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Effect of thyroxine and thiourea on cholesterol total lipid and glycogen contents of brain of Singi fish (Heteropneustes fossilis bloch)
Neurochem. Int. Vol. 6, No. 1, pp. 97 101, 1984 Printed in Great Britain. All rights reserved
01974)186/84 $3.00+0.00 Copyright © 1984 Pergamon Press Ltd
EFFECT OF THYROXINE AND THIOUREA ON CHOLESTEROL, TOTAL LIPID AND GLYCOGEN CONTENTS OF BRAIN OF SINGI FISH (HETEROPNEUSTES FOSSILIS BLOCH) RANJIT KUMAR GHOSH and AJIT KUMAR MEDDA Department of Animal Physiology, Bose Institute, Kankurgachi, Calcutta-700054, India
(Receil~ed 31 March 1983; accepted 9 June 1983) Abstract--Three consecutive days injections of thyroxine of different doses (1, 2 and 4#g/g of body weight) caused significant increase in cholesterol content of cerebrum of Singi fish at 25~C in comparison to the control. The cholesterol content of cerebellum, midbrain and medulla oblongata was enhanced significantlywith higher doses of 2 and 4/~g of thyroxine per g of body weight. The lipid and glycogen contents of whole brain were also found to increase with different doses of thyroxine after three consecutive days injections. These cellular constituents decreased in hypothyroid condition induced by thiourea treatment. The results indicate the thyroid hormonal regulation of lipid and carbohydrate metabolism in brain of Singi fish.
In higher vertebrates, the effects of thyroid hormones on the promotion of growth, protein and nucleic acid synthesis and their involvement in other metabolic processes are well known (Tara, 1974; Hoch, 1974; Eberhardt et al., 1980), but the metabolic actions of these hormones in vast number of fishes having different adaptive mechanisms are not so much established (Eales, 1979). However, earlier reports present some evidences on the influences of thyroid hormones on various biological processes, viz protein, nucleic acid, glycogen, lipid and nitrogen metabolism, in some fish (Ray et al., 1975; Ray and Medda, 1976, 1977; Medda and Ray, 1979; Eales, 1979). The cellular changes induced by thyroid hormone treatment are found to be mostly reversed in hypothyroidism or in animals treated with antithyroid drugs (Ray and Medda, 1977; Eales, 1979; Eberhardt et aL, 1980). In lata fish (Ophicephalus punctatus), thiourea treatment produces goitre characterised by complete depletion of colloid from the gland and packed mass of thyroidal epithelial cells mostly of columner type (Ray and Medda, 1972). The fish brain remained mostly unexplored with respect to its responsiveness to thyroid hormone
treatment. Recently it has been reported from our laboratory that thyroid hormone enhances the protein and nucleic acid contents of different parts of brain (Ghosh and Medda, 1982), and also increases the mitochondrial ~-glycerophosphate dehydrogenase (~-GPD) activity, mitochondrial and total protein content of brain of Singi fish (Ghosh et al., in press). In continuation of the investigation on the effects of thyroid hormones on the fish brain, the present study has been undertaken to show the influence of thyroxine and antithyroid drug, viz thiourea, on the cholesterol, total lipid and glycogen metabolism in brain of Singi fish (Heteropneustes fossilis Bloch). EXPERIMENTAL PROCEDURES
Singi fish (Heteropneustes fossilis Bloch), body weight 8 _ 2 g, length 10-12 cm, were purchased from the local supplier and acclimated in laboratory conditions at 25°C for about a week before experiments. They were fed ad libitum with Tubifex tubifex during acclimatized period. The animals were distributed randomly in different groups. Each fish was kept in 1 1. of tap water in a glass jar at 25°C. The medium was changed daily. L-thyroxine-Na-pentahydrate (T4) obtained from Sigma Chemical Company, U.S.A., was dissolved with a minimum amount of
Address ./or correspondence: Professor A. K. Medda, Department of Animal Physiology, Bose Institute, P-l/12 CIT Scheme VII M, Kankurgachi, Calcutta 700054, India. 97 NC I. 6/I (3
98
RANJIT KUMAR GHOSH and AJIT KUMAR MEDI)A
0.1 M N a O H a n d the required volume was made by 0.65'!~o saline. Various doses (1, 2 and 4/~g/g body weight) of thyroxine were injected intraperitoneally for three consecutive days. The volume injected did not exceed 40/~1 per animal. The same volume of alkaline 0.65'Yo saline was injected into the control animals. The fishes were kept in fasting condition during the experimental period. F o r the t r e a t m e n t with thiourea, each fish was immersed in 1 I. of thiourea (E. Merck AG. D a r m s t a d t ) - c o n t a i n i n g medium (1 mg/ml) for 45 days. C o r r e s p o n d i n g control fishes were kept in the same a m o u n t of tap water. The fishes were fed ad libitum with Tub([ex tubijex during the period of thiourea treatment. The T 4 injected animals along with their controls were killed on the 7th day after the first injection. The thiourea treated and the corresponding control animals were sacrificed just after 45 days of treatment. The brain from each animal was excised and the different parts of brain, viz cerebrum, cerebellum, m i d b r a i n and medulla oblongata, were quickly dissected out, weighed and taken for the estimation o f cholesterol, total lipid and glycogen. F o r the determination of total lipid and glycogen, the whole brain from each fish was used in order to get a detectable a m o u n t of these cellular constituents. The lipid content of brain was estimated by the m e t h o d o f Folch et al. (1957), and glycogen by the m e t h o d of Carrol et al. (1956). Cholesterol was determined according to the m e t h o d of King and W o t t o n (1956). Each of the treated and control groups contained 20 animals. All data were statistically analyzed by using Student's t-test. Values of P < 0.05 were taken as significant. RESULTS
Effect o/T4 on the cholesterol content o f different parts o/" brain (Table 1) Cerebrum. T 4 of different doses (1, 2 a n d 4/~ g/g, 3
consecutive days injections) caused significant increase in cholesterol content of cerebrum of Singi fish in comparison to the control. A l t h o u g h the cholesterol content tended to increase more with 2 itg of T 4 per g, there were no statistical differences in results with the different doses used. Cerebellum. In comparison to the control, the cholesterol content of cerebellum increased to a b o u t the same level with higher doses of T4 (2 or 4 Itg/g). T4 at the dose of 1/~g/g failed to alter the cholesterol content of this region of brain. Midbrain. The cholesterol content of m i d b r a i n tended to increase with l l~g of T 4 per g, but the difference was not significant in comparison to the control. The higher doses of T4 (2 and 4/zg/g) significantly increased the cholesterol content of midbrain to a b o u t the same level. Medulla oblongata. The changes in the cholesterol content of medulla o b l o n g a t a in T4 injected fish were of a b o u t the same nature as occurred in midbrain. A significant increase in cholesterol content was found with 2 and 4 # g of T4 per g, while the lower dose of 1 /Lg/g did not cause any significant change in comparison to the control.
E[:[~'ct ~)[ 74 on total lipid and glycogen contents q/ brain ( Tabh' 1) The respective different parts of brain, viz cerebrum, cerebellum, m i d b r a i n and medulla oblongata. contained lipid and glycogen too small in a m o u n t to be detectable. But when the whole brain was taken, both lipid and glycogen contents were found to be enhanced after three consecutive days injections of T4 (1, 2 a n d 4 ~ g). M a x i m u m increase in lipid content of the whole brain occurred with 2 p g of T 4 per g, while the other two doses (1 a n d 4/~g/g) caused a b o u t the same level of increase but less than that produced by 2 #g/g. In case of glycogen, all doses of T 4 elevated the level to a b o u t the same extent.
Table I. Effect of thyroxine (1, 2 and 4,ug/g, 3 consecutive days rejections) on cholesterol content of different parts of brain (cerebrum, cerebellum, midbrain and medulla oblongata) and total lipid and glycogen contents of whole brain o f Singi fish (Heteropneustes /bssilis Btoch) Dose of T~ (#g/g) Control 1 2 4
Cholesterol ( ~ g / m g tissue) Mean ± SE Cerebrum 12.3+0.5 14.7+0.9 ~ 15.6 + 0.9 b 14.4_+0.7 ~
Cerebellum 18.6_+0.2 17.5_+0.9 2 0 . 7 + 0 . 9 ~A 20.1 ± 0.& A
Midbrain 18.7_+0.4 19.5_+0.8 20.1 + 0 . 4 ~ 21.0+0.5 ~
Medulla oblongata 32.5_+0.7 34.5+1.5 36.8-+ 1.5 ~' 37.3_+2.0 ~'
Whole brain lipid ( m g / 1 0 0 m g tissue) Mean _+ SE 8.68_+0.32 13.06 _+ 0.39 b 14.09 _+ 0.25 bA 12.85 +_ O. lS b/~
Whole brain glycogen (/~g/100mg tissue) Mean _+ SE 175.1 242.2 291.0 283.9
+ 19.8 _+ 16.1 ~ + 19.7 b _+ 28.5 b
SE - Standard error, t-test probability differences: P value: aA - P < 0.05, b,B - P < 0.01, where lower case letters denote comparison between the control and 1, 2 or 4 pg/g; capital letter between 1 and 2 or 4 l~g/g and Greek letter between 2 and 4,ug/g. The fishes were kept at 25 C in fasting condition and sacrificed on the 7th day after the first injection (the day of injection was taken as zero day). Each group (treated or control) consisted of 20 animals.
Thyroid hormone-induced changes in brain lipid and glycogen
99
Table2. Effectof thioureaon the cholesterolcontentof differentpartsof brain(cerebrum,cerebellum,midbrainand medulla oblongata)and total lipidand glycogencontentsof wholebrain of Singifish (Heteropneustesfossilis Bloch) Cholesterol (,ug/mgtissue) Mean ± SE Whole brain lipid Wholebrain glycogen (mg/100mg tissue) (/~g/100mgtissue) Cerebrum Cerebellum Midbrain Medullaoblongata Mean _ SE Mean + SE Control 14.3+_0.5 18.6_+0.1 20.1 + 0 . 4 33.5+-0.5 8.86_+0.32 246.6_+ 17.2 Thiourea 11.6_+0.8" 17.5_+0.5" 17.6-+0.5t 28.3 -+ 1 . 0 t 7.53_+0.43* 194.4_+10.5" SE = Standard error, t-test probabilitydifferences:P value: * = P < 0.05, t = P < 0.01. The fisheswere immersedin thiourea-containingmedium(1 mg/ml)for 45 days at 25°Cand fed. Each group (treated or control) consistedof 20 animals.
Effect o f thiourea on cholesterol, total lipid and glycogen contents o f brain (Table 2) Thiourea treatment by immersion for 45 days caused a decrease in cholesterol content of different substructures (cerebrum, cerebellum, midbrain and medulla oblongata) of brain. The total lipid and glycogen contents of whole brain were also reduced after 45 days of thiourea treatment (Table 2). DISCUSSION As a sequal of the investigations on the thyroid hormone-induced responsiveness of the fish brain with regard to the changes in protein and nucleic acid metabolism (Ghosh and Medda, 1982) and specific enzyme activity (Ghosh et aL, in press), the present experiments were undertaken to provide evidences on the effects of thyroxine and an antithyroid drug, thiourea, on the alteration in lipid and carbohydrate metabolism in brain of Singi fish, since it is known that thyroid hormones have marked controlling influence both on the synthesis and breakdown of these two cellular constituents (Hoch, 1974; Ray et al., 1975; Eberhardt et al., 1980). The amounts of cholesterol in different parts of brain (cerebrum, cerebellum, midbrain and medulla oblongata) and total lipid and glycogen of whole brain were determined because the net amount under experimental conditions was thought to represent the sum total effect of both synthesis and degradation. It is to be pointed out that the amount of lipid and glycogen in cerebrum, cerebellum, midbrain or medulla oblongata was very small that could not be detected. For this reason, whole brain was taken to show the changes in total lipid and glycogen contents after T4 injections. The cholesterol content of cerebrum, cerebellum, midbrain and medulla oblongata and the total lipid and glycogen contents of the whole brain increased after T4 injections, while thiourea treatment decreased the amounts of these cellular constituents in the brain of Singi fish. The enhancement in choles-
terol, total lipid or glycogen content may be due to increased synthesis and decreased degradation, and the reduction in the amount of these substances is naturally supposed to be due to the opposite changes. In thiourea treated fish, the decrease in cholesterol, total lipid and glycogen contents of brain is likely to be due to thyroid hormone deficiency, as suggested by the production of goitrous gland characterised by packed mass of thyroidal epithelial cells with little or no colloid in the gland of fish (Ray and Medda, 1972). The various literatures, both on in vitro and in vivo studies, show that the nature of thyroid hormoneinduced changes in lipid or cholesterol metabolism depends on the dose of the hormone, substrate used for in vitro experiments, organs and species of the experimental animals (Strand 1963; Eskelson et al., 1970; Tata, 1965; Liu and Lee, 1972; Raheja and Snedecor, 1971; Thapliyal et al., 1975; Sokoloff and Kennedy, 1973). Variable results have also been obtained with fish (Eales, 1979). The lipid content of liver of Lata fish undergoes a biphasic nature of changes after a single injection of low (0.5, 1 and 2/1g/g) and high (4#g/g) doses of T 4 (Medda and Ray, in press). But the more pertinent findings with our results are the stimulation of the process of myelination by thyroid hormones (Eayrs, 1964; Hamburgh, 1966) and a positive indication of the thyroid hormonal role in the formation of a number of lipids that are constituents of myelin (Sokoloff and Kennedy, 1973). It has been further reported that neonatal thyroidectomy retards myelination associated with the reduced synthesis of cholesterol, cerebrosides and sulphatides (Cuaron et al., 1963; Walravens and Chase, 1969). The synthesis of saturated fatty acids and also total fatty acids in brain microsomes of new born rat are enhanced after daily injection of 60/~g of triiodothyronine (T3) per 100 g of body weight (Grippo and Menkes, 1971). But the mitochondrial fraction does not show any increase in synthesis of saturated and unsaturated fatty acids with T 3. Moreover, the fatty acid synthesis is reduced
100
RANJIT KUMAR GHOSH a n d A n T KUMAR MEDDA
in the mitochondrial and particularly in the microsomal fraction in animals made hypothyroid at birth (Grippo and Menkes, 1971). Although all these aspects have not been studied in Singi fish, it may be presumed that thyroid hormones have profound influence on the lipid metabolism, particularly on the synthesis in fish brain. The carbohydrate metabolism including glucose transport, glycolysis and glycogen synthesis is markedly influenced by thyroid hormones (Eberhardt et al., 1980). It is also known that thyroid hormones influence glycogen synthesis in a biphasic pattern in rat (Wertheimer and Bentor, 1953; Eberhardt et al., 1980). The biphasic nature of thyroid hormone action on the glycogen content of fish liver i.e. increase with lower doses (0.5-2 #g T4/g) and decrease with higher dose (4 ~g T4/g), has also been reported (Ray et al., 1975). In spotted Munia (Lonchura punctulata), however, 30 days injections of total doses of 0.05 rag, 0.1 mg and 0.2 mg of T4 decreased the hepatic glycogen content (Thapliyal et al., 1975). No biphasic response with respect to glycogen content (and lipid content also) after T 4 injections (1-4 #g/g) was found in Singi fish brain. All these doses of T 4 significantly increased the glycogen content of brain. It may be assumed that increased synthesis of glycogen and thereby the enhancement of glycogen content of brain took place with the doses of T4 used. This was supported by the fact that there was significant reduction in glycogen content of whole brain of thiourea treated hypothyroid fish. Our results do not support that thyroid hormones have no effect on brain glycogen of fish (see Review by Eales, 1979). The activity of glycogen synthetase-a in rat liver is increased by T~ injections at the dose of 30 #g/100 g/day for 4 days (Taningher et al., 1974). The changes in the activities in glucose6-phosphate dehydrogenase, 6-phosphogtuconate dehydrogenase, D-fl-hydroxybutyrate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase and also a number of other enzymes in developing rat brain in altered thyroid hormonal state (Sokoloff and Kennedy, 1973; Eberhardt et al., 1980) also indicate the involvement of the hormone, directly or indirectly, in the metabolism of carbohydrate, lipid and protein in the nervous system. |t may be that similar mechanisms in fish brain are also under the control of thyroid hormone. It may be questioned that the doses of T 4 (1-4/~g/g) used for the present experiments in addition to the endogenous level are very high. Whatever may be the total amount of the exogenous and endogenous hormone available for physiological actions, as evidenced in course of our investigations, it
is true that no side toxic effect of thyroid hormone was observed and there was no change in body weight of the Singi fish after injections of T~ (1 4 #g/g). The fish kept at 2 5 C requires higher dose of T 4 for manifestations of some physiological actions (Ray and Medda, 1975, 1976; Medda and Ray, 1979) and these effects cannot be comparable to mammals whose body temperature is much higher than fish. Thyroxine doses of 0.01 and 0.1 #g/g injected for 5 consecutive days have failed to cause any change in liver of Lata fish (Paul and Medda. 1983). We have also observed that T 4 dose of 0.25 ~ g/g (3 consecutive days injections) is ineffective in producing any change in Singi fish brain (unpublished observation). Although the endogenous T 4 level of Singi fish is not known, it has been reported that some teleosts have T 4 concentrations in the range of 8.4 17.0 ng/ml or more depending on the season of the year (Dickhofl et al., 1978). Other reports indicate T~ o r T 4 level of about 500 ngdl ~ or more (see Review by Eales, 1979). Moreover, it has been reported that due to binding to plasma proteins about 50~, of the total endogenous T4 or T~ in fish is free (Falkner and Eales, 1973). The nuclear T~-binding affinity as well as capacity in some fish liver is much less than that measured with rat liver nuclei (Darling et al., 1982). The T4 T~ conversion takes place in fish (Eales, 1979) and other T4 degradation products, viz diiodothyronine (Osborn and Simpson, 1969) and monoiodothyronine (Eales. 1972) have also been found in the plasma of teleosts. Moreover, there should be a preferential distribution of free thyroid hormones in different organs of fish according to the number or capacity of the binding sites. It is known that T3-binding to fish brain nuclei is less than to nuclei from fish liver (Darling et aL, 1982). Naturally, it is reasonable to assume that the whole injected T4 will not be available to Singi fish brain to elicit the changes as observed during the present investigations. Thus, the changes in protein, RNA and DNA contents (Ghosh and Medda, 1982), :¢-GPD activity (Ghosh et al., in press) and the present results with respect to increase in cholesterol, total lipid and glycogen contents are physiological effects and these are clear indications of the thyroid hormone-induced metabolic alterations in fish brain.
REFERENCES
Carrol N. V., Longley R. W. and Roe J. H. (1956) The determination of glycogen in liver and muscle by use of anthrone reagent. J, bioL Chem. 220, 583-593.
Thyroid hormone-induced changes in brain lipid and glycogen Cuaron A., Gamble J., Myant N. B. and Osorio C. (1963) The effect of thyroid deficiency on the growth of the brain and on the deposition of brain phospholipids in foetal and newborn rabbits. J. Physiol. 168, 613-630. Darling D. S., Dickhoff W. W. and Gorbman A. (1982) Comparison of thyroid hormone binding to hepatic nuclei of the rat and a teleost (Oncorhynchus kisutch). Endocrinology 111, 1936--1943. Dickhoff W. W., Folmar L. C. and Gorbman A. (1978) Changes in plasma thyroxine during smoltification of coho salmon ( Oncorhynchus kisutch ). Gen. Comp. Endocr. 36, 229-232. Eales J. G. (1972) Radiothyroxine metabolism in several freshwater teleost fishes. Can. J. Zool. 50, 623-631. Eales J. G. (1979) Thyroid functions in cyclostomes and fishes. In: Hormones and Evolution (Barrington E. J. W., ed.). Vol. 1, pp. 341-436. Academic Press, London. Eayrs J. T. (1964) Endocrine influence on cerebral development. Arch. Biol. 75, 529-565. Eberhardt N. L., Apriletti J. W. and Baxter J. D. (1980) The molecular biology of thyroid hormone action. In: Biochemical Actions of Hormones (Litwack G., ed.). Vol. 7, pp. 311-394. Academic Press, New York. Eskelson C. D., Cazee C. R., Anthony W., Towne J. C. and Walske B. R. (1970) In vitro inhibition of cholesterolgenesis by various thyroid hormone analogs. J. reed. Chem. 13, 215-220. Falkner N. W. and Eales J. G. (1973) Investigation of iodothyronine binding to plasma proteins in brook trout, Sah,elinusfontinalis, using precipitation, dialysis and eleco trophoretic methods. J. Fish. Res. Bd. Can. 30, 1131 1140. Folch J., Lees M. and Stanley G. H. S. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497 509. Ghosh R. K. and Medda A. K. (1982) Effect of thyroxine on protein and nucleic acid contents of different parts of brain of Singi fish (Heteropneustes fossilis Bloch). Endokrinologie 79, 355-361. Ghosh R. K., Ghosh N., De S., Ray A. K. and Medda A. K. (1983) Effect of L-triiodothyronine on the mitochondrial ct-glycerophosphate dehydrogenase activity, mitochondrial and total protein contents of brain of singi fish (Heteropneustes fossilis Bloch). Neurochem. Int. 5, 635-640. Grippo J. and Menkes J. H. (1971 ) Effect of thyroid on fatty acid biosynthesis in brain. Pediat. Res. 5, 466-471. Hamburgh M. (1966) Evidence for a direct effect of temperature and thyroid hormone on myelinogenesis in vitro. Devl. Biol. 13, 15-30. Hoch F. L. (1974) Metabolic effects of thyroid hormone. In Handbook of Physiology (Greer M. A. and Solomon D. H., eds.). Sect. 7, Vol. 3, pp. 391-412. American Physiological Society, Washington, D.C. King E. J. and Wotton I. D. P. (1956) In: Microanalysis in Medical Biochemistry, 3rd Edn. (Churchill J. and Churchill A., eds) pp. 42. London. Liu C-Y. and Lee Y-P (1972) A comparison of the effect of L-triiodothyronine on the plasma lipid and cholesterol concentration of the rat, chicken and guinea pig. Endocrinology 90, 830-834. Medda A. K. and Ray A. K. (1979) Effect of thyroxine and
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analogs on protein and nucleic acid contents of liver and muscle of lata fish (Ophicephalus punctatus). Gen. Comp. Endocr. 37, 74-80. Medda A. K. and Ray A. K. (1983) Thyroxine-induced changes in lipid content of liver of fish and amphibia. Indian J. Physiol. all. Sci. In press. Osborn R. H. and Simpson T. H. (1969) Thyroxine metabolism in the plaice, Pleuronectes platessa L. Gen. Comp. Endocr. 13, 524. Paul A. K. and Medda A. K. (1983) Comparative study on the effects of thyroxine on protein, RNA and DNA contents of liver of different vertebrates at different stages of life. Morph. Jb. Leipzig 129, 240-256. Raheja K. L. and Snedecor J. G. (1971) Some effects of single doses of triiodothyronine and thyroxine in hypothyroid chick. Gen. Comp. Endocr. 16, 97-104. Ray A. K. and Medda A. K. (1972) Production of goitre in lata fish (Ophicephalus punctatus). Indian J. Physiol. all. Sci. 26, 83-86. Ray A. K., Bhattacharjee S. S. and Medda A. K. (1975) Histochemical studies on the effect of thyroid hormone on the glycogen content of liver of fish and amphibia. Indian. J. Physiol. all. Sci. 29, 121-126. Ray A. K. and Medda A. K. (1976) Effect of thyroid hormones and analogues on ammonia and urea excretion in lata fish (Ophicephalus punctatus ). Gen. Comp. Endocr. 29, 190-197. Ray A. K. and Medda A. K. (1977) Changes in nitrogen and nucleic acid metabolism in lata fish (Ophicephalus punctatus) after thiourea and thyroxine treatments by immersion. Indian J. Physiol. all. Sci. 31, 58-65. Sokoloff L. and Kennedy C. (1973) The action of thyroid hormones and their influence on brain development and function. In: Biology of Brain Dysfunction (Gaull G. E., ed.). Vol. 2, pp. 295-332. Plenum Press. Strand O. (1963). Effects of D- and L-triiodothyronine and of propylthiouracil on the production of bile acids in the rat. J. Lipid Res. 4, 305-311. Taninger M., Gallo G., Fugassa E. and Pranzetti P. (1974) Activation of hepatic glycogen synthetase in rats treated with thyroid hormone. Boll. Soc. ital. Biol. sper. 50, 1911-1917. Tata J. R. (1965) Biological action of thyroid hormones at the cellular and molecular levels. In: Action of Hormones on Molecular Processes (Litwack G. and Kritchevsky D., eds.), pp. 55-131. Wiley, London. Tata J. R. (1974) Growth and developmental action of thyroid hormones at the cellular level. In: Handbook of Physiology (Greer M. A. and Solomon D. H., eds.). Sect. 7, Vol. 3, pp. 469-478. American Physiological Society, Washington, D.C. Thapliyal J. P., Garg R. K., Murty G. S. R. C. and Gupta S. C. (1975) Effects of L-thyroxine on the intermediary metabolism of the Spotted Munia, Lonchura punctulata. Gen. Comp. Endocr. 25, 31-35. Walravens P. and Chase H. P. (1969) Influence of thyroid on formation of myelin lipids. J. Neurochem. 16, 1477 1484. Wertheimer E. and Bentor V. (1953) Metabolic changes in the rat diaphragm during heat regulation, as a thyroxine effect. Metabolism 2, 536-545.
01974)186/84 $3.00+0.00 Copyright © 1984 Pergamon Press Ltd
EFFECT OF THYROXINE AND THIOUREA ON CHOLESTEROL, TOTAL LIPID AND GLYCOGEN CONTENTS OF BRAIN OF SINGI FISH (HETEROPNEUSTES FOSSILIS BLOCH) RANJIT KUMAR GHOSH and AJIT KUMAR MEDDA Department of Animal Physiology, Bose Institute, Kankurgachi, Calcutta-700054, India
(Receil~ed 31 March 1983; accepted 9 June 1983) Abstract--Three consecutive days injections of thyroxine of different doses (1, 2 and 4#g/g of body weight) caused significant increase in cholesterol content of cerebrum of Singi fish at 25~C in comparison to the control. The cholesterol content of cerebellum, midbrain and medulla oblongata was enhanced significantlywith higher doses of 2 and 4/~g of thyroxine per g of body weight. The lipid and glycogen contents of whole brain were also found to increase with different doses of thyroxine after three consecutive days injections. These cellular constituents decreased in hypothyroid condition induced by thiourea treatment. The results indicate the thyroid hormonal regulation of lipid and carbohydrate metabolism in brain of Singi fish.
In higher vertebrates, the effects of thyroid hormones on the promotion of growth, protein and nucleic acid synthesis and their involvement in other metabolic processes are well known (Tara, 1974; Hoch, 1974; Eberhardt et al., 1980), but the metabolic actions of these hormones in vast number of fishes having different adaptive mechanisms are not so much established (Eales, 1979). However, earlier reports present some evidences on the influences of thyroid hormones on various biological processes, viz protein, nucleic acid, glycogen, lipid and nitrogen metabolism, in some fish (Ray et al., 1975; Ray and Medda, 1976, 1977; Medda and Ray, 1979; Eales, 1979). The cellular changes induced by thyroid hormone treatment are found to be mostly reversed in hypothyroidism or in animals treated with antithyroid drugs (Ray and Medda, 1977; Eales, 1979; Eberhardt et aL, 1980). In lata fish (Ophicephalus punctatus), thiourea treatment produces goitre characterised by complete depletion of colloid from the gland and packed mass of thyroidal epithelial cells mostly of columner type (Ray and Medda, 1972). The fish brain remained mostly unexplored with respect to its responsiveness to thyroid hormone
treatment. Recently it has been reported from our laboratory that thyroid hormone enhances the protein and nucleic acid contents of different parts of brain (Ghosh and Medda, 1982), and also increases the mitochondrial ~-glycerophosphate dehydrogenase (~-GPD) activity, mitochondrial and total protein content of brain of Singi fish (Ghosh et al., in press). In continuation of the investigation on the effects of thyroid hormones on the fish brain, the present study has been undertaken to show the influence of thyroxine and antithyroid drug, viz thiourea, on the cholesterol, total lipid and glycogen metabolism in brain of Singi fish (Heteropneustes fossilis Bloch). EXPERIMENTAL PROCEDURES
Singi fish (Heteropneustes fossilis Bloch), body weight 8 _ 2 g, length 10-12 cm, were purchased from the local supplier and acclimated in laboratory conditions at 25°C for about a week before experiments. They were fed ad libitum with Tubifex tubifex during acclimatized period. The animals were distributed randomly in different groups. Each fish was kept in 1 1. of tap water in a glass jar at 25°C. The medium was changed daily. L-thyroxine-Na-pentahydrate (T4) obtained from Sigma Chemical Company, U.S.A., was dissolved with a minimum amount of
Address ./or correspondence: Professor A. K. Medda, Department of Animal Physiology, Bose Institute, P-l/12 CIT Scheme VII M, Kankurgachi, Calcutta 700054, India. 97 NC I. 6/I (3
98
RANJIT KUMAR GHOSH and AJIT KUMAR MEDI)A
0.1 M N a O H a n d the required volume was made by 0.65'!~o saline. Various doses (1, 2 and 4/~g/g body weight) of thyroxine were injected intraperitoneally for three consecutive days. The volume injected did not exceed 40/~1 per animal. The same volume of alkaline 0.65'Yo saline was injected into the control animals. The fishes were kept in fasting condition during the experimental period. F o r the t r e a t m e n t with thiourea, each fish was immersed in 1 I. of thiourea (E. Merck AG. D a r m s t a d t ) - c o n t a i n i n g medium (1 mg/ml) for 45 days. C o r r e s p o n d i n g control fishes were kept in the same a m o u n t of tap water. The fishes were fed ad libitum with Tub([ex tubijex during the period of thiourea treatment. The T 4 injected animals along with their controls were killed on the 7th day after the first injection. The thiourea treated and the corresponding control animals were sacrificed just after 45 days of treatment. The brain from each animal was excised and the different parts of brain, viz cerebrum, cerebellum, m i d b r a i n and medulla oblongata, were quickly dissected out, weighed and taken for the estimation o f cholesterol, total lipid and glycogen. F o r the determination of total lipid and glycogen, the whole brain from each fish was used in order to get a detectable a m o u n t of these cellular constituents. The lipid content of brain was estimated by the m e t h o d o f Folch et al. (1957), and glycogen by the m e t h o d of Carrol et al. (1956). Cholesterol was determined according to the m e t h o d of King and W o t t o n (1956). Each of the treated and control groups contained 20 animals. All data were statistically analyzed by using Student's t-test. Values of P < 0.05 were taken as significant. RESULTS
Effect o/T4 on the cholesterol content o f different parts o/" brain (Table 1) Cerebrum. T 4 of different doses (1, 2 a n d 4/~ g/g, 3
consecutive days injections) caused significant increase in cholesterol content of cerebrum of Singi fish in comparison to the control. A l t h o u g h the cholesterol content tended to increase more with 2 itg of T 4 per g, there were no statistical differences in results with the different doses used. Cerebellum. In comparison to the control, the cholesterol content of cerebellum increased to a b o u t the same level with higher doses of T4 (2 or 4 Itg/g). T4 at the dose of 1/~g/g failed to alter the cholesterol content of this region of brain. Midbrain. The cholesterol content of m i d b r a i n tended to increase with l l~g of T 4 per g, but the difference was not significant in comparison to the control. The higher doses of T4 (2 and 4/zg/g) significantly increased the cholesterol content of midbrain to a b o u t the same level. Medulla oblongata. The changes in the cholesterol content of medulla o b l o n g a t a in T4 injected fish were of a b o u t the same nature as occurred in midbrain. A significant increase in cholesterol content was found with 2 and 4 # g of T4 per g, while the lower dose of 1 /Lg/g did not cause any significant change in comparison to the control.
E[:[~'ct ~)[ 74 on total lipid and glycogen contents q/ brain ( Tabh' 1) The respective different parts of brain, viz cerebrum, cerebellum, m i d b r a i n and medulla oblongata. contained lipid and glycogen too small in a m o u n t to be detectable. But when the whole brain was taken, both lipid and glycogen contents were found to be enhanced after three consecutive days injections of T4 (1, 2 a n d 4 ~ g). M a x i m u m increase in lipid content of the whole brain occurred with 2 p g of T 4 per g, while the other two doses (1 a n d 4/~g/g) caused a b o u t the same level of increase but less than that produced by 2 #g/g. In case of glycogen, all doses of T 4 elevated the level to a b o u t the same extent.
Table I. Effect of thyroxine (1, 2 and 4,ug/g, 3 consecutive days rejections) on cholesterol content of different parts of brain (cerebrum, cerebellum, midbrain and medulla oblongata) and total lipid and glycogen contents of whole brain o f Singi fish (Heteropneustes /bssilis Btoch) Dose of T~ (#g/g) Control 1 2 4
Cholesterol ( ~ g / m g tissue) Mean ± SE Cerebrum 12.3+0.5 14.7+0.9 ~ 15.6 + 0.9 b 14.4_+0.7 ~
Cerebellum 18.6_+0.2 17.5_+0.9 2 0 . 7 + 0 . 9 ~A 20.1 ± 0.& A
Midbrain 18.7_+0.4 19.5_+0.8 20.1 + 0 . 4 ~ 21.0+0.5 ~
Medulla oblongata 32.5_+0.7 34.5+1.5 36.8-+ 1.5 ~' 37.3_+2.0 ~'
Whole brain lipid ( m g / 1 0 0 m g tissue) Mean _+ SE 8.68_+0.32 13.06 _+ 0.39 b 14.09 _+ 0.25 bA 12.85 +_ O. lS b/~
Whole brain glycogen (/~g/100mg tissue) Mean _+ SE 175.1 242.2 291.0 283.9
+ 19.8 _+ 16.1 ~ + 19.7 b _+ 28.5 b
SE - Standard error, t-test probability differences: P value: aA - P < 0.05, b,B - P < 0.01, where lower case letters denote comparison between the control and 1, 2 or 4 pg/g; capital letter between 1 and 2 or 4 l~g/g and Greek letter between 2 and 4,ug/g. The fishes were kept at 25 C in fasting condition and sacrificed on the 7th day after the first injection (the day of injection was taken as zero day). Each group (treated or control) consisted of 20 animals.
Thyroid hormone-induced changes in brain lipid and glycogen
99
Table2. Effectof thioureaon the cholesterolcontentof differentpartsof brain(cerebrum,cerebellum,midbrainand medulla oblongata)and total lipidand glycogencontentsof wholebrain of Singifish (Heteropneustesfossilis Bloch) Cholesterol (,ug/mgtissue) Mean ± SE Whole brain lipid Wholebrain glycogen (mg/100mg tissue) (/~g/100mgtissue) Cerebrum Cerebellum Midbrain Medullaoblongata Mean _ SE Mean + SE Control 14.3+_0.5 18.6_+0.1 20.1 + 0 . 4 33.5+-0.5 8.86_+0.32 246.6_+ 17.2 Thiourea 11.6_+0.8" 17.5_+0.5" 17.6-+0.5t 28.3 -+ 1 . 0 t 7.53_+0.43* 194.4_+10.5" SE = Standard error, t-test probabilitydifferences:P value: * = P < 0.05, t = P < 0.01. The fisheswere immersedin thiourea-containingmedium(1 mg/ml)for 45 days at 25°Cand fed. Each group (treated or control) consistedof 20 animals.
Effect o f thiourea on cholesterol, total lipid and glycogen contents o f brain (Table 2) Thiourea treatment by immersion for 45 days caused a decrease in cholesterol content of different substructures (cerebrum, cerebellum, midbrain and medulla oblongata) of brain. The total lipid and glycogen contents of whole brain were also reduced after 45 days of thiourea treatment (Table 2). DISCUSSION As a sequal of the investigations on the thyroid hormone-induced responsiveness of the fish brain with regard to the changes in protein and nucleic acid metabolism (Ghosh and Medda, 1982) and specific enzyme activity (Ghosh et aL, in press), the present experiments were undertaken to provide evidences on the effects of thyroxine and an antithyroid drug, thiourea, on the alteration in lipid and carbohydrate metabolism in brain of Singi fish, since it is known that thyroid hormones have marked controlling influence both on the synthesis and breakdown of these two cellular constituents (Hoch, 1974; Ray et al., 1975; Eberhardt et al., 1980). The amounts of cholesterol in different parts of brain (cerebrum, cerebellum, midbrain and medulla oblongata) and total lipid and glycogen of whole brain were determined because the net amount under experimental conditions was thought to represent the sum total effect of both synthesis and degradation. It is to be pointed out that the amount of lipid and glycogen in cerebrum, cerebellum, midbrain or medulla oblongata was very small that could not be detected. For this reason, whole brain was taken to show the changes in total lipid and glycogen contents after T4 injections. The cholesterol content of cerebrum, cerebellum, midbrain and medulla oblongata and the total lipid and glycogen contents of the whole brain increased after T4 injections, while thiourea treatment decreased the amounts of these cellular constituents in the brain of Singi fish. The enhancement in choles-
terol, total lipid or glycogen content may be due to increased synthesis and decreased degradation, and the reduction in the amount of these substances is naturally supposed to be due to the opposite changes. In thiourea treated fish, the decrease in cholesterol, total lipid and glycogen contents of brain is likely to be due to thyroid hormone deficiency, as suggested by the production of goitrous gland characterised by packed mass of thyroidal epithelial cells with little or no colloid in the gland of fish (Ray and Medda, 1972). The various literatures, both on in vitro and in vivo studies, show that the nature of thyroid hormoneinduced changes in lipid or cholesterol metabolism depends on the dose of the hormone, substrate used for in vitro experiments, organs and species of the experimental animals (Strand 1963; Eskelson et al., 1970; Tata, 1965; Liu and Lee, 1972; Raheja and Snedecor, 1971; Thapliyal et al., 1975; Sokoloff and Kennedy, 1973). Variable results have also been obtained with fish (Eales, 1979). The lipid content of liver of Lata fish undergoes a biphasic nature of changes after a single injection of low (0.5, 1 and 2/1g/g) and high (4#g/g) doses of T 4 (Medda and Ray, in press). But the more pertinent findings with our results are the stimulation of the process of myelination by thyroid hormones (Eayrs, 1964; Hamburgh, 1966) and a positive indication of the thyroid hormonal role in the formation of a number of lipids that are constituents of myelin (Sokoloff and Kennedy, 1973). It has been further reported that neonatal thyroidectomy retards myelination associated with the reduced synthesis of cholesterol, cerebrosides and sulphatides (Cuaron et al., 1963; Walravens and Chase, 1969). The synthesis of saturated fatty acids and also total fatty acids in brain microsomes of new born rat are enhanced after daily injection of 60/~g of triiodothyronine (T3) per 100 g of body weight (Grippo and Menkes, 1971). But the mitochondrial fraction does not show any increase in synthesis of saturated and unsaturated fatty acids with T 3. Moreover, the fatty acid synthesis is reduced
100
RANJIT KUMAR GHOSH a n d A n T KUMAR MEDDA
in the mitochondrial and particularly in the microsomal fraction in animals made hypothyroid at birth (Grippo and Menkes, 1971). Although all these aspects have not been studied in Singi fish, it may be presumed that thyroid hormones have profound influence on the lipid metabolism, particularly on the synthesis in fish brain. The carbohydrate metabolism including glucose transport, glycolysis and glycogen synthesis is markedly influenced by thyroid hormones (Eberhardt et al., 1980). It is also known that thyroid hormones influence glycogen synthesis in a biphasic pattern in rat (Wertheimer and Bentor, 1953; Eberhardt et al., 1980). The biphasic nature of thyroid hormone action on the glycogen content of fish liver i.e. increase with lower doses (0.5-2 #g T4/g) and decrease with higher dose (4 ~g T4/g), has also been reported (Ray et al., 1975). In spotted Munia (Lonchura punctulata), however, 30 days injections of total doses of 0.05 rag, 0.1 mg and 0.2 mg of T4 decreased the hepatic glycogen content (Thapliyal et al., 1975). No biphasic response with respect to glycogen content (and lipid content also) after T 4 injections (1-4 #g/g) was found in Singi fish brain. All these doses of T 4 significantly increased the glycogen content of brain. It may be assumed that increased synthesis of glycogen and thereby the enhancement of glycogen content of brain took place with the doses of T4 used. This was supported by the fact that there was significant reduction in glycogen content of whole brain of thiourea treated hypothyroid fish. Our results do not support that thyroid hormones have no effect on brain glycogen of fish (see Review by Eales, 1979). The activity of glycogen synthetase-a in rat liver is increased by T~ injections at the dose of 30 #g/100 g/day for 4 days (Taningher et al., 1974). The changes in the activities in glucose6-phosphate dehydrogenase, 6-phosphogtuconate dehydrogenase, D-fl-hydroxybutyrate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase and also a number of other enzymes in developing rat brain in altered thyroid hormonal state (Sokoloff and Kennedy, 1973; Eberhardt et al., 1980) also indicate the involvement of the hormone, directly or indirectly, in the metabolism of carbohydrate, lipid and protein in the nervous system. |t may be that similar mechanisms in fish brain are also under the control of thyroid hormone. It may be questioned that the doses of T 4 (1-4/~g/g) used for the present experiments in addition to the endogenous level are very high. Whatever may be the total amount of the exogenous and endogenous hormone available for physiological actions, as evidenced in course of our investigations, it
is true that no side toxic effect of thyroid hormone was observed and there was no change in body weight of the Singi fish after injections of T~ (1 4 #g/g). The fish kept at 2 5 C requires higher dose of T 4 for manifestations of some physiological actions (Ray and Medda, 1975, 1976; Medda and Ray, 1979) and these effects cannot be comparable to mammals whose body temperature is much higher than fish. Thyroxine doses of 0.01 and 0.1 #g/g injected for 5 consecutive days have failed to cause any change in liver of Lata fish (Paul and Medda. 1983). We have also observed that T 4 dose of 0.25 ~ g/g (3 consecutive days injections) is ineffective in producing any change in Singi fish brain (unpublished observation). Although the endogenous T 4 level of Singi fish is not known, it has been reported that some teleosts have T 4 concentrations in the range of 8.4 17.0 ng/ml or more depending on the season of the year (Dickhofl et al., 1978). Other reports indicate T~ o r T 4 level of about 500 ngdl ~ or more (see Review by Eales, 1979). Moreover, it has been reported that due to binding to plasma proteins about 50~, of the total endogenous T4 or T~ in fish is free (Falkner and Eales, 1973). The nuclear T~-binding affinity as well as capacity in some fish liver is much less than that measured with rat liver nuclei (Darling et al., 1982). The T4 T~ conversion takes place in fish (Eales, 1979) and other T4 degradation products, viz diiodothyronine (Osborn and Simpson, 1969) and monoiodothyronine (Eales. 1972) have also been found in the plasma of teleosts. Moreover, there should be a preferential distribution of free thyroid hormones in different organs of fish according to the number or capacity of the binding sites. It is known that T3-binding to fish brain nuclei is less than to nuclei from fish liver (Darling et aL, 1982). Naturally, it is reasonable to assume that the whole injected T4 will not be available to Singi fish brain to elicit the changes as observed during the present investigations. Thus, the changes in protein, RNA and DNA contents (Ghosh and Medda, 1982), :¢-GPD activity (Ghosh et al., in press) and the present results with respect to increase in cholesterol, total lipid and glycogen contents are physiological effects and these are clear indications of the thyroid hormone-induced metabolic alterations in fish brain.
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