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Relationships between circadian cycles of rat adrenal cholesterol ester metabolizing enzymes, cholesterol, ascorbic acid, and corticosteroid secretion
0 0022-4731,82460817-06503.00 Copyright 0 1982 Pergamon Press Ltd
J. stemi$ Biochem. Vol. 16. pp. Xl7 IO 822. 1982 Printed in Great Britain. All rights reserved
RELATIONSHIPS BETWEEN CIRCADIAN CYCLES OF RAT ADRENAL CHOLESTEROL ESTER METABOLIZING ENZYMES, CHOLESTEROL, ASCORBIC ACID, AND CORTICOSTEROID SECRETION M. Ctv&*.
J. LEEB*
and R. J. MORIN:
*Medical Research Programs. VAMC. Long Beach. CA 90822 tDepartment of Physiology. University of California, Irvine. CA and ZDepartment of Pathology. Harbor-UCLA Medical Center Torrance. CA. U.S.A.
SUMMARY
The circadian changes in rat adrenal cholesterol ester (CE) metabolism have been studied and related to the circadian changes in adrenal ascorbic acid and corticosterone levels and plasma corticosterone levels. Significant declines in the ratio of CE/C. cholesterol esterase (CEase) and acyl CoA:cholesterol acyl transferase (ACAT) activities occur during the peak and declining phases of adrenal and plasma corticosteroids. suggesting that when there is a decreased need for steroid precursors the amount of stored CE is decreased. and that the enzymes involved in CE metabolism decline in activity. Adrenal ascorbic acid has a biphasic rhythm with peaks at 0900 and 2200 h. The rhythm of ascorbic acid appears to be inverse to that of ACAT activity. suggesting possible relationships between the two parameters.
INTRODUCTION
Steroidogenic large amounts
tissues such as adrenal cortex contain of cholesterol
esterified
to long chain
The cholesterol esters are localized mainly in cytoplasmic droplets where they appear to function as a reservoir for the free cholesterol required for steroid biosynthesis. Recently, evidence has been presented that indicates that the stress mediated hydrolysis of cholesterol esters may occur independently of ACTH’s effects on later steps in steroidogenesis [2], Two enzymes which play a major role in controlling the level of cholesteryl esters in steroid secretory tissue in the rat adrenal cortex are acyl coenzyme A : cholesteryl o-acyl transferase (ACAT) and cholesterol esterase. It has been shown that ACTH has a deactivating effect on ACAT activity [3]. while stress and the pituitary peptide, y-MSH, but not ACTH. activate cholesterol esterase [23. To further understand the regulatory roles of these two enzyme systems in adrenal cholesterol ester metabolism we have studied the circadian rhythms of these two enzyme systems. and correlated them with circadian changes in the adrenal free and esterified cholesterol. Circadian changes in plasma and adrenal corticosterone levels and adrenal ascorbic acid levels were measured fatty
acids [l].
Correspondence to be sent to: M. Civen. Ph.D.. Medical Research (151). VA Medical Center, Long Beach. CA 90822. U.S.A. \.B 166-14
817
as metabolic “bench marks” of the functional activity of the gland. Our results show significant circadian changes occur in the activity of both ACAT and cholesterol esterase. Changes in adrenal esterified and free cholesterol also occur. but are of a smaller order of magnitude. The above changes appear to bear functional relationships to the circadian changes in adrenal and plasma corticosterone and adrenal ascorbic acid. EXPERIMENTAL
Male Sprague-Dawley rats (approximately 350 g) were housed individually under conditions of uniform temperature (25 + 1°C) and illumination (08002000 h) during the study. Animals were maintained for one month with daily handling. At the time indicated, animals were stunned by a blow to the head and decapitated; trunk blood and adrenals were collected on ice. A maximum of 10s elapsed between removal of the animal from the cage and decapitation. Sucrose homogenates and chloroform-methanol (4: 1, v/v) extracts were prepared from the two adrenals as previously described [4]. Enzyme assays and plasma and adrenal cholesterol and corticosterone assays were as previously described [4]. Ascorbic acid was measured in the sucrose homogenate by the procedure of Zannoni et al.[S]. Each experimental group contained 6 animals. and statistical significance of the data were measured by one way analysis of variance F-test, Dunnett’s test and Schemes multiple contrast test [6].
818
M. GIVENef al.
cd . . urn . . .
1 .
camloooIamux,naolammm~moomooomorx,looo
.
.
.
. 1
Time (clockkOum)
Fig. 1. Circadian rhythm of plasma corticosteroncconcentration in male Sprague-Dawley rats. Data are +SD
of 6 rats for each point.
l
= Significant differences between O!WOh time point and * time point P < 0.001.
RESULTS AND DISCUSSION
test) lower than at 0900 h. Figure 5 shows that total adrenal ascorbic acid undergoes significant circadian Figure 1 shows the familiar circadian changes in and thus on balance the stronger effect on ACAT activity could be responsible for the depression of plasma and adrenal corticosterone. Plasma corticosterone levels increase 3-fold above the 0900 h mini- cholesterol ester levels. At present, there is no data on mum, reaching a maximum at 2200 h (P < 0.001, the circadian variation of lipoprotein uptake by the F-test). Adrenal corticosterone levels increased about rat adrenal cortex. It is known that rat plasma lipo40% above the 0900 h minimum at 2200 h (P e 0.01, proteins and adrenal free and ester&d cholesterol F-test). Figure 3 shows the changes in free and esteri- levels decline in parallel aRer administration of the fied cholesterol, these are relatively small. No signifi- lipoprotein biosynthetic inhibitor, aminopyrazolopy-rcant changes in ester&d cholesterol occurred when imidene (AAP)[7,8]. Conversely, when lipoprotein the minimum value at 1330h was compared with the was administered to AAP treated rats it produced a 0900 h point. Free cholesterol level was relatively con- marked increase in the depressed adrenal cholesterol stant with no significant circadian change. In Fig. 4 ester [7,8]. It has also been shown that when Y-l the data in Fig. 3 are expressed as the ratio of esteri- adrenal tumor cells are incubated with low-density tied to free cholesterol. When the data are expressed lipoprotein cellular free and ester&i cholesterol conin this way, there is a statistically significant de- centration and the activity of ACAT increases but pression at 13OOh,about 25”/, (P < 0.05, Dunnett’s cholesterol esterase does not [9]. Thus it appears that
Fig. 2. Circadian rhythm of adrenal cortiosterone concentration under same experunental conditions and animals as in Fig. I. l = Significant differences between 0900 h time point and * time point P < 0.01.
Rat adrenal cholesterol ester circadian rhythms
819
Fig. 3. Adrenal cholesterol concentration: esterified (EC) and free (FC). Same experimental conditions and animals as in Fig. 1.
is a linkage between cholesterol uptake from lipoprotein and ACAT activity. In the studies reported here we observed that at 2200 h, the time at which cholesterol ester levels begin to decline. ACAT activity is already at its minimum. The changes in cholesterol ester and free cholesterol levels are relatively small as compared to the changes in plasma corticosteroids. however in absolute terms they are several times larger than the changes in the levels of plasma and adrenal corticosteroids in the case of cholesterol ester, and twice as large in the case of the free cholesterol. The fact that the changes in cholesterol levels are greater than corticosteroid changes is probably due to catabolism and excretion of corticosteroids, unaccounted for in our studies. Although the changes in the total adrenal choksterol ester and free cholesterol are generally small, it will be of interest to examine the circadian changes of there
these two parameters in the different adrenal intracellular fractions, to determine if there is metabolic heterogeneity in the different cholesterol pools of the adrenal cell. Of the two enzymes, ACAT and cholesterol esterase, the former appears to undergo the greatest circadian change in its activity. As mentioned above, the rhythm is biphasic, with its first peak occurring at the midpoint of the rising phase of the corticosteroid cyck, and its minimum level at the peak of the corticosteroid cycle. The secondary peak which is lower, occurs during the declining phase of the corticosteroid cycle, and the second minimum occurs at the minimum for plasma corticosteroids. Balasubramaniam et aQ7-J observed that the circadian peaks in rat adrenal hydroxymethylglutaryl CoA reductase and hydroxymethyl-glutaryl CoA spthase (rate limiting enzymatic steps in cholesterol biosynthesis) occurs at 0200 (corrected to our lighting schedule) suggesting
Fig. 4. Adrenal cholesterol. ratio of Esterified to Free. (EC/FC). Same experimental conditions and animals as in Fig. 1. l = Significant ditrerence between 0900 h time point and l time point at P < 0.05.
820
M. GIVENet al.
Fig. 5. Total adrenal ascorbic acid. Same experimental conditions and animals as in Fig. 1. cant differences between 0900 h time point and * time point at P < 0.01.
activation of cholesterol synthesis. Since these enzymes have a microsomal location, as does ACAT, it is possible that increase in microsomal cholesterol through biosynthesis may be involved in the induction of the secondary peak in’ ACAT activity. If ACAT levels are modulated at least in part by the influx and metabolism of lipoproteins, as is suggested by the work of Faust et al. and Balasubramaniam et al.[6.13], then lipoprotein uptake, as evidenced by changes in cellular cholesterol concentrations should parallel the changes in ACAT activity. These were not seen in our studies on total adrenal cholesterol levels. The enhancement of cholesterol ester and free cholesterol may only be evident in certain intracellular fractions. such as in the microsomes where a large part of the ACAT activity occurs. It is of interest that adrenal ascorbic acid levels show circadian changes that are inverse to the changes in ACAT activity. A similar biphasic rhythm of adrenal ascorbic acid has been observed in the
l
= Signili-
male rat by GaggiClO]. In previously published work [I l] we found that when rats were fed a diet containing high levels of ascorbic acid and exposed to ether stress for a short period, ACAT activity increased several fold 15 min after stress. At the same time, ascorbic acid levels were depressed from the abnormally high adrenal levels produced by feeding the high ascorbic acid diet. In the experiments described in this paper, adrenal ascorbic acid levels were also depressed when ACAT was maximal. Thus the changes in adrenal ascorbic acid levels may play a role in the modulation of the adrenal ACAT activity. Cytoplasmic cholesterol esterase begins to decline in activity at the same time as ACAT, and reaches its minimum at the same time as ACAT. However in contrast to ACAT, cholesterol esterase remains suppressed through the declining phase of the corticosteroid rhythms. The lowered cholesterol esterase activity presumably may result in less free cholesterol being available for metabolic conversion to steroids.
Fig. 6. Adrenal microsomal acyl CoA = cholesterol acyl transferase activity (ACAT). Same experimental conditions and animals as in Fig. I. * = Significant differences between O!MOh time point and 1700. P < 0.05. ** = Significant ditkrences exist between 1700 and 2200. P < 0.05 and 1700 and 0500.
P < 0.05.
821
Rat adrenal cholesterol ester circadian rhythms
(clock hours)
Tfme
Fig. 7. Adrenal cytosolic cholesterol esterase activity. Same experimental conditions and animals as in Fig. I. * = Significant diKerences between 0900 h time point and * time points at P < 0.005.
to the decline in corticosterone production during this period. The rhythm for hydroxymethylglutaryl CoA reductase and synthase [12] is roughly inverse to that of cholesterol esterase. which suggests that the decrease in cholesterol esterase results in a decrease in free cholesterol at some crucial site in the cell. which in turn stimulates cholesterol biosynthesis at these times. It is of interest that the decline in cholesterol esterase precedes the maximum of the corticosteroid circadian curves. and thus may be part of the braking action involved in the declining phase of the corticosterone cycle. The decrease in cholesterol esterase activity is not reflected in a decrease in total free cholesterol levels in the adrenal cell. since free cholesterol levels appear to remain relatively constant in the adrenal cell. while cholesterol ester levels change in response lo physiological stimuli. Again. the changes in free cholesterol may be localized in certain parts of the cell. There appears to be some controversy as to whether cholesterol esterase activity undergoes circadian changes. and the magnitude of these changes. Pedersen and Brownie[lZ] have reported the rhythm to be absent or very modest in female rats. Klemcke[13] has observed rhythms in both adrenal and luteal cholesterol esterase of pseudopregnant female rats. The differences in these observations could be due to sex andlor methodological differences. In summary. we have observed significant circadian changes in the following parameters of the adrenal gland: Esterified cholesterol levels. ascorbic acid and the cholesterol esterase and ACAT activity. The declining levels in esterified cholesterol and of cholesterol esterase and ACAT activity occur during the peak and declining phase of adrenal and plasma corticosteroids. suggesting that when there is a decreased need for steroid precursor the amount of stored cholesterol is decreased and that the enzymes involved in cholesterol ester metabolism decline in activity. The and thus contributing
rhythm
of ascorbic
acid appears
to move inversely
to
that of ACAT, suggesting that ascorbic acid may be involved in inverse feedback regulation of ACAT’s activity. Acknowlrdyemcnr-The authors are grateful to Henry Batsel Ph.D., of the V.A. Medical Center. Long Beach for his help in the statistical analysis of the data. REFERENCES
I. Goodman D. S.: Cholesterol ester metabolism. Physiol. Rcr. 45. (1965) 747-839. 2. Pedersen R. C.. Brownie A. C. and Ling N.: Proadrenocorticotrophin/endorphin-derived peptides: Coordinate action on adrenal steroidogenesis. Science>208 (1980) 1044-1045. 3. Shima S.. Mitsunaga M. and Takeshi H. N.: Effect of ACTH on cholesterol dynamics in rat adrenal tissue. Endocrinology !JO(1972) 808-814. 4. Civen M.. Leeb J. E.. Wishnow R. M., Wolfsen A. and Morin R. J.: Effects of low level administration of dichlorvos on adrenocorticotrophic hormone secretion. adrenal cholesteryl ester and steroid metabolism. Biochrm. Phurmucol. 29 (1980) 635-641. 5. Zannoni V.. Lynch M.. Goldstein S. and Sato P.: A rapid micromethod for the determination of ascorbic acid in plasma and tissue. Biochem. Med. II (1974) 41-48. 6. Zar J. R.: Biostaristical Anulysis. Prentice-Hall. Englewood Cliffs. N.J. U.S.A. (1974). 7. Balasubramanian S.. Goldstein J. L.. Faust J. R.. Brunschede G. Y. and Brown M. S.: Lipoprotein mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat. J. ho/. Chcm. 252 (1977) 1771-1779. 8. Andersen J. M. and Dietschy J. M.: Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal eland. ovary and testis of the rat. J. hiol. Chem 252 71978) 9024-9037. 9. Faust J. R.. Goldstein J. L. and Brown M. S.: Receptor-mediated uptake of low density lipoprotein and utilization of this cholesterol for cultured mouse adrenal cells. J. hiol. Churn. 252 (1977) 4861-4871. 10. Gaggi R.: Ritmo circadian0 del colesterolo. dell’acido ascorbic0 e del plicogeno della surrenale e secrezione del Corticosterone ndl ratto. Arch. sci. Biol. (Bologna) 58 (1974) 71-77.
a22
bf.
GIVEN
11. Civen M., Leeb J. E., Wishnow R. M. and Morin R. J.: Effects of dietary ascorbic acid and vitamin E deficiency on rat adrenal cholesterol ester metabolism and corticosteroidogenesis. Inr. J. Fit. Nutr. Res. SO (1980) 79-88. 12. Pedersen R. C. and Brownie A. C.: Failure of ACTH to mimic the stress-induced activation of rat adrenocorti-
et 01.
cal cholesterol ester hydrolase in aico. J. sreroid Biothem. 11 (1979) 1393-1400. 13. Klemckt H. G. and Brinkley H. J.: Endogenous rhythms of iuteal and adrenal cholesterol ester hydrolast and serum Prl, LH and progesterone in mature pseudopregnant rats. Biol. Reprod. 22 (1980) 1022-1028.
J. stemi$ Biochem. Vol. 16. pp. Xl7 IO 822. 1982 Printed in Great Britain. All rights reserved
RELATIONSHIPS BETWEEN CIRCADIAN CYCLES OF RAT ADRENAL CHOLESTEROL ESTER METABOLIZING ENZYMES, CHOLESTEROL, ASCORBIC ACID, AND CORTICOSTEROID SECRETION M. Ctv&*.
J. LEEB*
and R. J. MORIN:
*Medical Research Programs. VAMC. Long Beach. CA 90822 tDepartment of Physiology. University of California, Irvine. CA and ZDepartment of Pathology. Harbor-UCLA Medical Center Torrance. CA. U.S.A.
SUMMARY
The circadian changes in rat adrenal cholesterol ester (CE) metabolism have been studied and related to the circadian changes in adrenal ascorbic acid and corticosterone levels and plasma corticosterone levels. Significant declines in the ratio of CE/C. cholesterol esterase (CEase) and acyl CoA:cholesterol acyl transferase (ACAT) activities occur during the peak and declining phases of adrenal and plasma corticosteroids. suggesting that when there is a decreased need for steroid precursors the amount of stored CE is decreased. and that the enzymes involved in CE metabolism decline in activity. Adrenal ascorbic acid has a biphasic rhythm with peaks at 0900 and 2200 h. The rhythm of ascorbic acid appears to be inverse to that of ACAT activity. suggesting possible relationships between the two parameters.
INTRODUCTION
Steroidogenic large amounts
tissues such as adrenal cortex contain of cholesterol
esterified
to long chain
The cholesterol esters are localized mainly in cytoplasmic droplets where they appear to function as a reservoir for the free cholesterol required for steroid biosynthesis. Recently, evidence has been presented that indicates that the stress mediated hydrolysis of cholesterol esters may occur independently of ACTH’s effects on later steps in steroidogenesis [2], Two enzymes which play a major role in controlling the level of cholesteryl esters in steroid secretory tissue in the rat adrenal cortex are acyl coenzyme A : cholesteryl o-acyl transferase (ACAT) and cholesterol esterase. It has been shown that ACTH has a deactivating effect on ACAT activity [3]. while stress and the pituitary peptide, y-MSH, but not ACTH. activate cholesterol esterase [23. To further understand the regulatory roles of these two enzyme systems in adrenal cholesterol ester metabolism we have studied the circadian rhythms of these two enzyme systems. and correlated them with circadian changes in the adrenal free and esterified cholesterol. Circadian changes in plasma and adrenal corticosterone levels and adrenal ascorbic acid levels were measured fatty
acids [l].
Correspondence to be sent to: M. Civen. Ph.D.. Medical Research (151). VA Medical Center, Long Beach. CA 90822. U.S.A. \.B 166-14
817
as metabolic “bench marks” of the functional activity of the gland. Our results show significant circadian changes occur in the activity of both ACAT and cholesterol esterase. Changes in adrenal esterified and free cholesterol also occur. but are of a smaller order of magnitude. The above changes appear to bear functional relationships to the circadian changes in adrenal and plasma corticosterone and adrenal ascorbic acid. EXPERIMENTAL
Male Sprague-Dawley rats (approximately 350 g) were housed individually under conditions of uniform temperature (25 + 1°C) and illumination (08002000 h) during the study. Animals were maintained for one month with daily handling. At the time indicated, animals were stunned by a blow to the head and decapitated; trunk blood and adrenals were collected on ice. A maximum of 10s elapsed between removal of the animal from the cage and decapitation. Sucrose homogenates and chloroform-methanol (4: 1, v/v) extracts were prepared from the two adrenals as previously described [4]. Enzyme assays and plasma and adrenal cholesterol and corticosterone assays were as previously described [4]. Ascorbic acid was measured in the sucrose homogenate by the procedure of Zannoni et al.[S]. Each experimental group contained 6 animals. and statistical significance of the data were measured by one way analysis of variance F-test, Dunnett’s test and Schemes multiple contrast test [6].
818
M. GIVENef al.
cd . . urn . . .
1 .
camloooIamux,naolammm~moomooomorx,looo
.
.
.
. 1
Time (clockkOum)
Fig. 1. Circadian rhythm of plasma corticosteroncconcentration in male Sprague-Dawley rats. Data are +SD
of 6 rats for each point.
l
= Significant differences between O!WOh time point and * time point P < 0.001.
RESULTS AND DISCUSSION
test) lower than at 0900 h. Figure 5 shows that total adrenal ascorbic acid undergoes significant circadian Figure 1 shows the familiar circadian changes in and thus on balance the stronger effect on ACAT activity could be responsible for the depression of plasma and adrenal corticosterone. Plasma corticosterone levels increase 3-fold above the 0900 h mini- cholesterol ester levels. At present, there is no data on mum, reaching a maximum at 2200 h (P < 0.001, the circadian variation of lipoprotein uptake by the F-test). Adrenal corticosterone levels increased about rat adrenal cortex. It is known that rat plasma lipo40% above the 0900 h minimum at 2200 h (P e 0.01, proteins and adrenal free and ester&d cholesterol F-test). Figure 3 shows the changes in free and esteri- levels decline in parallel aRer administration of the fied cholesterol, these are relatively small. No signifi- lipoprotein biosynthetic inhibitor, aminopyrazolopy-rcant changes in ester&d cholesterol occurred when imidene (AAP)[7,8]. Conversely, when lipoprotein the minimum value at 1330h was compared with the was administered to AAP treated rats it produced a 0900 h point. Free cholesterol level was relatively con- marked increase in the depressed adrenal cholesterol stant with no significant circadian change. In Fig. 4 ester [7,8]. It has also been shown that when Y-l the data in Fig. 3 are expressed as the ratio of esteri- adrenal tumor cells are incubated with low-density tied to free cholesterol. When the data are expressed lipoprotein cellular free and ester&i cholesterol conin this way, there is a statistically significant de- centration and the activity of ACAT increases but pression at 13OOh,about 25”/, (P < 0.05, Dunnett’s cholesterol esterase does not [9]. Thus it appears that
Fig. 2. Circadian rhythm of adrenal cortiosterone concentration under same experunental conditions and animals as in Fig. I. l = Significant differences between 0900 h time point and * time point P < 0.01.
Rat adrenal cholesterol ester circadian rhythms
819
Fig. 3. Adrenal cholesterol concentration: esterified (EC) and free (FC). Same experimental conditions and animals as in Fig. 1.
is a linkage between cholesterol uptake from lipoprotein and ACAT activity. In the studies reported here we observed that at 2200 h, the time at which cholesterol ester levels begin to decline. ACAT activity is already at its minimum. The changes in cholesterol ester and free cholesterol levels are relatively small as compared to the changes in plasma corticosteroids. however in absolute terms they are several times larger than the changes in the levels of plasma and adrenal corticosteroids in the case of cholesterol ester, and twice as large in the case of the free cholesterol. The fact that the changes in cholesterol levels are greater than corticosteroid changes is probably due to catabolism and excretion of corticosteroids, unaccounted for in our studies. Although the changes in the total adrenal choksterol ester and free cholesterol are generally small, it will be of interest to examine the circadian changes of there
these two parameters in the different adrenal intracellular fractions, to determine if there is metabolic heterogeneity in the different cholesterol pools of the adrenal cell. Of the two enzymes, ACAT and cholesterol esterase, the former appears to undergo the greatest circadian change in its activity. As mentioned above, the rhythm is biphasic, with its first peak occurring at the midpoint of the rising phase of the corticosteroid cyck, and its minimum level at the peak of the corticosteroid cycle. The secondary peak which is lower, occurs during the declining phase of the corticosteroid cycle, and the second minimum occurs at the minimum for plasma corticosteroids. Balasubramaniam et aQ7-J observed that the circadian peaks in rat adrenal hydroxymethylglutaryl CoA reductase and hydroxymethyl-glutaryl CoA spthase (rate limiting enzymatic steps in cholesterol biosynthesis) occurs at 0200 (corrected to our lighting schedule) suggesting
Fig. 4. Adrenal cholesterol. ratio of Esterified to Free. (EC/FC). Same experimental conditions and animals as in Fig. 1. l = Significant ditrerence between 0900 h time point and l time point at P < 0.05.
820
M. GIVENet al.
Fig. 5. Total adrenal ascorbic acid. Same experimental conditions and animals as in Fig. 1. cant differences between 0900 h time point and * time point at P < 0.01.
activation of cholesterol synthesis. Since these enzymes have a microsomal location, as does ACAT, it is possible that increase in microsomal cholesterol through biosynthesis may be involved in the induction of the secondary peak in’ ACAT activity. If ACAT levels are modulated at least in part by the influx and metabolism of lipoproteins, as is suggested by the work of Faust et al. and Balasubramaniam et al.[6.13], then lipoprotein uptake, as evidenced by changes in cellular cholesterol concentrations should parallel the changes in ACAT activity. These were not seen in our studies on total adrenal cholesterol levels. The enhancement of cholesterol ester and free cholesterol may only be evident in certain intracellular fractions. such as in the microsomes where a large part of the ACAT activity occurs. It is of interest that adrenal ascorbic acid levels show circadian changes that are inverse to the changes in ACAT activity. A similar biphasic rhythm of adrenal ascorbic acid has been observed in the
l
= Signili-
male rat by GaggiClO]. In previously published work [I l] we found that when rats were fed a diet containing high levels of ascorbic acid and exposed to ether stress for a short period, ACAT activity increased several fold 15 min after stress. At the same time, ascorbic acid levels were depressed from the abnormally high adrenal levels produced by feeding the high ascorbic acid diet. In the experiments described in this paper, adrenal ascorbic acid levels were also depressed when ACAT was maximal. Thus the changes in adrenal ascorbic acid levels may play a role in the modulation of the adrenal ACAT activity. Cytoplasmic cholesterol esterase begins to decline in activity at the same time as ACAT, and reaches its minimum at the same time as ACAT. However in contrast to ACAT, cholesterol esterase remains suppressed through the declining phase of the corticosteroid rhythms. The lowered cholesterol esterase activity presumably may result in less free cholesterol being available for metabolic conversion to steroids.
Fig. 6. Adrenal microsomal acyl CoA = cholesterol acyl transferase activity (ACAT). Same experimental conditions and animals as in Fig. I. * = Significant differences between O!MOh time point and 1700. P < 0.05. ** = Significant ditkrences exist between 1700 and 2200. P < 0.05 and 1700 and 0500.
P < 0.05.
821
Rat adrenal cholesterol ester circadian rhythms
(clock hours)
Tfme
Fig. 7. Adrenal cytosolic cholesterol esterase activity. Same experimental conditions and animals as in Fig. I. * = Significant diKerences between 0900 h time point and * time points at P < 0.005.
to the decline in corticosterone production during this period. The rhythm for hydroxymethylglutaryl CoA reductase and synthase [12] is roughly inverse to that of cholesterol esterase. which suggests that the decrease in cholesterol esterase results in a decrease in free cholesterol at some crucial site in the cell. which in turn stimulates cholesterol biosynthesis at these times. It is of interest that the decline in cholesterol esterase precedes the maximum of the corticosteroid circadian curves. and thus may be part of the braking action involved in the declining phase of the corticosterone cycle. The decrease in cholesterol esterase activity is not reflected in a decrease in total free cholesterol levels in the adrenal cell. since free cholesterol levels appear to remain relatively constant in the adrenal cell. while cholesterol ester levels change in response lo physiological stimuli. Again. the changes in free cholesterol may be localized in certain parts of the cell. There appears to be some controversy as to whether cholesterol esterase activity undergoes circadian changes. and the magnitude of these changes. Pedersen and Brownie[lZ] have reported the rhythm to be absent or very modest in female rats. Klemcke[13] has observed rhythms in both adrenal and luteal cholesterol esterase of pseudopregnant female rats. The differences in these observations could be due to sex andlor methodological differences. In summary. we have observed significant circadian changes in the following parameters of the adrenal gland: Esterified cholesterol levels. ascorbic acid and the cholesterol esterase and ACAT activity. The declining levels in esterified cholesterol and of cholesterol esterase and ACAT activity occur during the peak and declining phase of adrenal and plasma corticosteroids. suggesting that when there is a decreased need for steroid precursor the amount of stored cholesterol is decreased and that the enzymes involved in cholesterol ester metabolism decline in activity. The and thus contributing
rhythm
of ascorbic
acid appears
to move inversely
to
that of ACAT, suggesting that ascorbic acid may be involved in inverse feedback regulation of ACAT’s activity. Acknowlrdyemcnr-The authors are grateful to Henry Batsel Ph.D., of the V.A. Medical Center. Long Beach for his help in the statistical analysis of the data. REFERENCES
I. Goodman D. S.: Cholesterol ester metabolism. Physiol. Rcr. 45. (1965) 747-839. 2. Pedersen R. C.. Brownie A. C. and Ling N.: Proadrenocorticotrophin/endorphin-derived peptides: Coordinate action on adrenal steroidogenesis. Science>208 (1980) 1044-1045. 3. Shima S.. Mitsunaga M. and Takeshi H. N.: Effect of ACTH on cholesterol dynamics in rat adrenal tissue. Endocrinology !JO(1972) 808-814. 4. Civen M.. Leeb J. E.. Wishnow R. M., Wolfsen A. and Morin R. J.: Effects of low level administration of dichlorvos on adrenocorticotrophic hormone secretion. adrenal cholesteryl ester and steroid metabolism. Biochrm. Phurmucol. 29 (1980) 635-641. 5. Zannoni V.. Lynch M.. Goldstein S. and Sato P.: A rapid micromethod for the determination of ascorbic acid in plasma and tissue. Biochem. Med. II (1974) 41-48. 6. Zar J. R.: Biostaristical Anulysis. Prentice-Hall. Englewood Cliffs. N.J. U.S.A. (1974). 7. Balasubramanian S.. Goldstein J. L.. Faust J. R.. Brunschede G. Y. and Brown M. S.: Lipoprotein mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat. J. ho/. Chcm. 252 (1977) 1771-1779. 8. Andersen J. M. and Dietschy J. M.: Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal eland. ovary and testis of the rat. J. hiol. Chem 252 71978) 9024-9037. 9. Faust J. R.. Goldstein J. L. and Brown M. S.: Receptor-mediated uptake of low density lipoprotein and utilization of this cholesterol for cultured mouse adrenal cells. J. hiol. Churn. 252 (1977) 4861-4871. 10. Gaggi R.: Ritmo circadian0 del colesterolo. dell’acido ascorbic0 e del plicogeno della surrenale e secrezione del Corticosterone ndl ratto. Arch. sci. Biol. (Bologna) 58 (1974) 71-77.
a22
bf.
GIVEN
11. Civen M., Leeb J. E., Wishnow R. M. and Morin R. J.: Effects of dietary ascorbic acid and vitamin E deficiency on rat adrenal cholesterol ester metabolism and corticosteroidogenesis. Inr. J. Fit. Nutr. Res. SO (1980) 79-88. 12. Pedersen R. C. and Brownie A. C.: Failure of ACTH to mimic the stress-induced activation of rat adrenocorti-
et 01.
cal cholesterol ester hydrolase in aico. J. sreroid Biothem. 11 (1979) 1393-1400. 13. Klemckt H. G. and Brinkley H. J.: Endogenous rhythms of iuteal and adrenal cholesterol ester hydrolast and serum Prl, LH and progesterone in mature pseudopregnant rats. Biol. Reprod. 22 (1980) 1022-1028.