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Tissue dolichol levels in rats fed a diet rich in vitamin A
NUTRITION RESEARCH, Vol. 6, pp. 1429-1432, 1986 0271-5317/86 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
TISSUE DOLICHOL LEVELS IN RATS FED A DIET RICH IN VITAMIN A N.M.K. Ng Ying Kin, Ph.D.*, and J.J. Gauthier, M.Sc., D~partement de P~dlatrie, Facult~ de M~decine, Universlt~ de Sherbrooke (Quebec) Canada JIH 5N4 and R.A. Sikstrom, Ph.D. Clinical Research Institute of Montreal, Montreal (Quebec).
Sherbrooke,
ABSTRACT Dolichol content of brain and liver tissues were determined by high performance liquid chromatography in rats fed a normal diet and one wlth an excessive dose of Vitamin A (20 mg/kg/day for 14 days). Significant decreases of up to 50% in liver dollchols were found in the hypervitamlnosed rats when compared with the normal s. In the brain, however, the dollchol levels were not altered significantly. The changes observed in the liver are thought to be due to alterations in both membrane structures and the metabolism of dollchols, caused by storage of the excess vitamin in that organ. KEY WORDS: Vitamin A, Dollchols, Glycoprotein Synthesis, Membrane Modification. INTRODUCTION Hypervltamlnosls A has been shown to affect the nature and composition of llpids in tissues and sera of animals (1-4). Also, the biosynthesis of glycoproteins, as evidenced by the incorporation of [14C] mannose, is enhanced (5). On the other hand, in animals fed a Vitamin A deficient diet, label incorporation was decreased (6), suggesting a role for the Vitamin in such metabolic processes. Since dolichols are known to be key intermediates in the biosynthesis of glycoproteins (7), it would be of interest to examine the effects of excess Vitamin A intake on levels of dolichols in tissues from the brain and liver. Such studies may also be u s e f u l in our understanding of the pathophysiology of Neuronal Cerold Lipofuscinosis, an inherited neurological disorder, in which both retinoids and dolichols have been found to accumulate
(8). MATERIAL AND METHODS
Groups of 6 age-matched rats with an average weight of 120 grams were palr-fed with a normal diet of Ralston-Purina rat chow (I0 grams/rat/day) and one supplemented with 2 mg of retinol in the form of Vitamin A palmltate beads Hoffman La Roche), respectively. Water was given ad libltum. Subsequently, weanling rats weighing 50 grams were also used. Following completion of the *To whom correspondance
should be addressed.
1429
1430
N.M.K. NG YING KIM et al.
dietary treatment for 14 days (9)~ the animals were sacrificed by decapitation and the whole brain and liver isolated and weighed. Little difference was found in the weights of the whole rats as well as of the two organs when the two groups of animals were compared. For the determination of the brain doliehols, cortical matter from half a lobe (0.5 g) of the whole cerebrum was used, and for the liver, pieces weighing 0.5 g each. The tissues were saponified in 5M KOH in 50% aqueous ethanol and the dolichols determined by high performance liquid chromatography (HPLC) as described by Ng Ying Kin et al. (I0). RESULTS AND DISCUSSION The levels of dolichols obtained by HPLC analysis, after strong saponification of liver tissues from the hypervitaminosed and the normal rats respectively are summarised in the following Table. TABLE Liver dolichol levels in rats fed a normal diet (controls) and one supplemented with 20 mg Vitamin A /kg body weight for 14 days (hypervitaminosed rats).
~g dolichols/g, wet weight
Adult rats
Weanling rats
Controls n = 6
22.5 • 2.7
9.0 + 0.6
Hypervitaminosed rats n = 6
17.7 • 1.8
4.3 -+ 0.7
Values are mean • S.D.
p < 0.01
There is clearly a significant decrease (slightly more than 20 percent) in the levels of saponifiable dolichols extracted from the liver of the older rats treated with large doses of Vitamin A; but these levels were not significantly altered in the brains of the two age groups. On the other hand, the effects of the vitamih on the liver dolichols in the weanling rats were even more pronounced, with an observed two-fold decrease. It is also of interest to note that the dolichol levels in the control rats are age-dependantj as has previously been observed in the brain (I0). The changes observed in the liver reflect the importance of this organ as the major metabolic site for Vitamin A as well as for its storage (II). The action of the vitamin is probably mediated in part by the cellular retlnol-binding protein which is present in high concentration in the liver (12). Thus, in rats treated with retinoic acid, the levels of dolichols in the liver were not affected (13). Indeed, in the mature rat liver, cellular retlnoic acld-binding protein is not detectable (12). The sparing of the brain is probably also due to the very low concentrations of both cellular retinol-binding protein and the vitamin itself in cerebral tissues (12).
DOLICHOLS IN HYPERVITAMINOSIS A
1431
In studies on the effects of excess Vitamin A on the release of lysosomal enzymes, Dingle (14) suggested that the lysosomal membranes became labile. Now, since dolichols have been found to be concentrated mostly in the lysosomes and also the Golgi apparatus (15, 16), it is proposed that the decrease in the levels of dolichols observed in the liver after Vitamin A treatment could occur as a result of modifications to membranes associated with the Golgi and/or lysosomes. Morr~ et.al, recently showed morphological as well as physiological changes in the Golgi apparatus of liver slices treated with excess Vitamin A (17). Consequently, the dolichols, instead of concentrating within these altered structures, would probably associate with the more favourable environment offered by other subcellular organelles. Preliminary studies on the metabolism of 14C labelled dolichols appear to support this hypothesis. Modifications of the Golgi would most likely have effects on the translocations of enzymes involved in the processing of oligosaccharides in glycoprotein synthesis, known to occur in the Golgi-Endoplasmic Reticulum-Lysosome region (7). In hypervitaminosis A, the apparent increase in glycoprotein systhesis, as indicated by mannose incorporation, is probably due to incomplete processing of the high mannose oligosaccharide. On the other hand, levels of dolichol phosphate and its carbohydrate derivatives may well be increased under such conditions. Studies along these lines are now under way. Finally, it is of interest to note that, in contrast to dolichols, the levels of cholesterol are increased in the liver. Since these compounds share some common metabolic parthways via the regulating enzyme HMG CoA reductase, the in vitro biosynthesis of both dolichol phosphate and dolichols should be investigated. The altered synthesis of these compounds may affect the integrity of cellular membranes (18). ACKNOWLEDGEMENTS This research work is supported by grants from the Fonds de la Recherche en Sant~ du Quebec, Quebec, Canada, and the Medical Research Council of Canada, Canada. The authors are grateful to Ms. Huguette Savoie for providing excellent technical help and to Mrs. P. Cotnoir for skilful preparation of the manuscript. Thanks are also due to Drs. D. Shapcott and B. Preiss for helpful discussions. REFERENCES I.
DONOGHUE S, KRONFELD DS, SKLAN D. Retinol homeostasis in lambs given low and high intakes of Vitamin A. Br. J. Nutr. 1983; 5 0 : 2 3 5 - 128.
2.
SKLAN D. Effects of high Vitamin A or tocopherol intake on hepatic lipid metabolism and intestinal absorption and secretion of lipids and bile acids in the chick. Br. J. Nutr. 1983; 5 0 : 4 0 9 - 416.
3.
HUANG YS, MANKU MS, MITCHELL J, HORROBIN DF. Effects of high Vitamin A intake on plasma and liver phospholipid and triglyceride fatty acid compositions in hamsters. Nutr. Res. 1985; 5 : 9 5 - I00.
4.
MISRA UK. 911.
Lipid metabolism in hypervitaminosis A.
Nature 1986; 2 0 9 : 9 1 0
-
1432
N.M.K. NG YING KIM et al.
5.
HASSELL JR, SlLVERMAN-JONES CS, DE LUCA LM. The in vivo stimulation of mannose incorporation into mannosylated phosphate, dolichyl mannosyl phosphate and specific glyeopeptides of rat liver by high doses of retinyl palmitate. J. Biol. Chem. 1978; 2 5 3 : 1 6 2 7 - 1631.
6.
WOLF G, KIORPES TC, MASUSHIGE S, SCHREIBER JB, SMITH MJ, ANDERSON RS. Recent evidence for the participation of Vitamin A in glycoprotein synthesis. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1979; 3 8 : 2 5 4 0 - 2543.
7.
HUBBARD SC, IVATT RJ. Synthesis and processing of asparagine oligosaccharides. Ann. Rev. Biochem. 1981; 5 0 : 5 5 5 - 583.
8.
WOLFE LS, NG YING KIN NMK, BAKER RR. Batten disease and related disorders: New findings on the chemistry of the storage material. In: Callahan JW, Lowden JA Eds: Lysosomes and Lysosomal storage diseases, New York: Raven Press, 1981, 315 - 330.
9.
FURR HC, BALL MD, OLSON JA. The effect of high Vitamin A intake on rat liver retinyl ester composition and fatty acid composition. Fed. Proc. Fed. Am. Soc. Exp. Biol. Abs: 1985; 44 (3): 772.
I0.
NG YING KIN NMK, PALO J, HALTIA M, WOLFE LS. High levels of brain doliehols in neuronal ceroid lipofuscinosis and senescence. J. Neurochem. 1983; 40: 1465 - 1473.
II.
GOODMAN DS. Vitamin A and retinoids Med. 1985; 310: 1023 - 1031.
12.
ONG DE, CROW JA, CHYTIL F. Radioimmunochemical determination of cellular retinol- and retinoic acid-binding proteins in cytosols of rat tissues. J. Biol. Chem. 1982; 257: 13385-13389.
13
NG YING KIN NMK, GAUTHIER JJ, SlKSTROM RA.
14.
DINGLE JT. Studies on the mode of action of excess Vitamin A.3. a bound protease by the action of Vitamin A. Biochem. J. 1961; 512.
15.
WONG TK, DECKER GL, LENNARZ WJ. Localisation of dolichol in the fraction of rat liver. J. Biol. Chem. 1982; 2 5 7 : 6 6 1 4 - 6618.
16.
RIP JW, CHAUDHARY N, CARROLL KK. Distribution and metabolism of dolichol phosphate in rat liver. Can. J. Bioehem. Cell Biol. 1983; 6 1 : 1 0 2 5 - 1031.
17.
MORRE DMM, MORRE DJ, REUTTER W, MOLLENHAEUR HH. Comparison of monensin and Vitamin A excess on Golgi apparatus morphology and short-term labeling and turnover of Golgi apparatus from slices of rat liver. J. Cell. Biol. 1985; I 0 1 : 6 0 a Abs.
18.
VIGO C, GROSSMAN SH, DROST-HANSEN W. phosphate with phospholipid bilayers. 221 - 226.
Accepted for publication October i0, 1986.
in health and disease.
linked
New Engl.
J.
Unpublished data. Release of 79: 509 -
lysosomal
Interaction of dolichol and dolichyl Biochem. Biophys. Acta 1984; 774:
TISSUE DOLICHOL LEVELS IN RATS FED A DIET RICH IN VITAMIN A N.M.K. Ng Ying Kin, Ph.D.*, and J.J. Gauthier, M.Sc., D~partement de P~dlatrie, Facult~ de M~decine, Universlt~ de Sherbrooke (Quebec) Canada JIH 5N4 and R.A. Sikstrom, Ph.D. Clinical Research Institute of Montreal, Montreal (Quebec).
Sherbrooke,
ABSTRACT Dolichol content of brain and liver tissues were determined by high performance liquid chromatography in rats fed a normal diet and one wlth an excessive dose of Vitamin A (20 mg/kg/day for 14 days). Significant decreases of up to 50% in liver dollchols were found in the hypervitamlnosed rats when compared with the normal s. In the brain, however, the dollchol levels were not altered significantly. The changes observed in the liver are thought to be due to alterations in both membrane structures and the metabolism of dollchols, caused by storage of the excess vitamin in that organ. KEY WORDS: Vitamin A, Dollchols, Glycoprotein Synthesis, Membrane Modification. INTRODUCTION Hypervltamlnosls A has been shown to affect the nature and composition of llpids in tissues and sera of animals (1-4). Also, the biosynthesis of glycoproteins, as evidenced by the incorporation of [14C] mannose, is enhanced (5). On the other hand, in animals fed a Vitamin A deficient diet, label incorporation was decreased (6), suggesting a role for the Vitamin in such metabolic processes. Since dolichols are known to be key intermediates in the biosynthesis of glycoproteins (7), it would be of interest to examine the effects of excess Vitamin A intake on levels of dolichols in tissues from the brain and liver. Such studies may also be u s e f u l in our understanding of the pathophysiology of Neuronal Cerold Lipofuscinosis, an inherited neurological disorder, in which both retinoids and dolichols have been found to accumulate
(8). MATERIAL AND METHODS
Groups of 6 age-matched rats with an average weight of 120 grams were palr-fed with a normal diet of Ralston-Purina rat chow (I0 grams/rat/day) and one supplemented with 2 mg of retinol in the form of Vitamin A palmltate beads Hoffman La Roche), respectively. Water was given ad libltum. Subsequently, weanling rats weighing 50 grams were also used. Following completion of the *To whom correspondance
should be addressed.
1429
1430
N.M.K. NG YING KIM et al.
dietary treatment for 14 days (9)~ the animals were sacrificed by decapitation and the whole brain and liver isolated and weighed. Little difference was found in the weights of the whole rats as well as of the two organs when the two groups of animals were compared. For the determination of the brain doliehols, cortical matter from half a lobe (0.5 g) of the whole cerebrum was used, and for the liver, pieces weighing 0.5 g each. The tissues were saponified in 5M KOH in 50% aqueous ethanol and the dolichols determined by high performance liquid chromatography (HPLC) as described by Ng Ying Kin et al. (I0). RESULTS AND DISCUSSION The levels of dolichols obtained by HPLC analysis, after strong saponification of liver tissues from the hypervitaminosed and the normal rats respectively are summarised in the following Table. TABLE Liver dolichol levels in rats fed a normal diet (controls) and one supplemented with 20 mg Vitamin A /kg body weight for 14 days (hypervitaminosed rats).
~g dolichols/g, wet weight
Adult rats
Weanling rats
Controls n = 6
22.5 • 2.7
9.0 + 0.6
Hypervitaminosed rats n = 6
17.7 • 1.8
4.3 -+ 0.7
Values are mean • S.D.
p < 0.01
There is clearly a significant decrease (slightly more than 20 percent) in the levels of saponifiable dolichols extracted from the liver of the older rats treated with large doses of Vitamin A; but these levels were not significantly altered in the brains of the two age groups. On the other hand, the effects of the vitamih on the liver dolichols in the weanling rats were even more pronounced, with an observed two-fold decrease. It is also of interest to note that the dolichol levels in the control rats are age-dependantj as has previously been observed in the brain (I0). The changes observed in the liver reflect the importance of this organ as the major metabolic site for Vitamin A as well as for its storage (II). The action of the vitamin is probably mediated in part by the cellular retlnol-binding protein which is present in high concentration in the liver (12). Thus, in rats treated with retinoic acid, the levels of dolichols in the liver were not affected (13). Indeed, in the mature rat liver, cellular retlnoic acld-binding protein is not detectable (12). The sparing of the brain is probably also due to the very low concentrations of both cellular retinol-binding protein and the vitamin itself in cerebral tissues (12).
DOLICHOLS IN HYPERVITAMINOSIS A
1431
In studies on the effects of excess Vitamin A on the release of lysosomal enzymes, Dingle (14) suggested that the lysosomal membranes became labile. Now, since dolichols have been found to be concentrated mostly in the lysosomes and also the Golgi apparatus (15, 16), it is proposed that the decrease in the levels of dolichols observed in the liver after Vitamin A treatment could occur as a result of modifications to membranes associated with the Golgi and/or lysosomes. Morr~ et.al, recently showed morphological as well as physiological changes in the Golgi apparatus of liver slices treated with excess Vitamin A (17). Consequently, the dolichols, instead of concentrating within these altered structures, would probably associate with the more favourable environment offered by other subcellular organelles. Preliminary studies on the metabolism of 14C labelled dolichols appear to support this hypothesis. Modifications of the Golgi would most likely have effects on the translocations of enzymes involved in the processing of oligosaccharides in glycoprotein synthesis, known to occur in the Golgi-Endoplasmic Reticulum-Lysosome region (7). In hypervitaminosis A, the apparent increase in glycoprotein systhesis, as indicated by mannose incorporation, is probably due to incomplete processing of the high mannose oligosaccharide. On the other hand, levels of dolichol phosphate and its carbohydrate derivatives may well be increased under such conditions. Studies along these lines are now under way. Finally, it is of interest to note that, in contrast to dolichols, the levels of cholesterol are increased in the liver. Since these compounds share some common metabolic parthways via the regulating enzyme HMG CoA reductase, the in vitro biosynthesis of both dolichol phosphate and dolichols should be investigated. The altered synthesis of these compounds may affect the integrity of cellular membranes (18). ACKNOWLEDGEMENTS This research work is supported by grants from the Fonds de la Recherche en Sant~ du Quebec, Quebec, Canada, and the Medical Research Council of Canada, Canada. The authors are grateful to Ms. Huguette Savoie for providing excellent technical help and to Mrs. P. Cotnoir for skilful preparation of the manuscript. Thanks are also due to Drs. D. Shapcott and B. Preiss for helpful discussions. REFERENCES I.
DONOGHUE S, KRONFELD DS, SKLAN D. Retinol homeostasis in lambs given low and high intakes of Vitamin A. Br. J. Nutr. 1983; 5 0 : 2 3 5 - 128.
2.
SKLAN D. Effects of high Vitamin A or tocopherol intake on hepatic lipid metabolism and intestinal absorption and secretion of lipids and bile acids in the chick. Br. J. Nutr. 1983; 5 0 : 4 0 9 - 416.
3.
HUANG YS, MANKU MS, MITCHELL J, HORROBIN DF. Effects of high Vitamin A intake on plasma and liver phospholipid and triglyceride fatty acid compositions in hamsters. Nutr. Res. 1985; 5 : 9 5 - I00.
4.
MISRA UK. 911.
Lipid metabolism in hypervitaminosis A.
Nature 1986; 2 0 9 : 9 1 0
-
1432
N.M.K. NG YING KIM et al.
5.
HASSELL JR, SlLVERMAN-JONES CS, DE LUCA LM. The in vivo stimulation of mannose incorporation into mannosylated phosphate, dolichyl mannosyl phosphate and specific glyeopeptides of rat liver by high doses of retinyl palmitate. J. Biol. Chem. 1978; 2 5 3 : 1 6 2 7 - 1631.
6.
WOLF G, KIORPES TC, MASUSHIGE S, SCHREIBER JB, SMITH MJ, ANDERSON RS. Recent evidence for the participation of Vitamin A in glycoprotein synthesis. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1979; 3 8 : 2 5 4 0 - 2543.
7.
HUBBARD SC, IVATT RJ. Synthesis and processing of asparagine oligosaccharides. Ann. Rev. Biochem. 1981; 5 0 : 5 5 5 - 583.
8.
WOLFE LS, NG YING KIN NMK, BAKER RR. Batten disease and related disorders: New findings on the chemistry of the storage material. In: Callahan JW, Lowden JA Eds: Lysosomes and Lysosomal storage diseases, New York: Raven Press, 1981, 315 - 330.
9.
FURR HC, BALL MD, OLSON JA. The effect of high Vitamin A intake on rat liver retinyl ester composition and fatty acid composition. Fed. Proc. Fed. Am. Soc. Exp. Biol. Abs: 1985; 44 (3): 772.
I0.
NG YING KIN NMK, PALO J, HALTIA M, WOLFE LS. High levels of brain doliehols in neuronal ceroid lipofuscinosis and senescence. J. Neurochem. 1983; 40: 1465 - 1473.
II.
GOODMAN DS. Vitamin A and retinoids Med. 1985; 310: 1023 - 1031.
12.
ONG DE, CROW JA, CHYTIL F. Radioimmunochemical determination of cellular retinol- and retinoic acid-binding proteins in cytosols of rat tissues. J. Biol. Chem. 1982; 257: 13385-13389.
13
NG YING KIN NMK, GAUTHIER JJ, SlKSTROM RA.
14.
DINGLE JT. Studies on the mode of action of excess Vitamin A.3. a bound protease by the action of Vitamin A. Biochem. J. 1961; 512.
15.
WONG TK, DECKER GL, LENNARZ WJ. Localisation of dolichol in the fraction of rat liver. J. Biol. Chem. 1982; 2 5 7 : 6 6 1 4 - 6618.
16.
RIP JW, CHAUDHARY N, CARROLL KK. Distribution and metabolism of dolichol phosphate in rat liver. Can. J. Bioehem. Cell Biol. 1983; 6 1 : 1 0 2 5 - 1031.
17.
MORRE DMM, MORRE DJ, REUTTER W, MOLLENHAEUR HH. Comparison of monensin and Vitamin A excess on Golgi apparatus morphology and short-term labeling and turnover of Golgi apparatus from slices of rat liver. J. Cell. Biol. 1985; I 0 1 : 6 0 a Abs.
18.
VIGO C, GROSSMAN SH, DROST-HANSEN W. phosphate with phospholipid bilayers. 221 - 226.
Accepted for publication October i0, 1986.
in health and disease.
linked
New Engl.
J.
Unpublished data. Release of 79: 509 -
lysosomal
Interaction of dolichol and dolichyl Biochem. Biophys. Acta 1984; 774: