDH activities in kidneys (Figs. 3 and 4).
3 . 2 . Effect of natural hibernation on GPDH acti 6 ity
Hibernation increased cGPDH enzyme activity in all tissues examined (Fig. 1). The increase observed in comparison with the euthermic jer- boas was about four-fold for liver, three-fold for BAT and brain, and two-fold for skeletal muscle and kidneys.
In contrast, mGPDH activity decreased in all
tissues examined except for the skeletal muscle.
4. Discussion
GPDH is implicated in thermogenesis (Kozak et al., 1991; Koza et al., 1996), gluconeogenesis (Yeh et al., 1995), lipid metabolism (Wise and Green, 1979), and the transfer of reducing equiva- lents to the electron transport chain; those are important factors in hibernation. Our results indi-
cate that cGPDH activity in the euthermic jerboa varies from a low specific activity in the brain to a high specific activity in the skeletal muscle. This result suggests an important role of cGPDH in skeletal muscle and is in agreement with earlier findings (Collier et al., 1976; Lin, 1977). Indeed, it was reported that cGPDH is implicated in energy production (Collier et al., 1976). On the other
Fig. 3. Effect of T4, PTU, and dexamethasone on cGPDH activity in BAT, skeletal muscle, liver, kidney and brain. Specific activities
(U/mg of protein) in , euthyroid; , hyperthyroid; b , hypothyroid jerboas and a , dexamethasone treated jerboas are shown. Data
are means 9 S.E.M., n=10 for each treatment group. Each sample was essayed in triplicate. The asterisk indicates a significant
difference from the control value (P B 0.05).
Fig. 4. Effect of T4, PTU, and dexamethasone on mGPDH activity in BAT, skeletal muscle, liver, kidney and brain. Specific activities (U/mg of protein) in , euthyroid; , hyperthyroid; b , hypothyroid jerboas and, a , dexamethasone treated jerboas are shown. Data are means 9 S.E.M., n=10 for each treatment group. Each sample was essayed in triplicate. The asterisk indicates a significant difference from the control value (P B 0.05).
hand, the highest activity of mGPDH was found in BAT and the lowest in the liver. Recently, it was reported that very high mGPDH activity and mRNA levels were found in rat BAT (Gong et al., 1998). The high mGPDH activity in BAT could be associated to the role of mGPDH and BAT in thermogenesis (Himms-Hagen, 1990; Kozak et al., 1991). Indeed, BAT is the main site of facultative
thermogenesis in small rodents. Furthermore, this
tissue is also traditionally regarded as being of
particular significance in hibernating species, prin-
cipally in relation to arousal from hibernation and
maintenance of the euthermic state in the cold
(Cannon et al., 1996). To understand the effect of
hibernation on cGPDH and mGPDH activities,
we have used natural and induced hibernating
jerboas. Our result indicates that both cGPDH and mGPDH activities were affected by natural hibernation and the same results were found with induced hibernation (data not shown). The cG- PDH activity was increased in all tissues exam- ined in comparison with euthermic jerboas. The high cGPDH activity observed during hibernation is probably due to a high utilization of lipids accumulated during the pre-hibernating period.
This use of lipids produces a high level of glycerol that is immediately phosphorylated by glycerol kinase. The subsequent accumulation of G3P is probably responsible of the induction of cGPDH activity. The increase of cGPDH activity suggests also an enhanced rate of gluconeogenesis via glyc- erol (Yeh et al., 1995). On the other hand, we have observed that mGPDH enzyme activity de- creases in all tissues examined except skeletal muscle. The most dramatic decrease was observed in BAT. We interpret this reduction of mGPDH enzyme activity by the fact that during hiberna- tion, mitochondria are relatively shrunken in comparison with mitochondria isolated from eu- thermic animals. Consequently, this shrinkage of mitochondria probably causes a decrease of the enzyme activity. Indeed, it was reported that the swollen mitochondria from euthermic ground squirrels show high oxidative activity, while the shrunken mitochondria from hibernating animals show low oxidative activity (Brustovetsky et al., 1993).
As reported elsewhere, lipids rather than carbo- hydrates are the principal substrate oxidized in the hibernating state (Willis, 1982). This low glu- cose oxidation might account of the decrease in mGPDH activity resulting from a low capacity of glycerophosphate shuttle during the hibernating state of jerboa. During hibernation of ground squirrels, the activity of many dehydrogenases implicated in the respiration chain was reduced (Fedotcheva et al., 1985). It is well known that profound metabolic changes accompany mam- malian hibernator with cellular respiratory and metabolic activity depression (Wang, 1989). These metabolic changes are in general controlled by many hormones including thyroid hormones (Hudson, 1973; Wang, 1982; Tomasi and Strib- ling, 1996; Tomasi et al., 1998). The role of thy- roid hormones in hibernating animals is controversial; the contradictions in the literature are innumerable and difficult to resolve. Some- times they are likely due to differences between
species (Hudson, 1973), and sometimes to differ- ent methodologies used. Indeed, many investiga- tors contend that thyroid gland is active during hibernation and that thyroidectomy prevents hi- bernation and T4 restores it (Malan and Canguil- hem, 1971; Damassa et al., 1995). Others concluded that hibernation does not occur until the thyroid gland becomes inactive (Hoffman, 1964; Tomasi et al., 1998) and hyperthyroidism delays hibernation in several species (Lachiver, 1969). In the present study, we observed that free T4 and free T3 in serum were depressed in the case of natural and induced hibernation and in hypothyroid jerboas. Our data confirm previous work showing that food restriction and/or physio- logical preparation for hibernation is coincident with depressed plasma concentration of thyroid hormones (Tomasi et al., 1998).
The influence of the thyroid status on mito- chondrial metabolism is widely supported (Op- penheimer et al., 1987; Samuels et al., 1988;
Dummler et al., 1996; Pillar and Seitz, 1997).
Mitochondrial GPDH is considered as a proto-
typical thyroid hormone responsive enzyme in rat
(Lee and Lardy, 1965; Dummler et al., 1996). The
most dramatic thyroid hormone effect occurs in
rat liver, where hyperthyroidism leads to an in-
crease in mGPDH activity and hypothyroidism
results in a decrease to barely detectable levels of
enzyme activity (Lee and Lardy, 1965; Wernette
et al., 1981). In the case of J. orientalis liver, T4
treatment increases mGPDH activity while the
opposite effect was observed in BAT. These re-
sults correspond closely to those reported by oth-
ers, namely that thyroid hormones increased
mGPDH mRNA levels in rat liver but not in
BAT and brain (Dummler et al., 1996; Gong et
al., 1998). Concerning cGPDH, T4 treatment in-
duces an increase of cGPDH activity only in
kidneys. In the case of brain, BAT and liver, an
increase of cGPDH activity was observed in jer-
boas treated by PTU. It was established that by
postnatal day 40, the activity of cGPDH was
significantly depressed in the cerebellum of ge-
netic-hypothyroid mutant mice and the exogenous
thyroid hormone T4 has a stimulatory effect on
mouse cGPDH activity (Sugisaki and Noguchi,
1992). Many hypotheses could be put forward to
explain these variations of cGPDH and mGPDH
activities in response to T4 and PTU treatment in
J. orientalis. The increase of enzymes activities
could be due to an augmentation of the rate of
synthesis of GPDH molecules, an activation of preexisting enzyme or an alteration in its rate of degradation. Thyroid hormone level seems to be a very important factor determining mGPDH activ- ity especially in the liver. The hormone controls the mGPDH activity mainly by enhancing the synthesis of the enzyme (Taylor and Ragan, 1986). Others reported that an increase in mG- PDH activity in response to thyroid hormones is due to induction of mGPDH mRNA after admin- istration of T3 (Dummler et al., 1996). The higher capacity of the glycerophosphate shuttle, resulting from this up-regulation, prevents the accumula- tion of reducing equivalents derived from T3- stimulated glycolysis. Mitochondrial oxidation of these reducing equivalents might account for a considerable part of the increased mGPDH activ- ity and mitochondrial oxygen uptake (Gregory and Berry, 1995). Nevertheless, additive factors like the effect of thyroid status on the physical properties of mitochondrial membranes and on lipid microenvironment of proteins might influ- ence mGPDH activity. This postulate was verified in mithochondrial rat liver (Beleznai et al., 1988).
Therefore, different hormone levels would affect the physical properties of membranes, lipid- protein interaction and consequently the mem- brane-bound enzyme activities.
In regard to the effect of dexamethasone on GPDH enzyme activity, earlier works demon- strated that cGPDH was the first enzyme to be identified as steroid inducible in brain (de Villis and Inglish, 1969; McGinnis and de Villis, 1977).
In addition, it was reported that in the rat glioma C6 cell line, the hydrocortisone increases the level of cGPDH activity (Kumar et al., 1985). It should be noted that the mouse brain cGPDH is not steroid inducible (McGinnis and de Villis, 1977).
The effects of glucocorticoid on cGPDH and mG- PDH activities in brain and BAT of jerboa, are reminiscent of those seen for rat brain (Kumar et al., 1985; Langley-Evans and Nwagwu, 1998).
Furthermore, in the skeletal muscle mGPDH and cGPDH activities are reduced by dexamethasone treatment. Others have reported that rat skeletal muscle cGPDH enzyme activity is not steroid inducible (McGinnis and de Villis, 1977). Several key observations have been made to understand the glucocorticoid regulation of cGPDH induc- tion in rat brain (Kumar et al., 1985). Collec- tively, these observations suggest that glucocor- ticoids mediate the transcription of the cGPDH
gene, accounting for it subsequent induction. The role of glucocorticoids in the metabolism of hiber- nating animals is discussed. Several studies have demonstrated that the level of glucocorticoids de- creases in hibernation and increases during arousal (Boswell et al., 1994). Glucocorticoids are also known to be important in the thermogenesis mechanisms (Steffen and Musacchia, 1985). The hormonal regulation of the GPDH activity is a complicated process, involving not only thyroid hormones and glucocorticoids, but also hormones of other endocrine glands (Lin, 1977).
Future studies with animals in prehibernating, deep hibernating and arousal state will pre- sumably give us further information of the impli- cation of these two enzymes in the mechanism of the induction of hibernation. One way to confirm these results at the molecular level, a necessary task, is to examine the effects of these treatments and hibernation on the gene transcription of mG- PDH and cGPDH in the different tissues used in the present study. These kinds of studies are at present in progress in our laboratory.
Acknowledgements
This work was supported by research grants from the Moroccan national research fund ‘Pro- gramme d’Appui a` la Recherche Scientifique (PARS-CNCPRST)’ and ‘CNR Convention’ at- tributed to the Unite´ de Biochimie. The authors thank Dr M. Kabine, Dr J. Fekkak, Dr M.
Kebbou and A. Moutaouakkil for their help dur- ing the realization of the present work.
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