Correlation of rat liver chromatin-bound free and esterified cholesterol with the circadian rhythm of cholesterol biosynthesis in the rat

59

Biochimica et Biophysics Acta, 409 (1975) 59-67 0 Elsevier Scientific Publishing Company, Amsterdam

- Printed in The Netherlands

BBA 56669

CORRELATION OF RAT LIVER CHROMATIN-BOUND ESTERIFIED CHOLESTEROL WITH THE CIRCADIAN CHOLESTEROL BIOSYNTHESIS IN THE RAT

FREE AND RHYTHM OF

SANDRA K. ERICKSON, AUDREY M. DAVISON and R. GORDON GOULD Department (U.S.A.)

of Medicine, Stanford

University School of Medicine, Stanford,

Calif. 94305

(Received March 18th, 1975)

Summary Cholesterol has been shown to be present. in rat liver chromatin isolated by methods designed to avoid contamination by membrane fragments. Evidence that the cholesterol was actually a component of chromatin includes (a) the constancy of the amount (1.30 + 0.14 pg per mg DNA), (b) the striking difference in the ratio of free (i.e. unesterified) to esterified cholesterol between that in chromatin and that in membranes, and (c) the rapid and marked changes which occurred in this ratio during the circadian cycle in chromatin but not in membranes. Although the total amount of chromatin-bound cholesterol did not change throughout the circadian cycle, the concentration of free cholesterol increased sharply a short time before the peak of cholesterol synthetic activity was reached at about midnight; it, reached a basal level about 6 h later at approximately the same time the rate of synthesis returned to its basal level. When labelled cholesterol was administered by stomach tube, it. was detectable within 2 h in whole nuclei and in chromatin, indicating that chromatin-bound cholesterol is rapidly exchangeable with that in liver cytoplasm and in blood plasma. Removal of basic proteins from chromatin did not result in the loss of any cholesterol, but removal of most, of the acidic as well as the basic proteins resulted in loss of most of the chromatin-bound cholesterol. These results indicate that cholesterol is bound either to the acidic proteins or to both the acidic proteins and DNA. The data ,qe compatible with the hypothesis that cholesterol biosynthesis is controlled at the nuclear level and suggest that the relative amounts of free and esterified cholesterol associated with chromatin may play a role.

60

Introduction The rate of cholesterol biosynthesis in liver varies over a very wide range in response to cholesterol feeding, starvation, and many other experimental procedures. Normal rats fed ad libitum show a pronounced circadian rhythm in synthetic rate with a rapid rise from the beginning of the dark phase to midnight, followed by an equally rapid fall to a basal level [l-3] . The overall rate of synthesis is controlled primarily by the activity of one enzyme, 3-hydroxy3-methylglutaryl-CoA reductase [4,5] which has a turn-over half time of 2--4 h [6,7] . It has been shown that increases in the rate of synthesis of cholesterol in vivo are usually dependent on increases in the rate of synthesis of the reductase as judged by the effects of inhibitors of protein and of RNA synthesis [6-691. Cholesterol in a variety of forms, including lipoproteins, has been found to have no effect on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase when added in vitro. Hypocholesterolemic drugs and certain hormones also have no effect, in vitro except in such high doses that non-specific effects are produced. When given to the intact animal, however, cholesterol [lO,ll] and a number of drugs and hormones produce changes in reductase activity and cholesterol synthesis within a few hours, probably due to increases or decreases in the rate of synthesis of 3-hydroxy-3-methylglutaryl-CoA reductase. Since changes in rate of synthesis have been reported to occur only in preparations containing a functioning nucleus, i.e. intact animals, perfused liver [12], hepatocyte suspensions [13,14] or cells in tissue culture [15-191, it is clearly of interest to investigate the possibility that cholesterol (or a metabolite) may be bound to chromatin in the nucleus and may be influencing the synthesis of the reductase through genetic control mechanisms. Recent work demonstrating that steroid hormones bind to chromatin [20-251 suggests that the structurally similar cholesterol molecule might bind to chromatin in an analogous fashion. Little work has been reported on chromatin-bound lipids, and much of that has been restricted to phospholipids [ 26--311. The present investigation deals with the presence of cholesterol in rat liver chromatin, its distribution between free* and esterified forms, and the changes in this distribution during the circadian cycle. A preliminary report of this work has appeared [32] . Experimental

Procedure

Materials and Methods Chemicals. Cholesterol,

obtained from Eastman Kodak, was purified by recrystallization from glacial acetic acid. It was checked for purity by melting point and by thin-layer chromatography. Bovine serum albumin was obtained from Sigma and calf thymus DNA from Calbiochem. All other chemicals used were reagent grade. [1,2-3Hz ] Cholesterol, obtained from New England Nuclear, had a specific activity of 39-52 Ci/mmol. It was in benzene solution and was stored at * Free cholesterol in this paper refers to unesterified cholesterol which mw. protein.

however. be bound to

61

15°C. Before use aliquots were chromatographed on thin-layer plates of silica gel HR with three systems; light petroleum/ethyl ether/glacial acetic acid (75 : 25 : 1, v/v), benzene/ethyl acetate (9 : 1, v/v), and benzene/ether (1 : 1, v/v). The cholesterol band was eluted and the 3H content found to be >90% of that applied. Animals. Sprague-Dawley male rats (150-220 g) fed Purina Rat Chow ad libitum were kept in the animal room for 3 weeks before use. Preparation of rut liver nuclei. Nuclei were isolated according to the method of Blobel and Potter [33] . The pelleted nuclei were washed once with 0.5 M sucrose buffer containing 0.05 M Tris - HCl, 0.025 M KCl, 0.005 M MgClz at pH 7.5, by gentle homogenization with a loose fitting teflon-glass homogenizer and centrifugation at 700 X g for 10 min. Microscopic examination of the washed nuclear suspension showed no evidence of whole cells or cellular debris. Preparation of chromatin. The outer membrane was removed by gentle homogenization of the nuclei in the 0.5 M sucrose buffer described above + 0.5% Triton X-100, followed by centrifugation at 700 X g for 10 min as described by Blobel and Potter [ 331. Chromatin was prepared from the resulting nuclei by homogenization in 0.01 M Tris buffer containing 8 mM EDTA, pH 8.0, and centrifugation through 1.7 M sucrose in 0.01 M Tris buffer, pH 8.0, for 90 min at 78 000 X g, as described by Swanek et al. [25]. The chromatin pellet was washed twice by homogenizing in 0.01 M Tris * HCl, pH 8.0, and centrifuging 10 min at 700 X g and then was resuspended in the same buffer for analysis. Microscopic examination showed no detectable nuclei or membrane fragments. Fractionation of chromatin. Chromatin was separated into histone and DNA - acidic protein fractions by the method of Spelsberg et al. [34] using 2.0 M NaCl in acetate buffer, pH 6.5. It was fractionated into a protein fraction containing all the histones and approx. 90% of the acidic proteins and a DNA fraction containing the remaining acidic proteins by the method of Spelsberg et al. [35] using 5.0 M urea, 2.0 M NaCl, 0.5 mM NaHS03, 0.5 mM EDTA in Tris buffer, pH 8.5. Preparation of liver microsomes and cytosol. Microsomes were prepared and microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity was assayed as described by Heller and Gould 1361. The supernatant from the microsome preparation (105 000 X g) was removed and recentrifuged 1 h at 105 000 X g to remove any remaining particulate material. The top one-fifth containing the fatty layer was removed and the upper two-thirds of the remaining supernatant was defined as the “cytosol fraction”. Analytical methods. Protein was estimated by the method of Lowry et al. [37] using bovine serum albumin as standard. DNA was determined by the diphenylamine method of Burton [38] using calf thymus DNA as standard. Total cholesterol was isolated by saponification and extraction with light petroleum [39]. Free and esterified cholesterol were isolated by extraction of tissue fractions with chloroform/methanol (2 : 1, v/v) and separation by thin-layer chromatography [ 401. Cholesterol was determined calorimetrically by the FeCl, method described by Leffler [41] for samples containing more than 20 pg cholesterol. For smaller samples, the FeC13 -fluorimetric micro-determination method of Solow and Freeman [42] was used except that an Aminco-

62

Bowman fluorimeter was found to be necessary for adequate sensitivity and reproducibility. This method gave accurate and reproducible values for a sample size of about l--15 pg cholesterol. Comparison with the Leffler [41] method on extracts of serum and liver homogenate showed no significant difference. Phospholipid analysis. Chromatin was extracted for total lipid with chloroform/methanol by the standard method [40]. Total phosphorus in the extract was determined by the method of Bartlett [43] and total phospholipid was estimated as 25 X [Pi]. Labelling of subcellular fractions of rat liver with [3H]cholesterol. [1,23 H2 ] Cholesterol, checked for radiochemical purity within 1 week, was dissolved in corn oil and given to rats by stomach tube at a standard dose of 0.7 pg cholesterol in 0.5 ml of corn oil per rat. The animals were killed 2 h later, the. livers excised and subcellular fractions prepared as described above. Aliquots of each fraction were extracted for total lipids, the lipids chromatographed on thin layer in the light petroleum/ethyl ether/glacial acetic acid system (75 : 25 : 1, v/v) and the bands corresponding to cholesterol and cholesterol esters scraped and eluted with chloroform (3 X 5 ml). Aliquots of these extracts were used to determine cholesterol fluorimetrically and to determine radioactivity by liquid scintillation counting in toluene/Liquifluor. Results Chromatin-bound cholesterol. Chromatin varies in composition depending on the method of preparation used and is best characterized by the protein : DNA ratio. Reported values of this ratio vary from 1.2 to 2.3 for chromatin isolated by usual methods with 1.6 as the generally accepted value [44-491. In this investigation the mean protein : DNA ratio was 1.28 f 0.06, (n = 30), a relatively low value indicating little if any contamination by protein such as membrane fragments (see Dice and Schimke [47] for discussion). Determinations of the total cholesterol present in chromatin per mg DNA on rats killed at various times during the circadian cycle showed no significant differences (Fig. 1, total cholesterol). The mean of all the values (n = 30) was 1.3 with SE. of 0.14. Gas-liquid chromatographic analysis of the total non-saponifiable fraction of chromatin demonstrated the presence of cholesterol as the only sterol constituent present in significant amount. Only trace amounts of other sterols could be detected*. Chromatin-bound phospholipids. The mean value for total phospholipids in pg/mg DNA was 2.26 f 0.25 (n = 13). The molar ratio of cholesterol : phospholipids was about 1.0 corresponding to a weight ratio of about 0.5. Rat liver microsomal, mitochondrial and nuclear membranes have been reported as

* Gas-liquid chromatography of chromatin extracts was kindly done by Dr R. Clayton. Practically all the material was found to have the retention time of cholesterol, but there was a small shoulder on the short retention side of the cholesterol peak, and a second minor constituent with about one-half the retention time of cholesterol. No evidence of any ‘T-k&o or 7-hydroxy-cholesterol was found.

63

I

I

-0

0

v

TOTAL

FREE

I

I

hd

0

“4

I

no ”

I

I

I

o”

4Y

0

,

0.0 =

50.6

5

:--i\::

I L-9

I L-12

I D-3

I D-6

TIME

OF

I D-9

I

I

I

D-12

L-3

L-6

DAY

Fig. 1. Variation with time of day of total and free cholesterol associated with rat liver chromatii from rats on a normal diet and comparison with reductase activity. The left-hand scale gives fig cholesterol/mg DNA for total cholesterol (0) and free cholesterol (01. The right-hand scale gives 3-hydroxy-3-methylglutaryl-CoA reducatse activities expressed as nmol/min per mg protein. This curve is a composite of results from this laboratory. Measurements of the reductase in animals killed at midnight and at noon from which chromatin was also isolated fell on this curve. At least two animals were assayed separately for each point.

having cholesterol : phospholipid molar ratios of about 0.1, and liver plasma membranes of 0.76 [ 12,50--521. Distribution of cholesterol in chromatin subfractions. Removal of histones from chromatin by treatment with NaCl resulted in no significant loss of cholesterol (Table I). Removal of the histones and most of the acidic proteins by treatment with NaCl and urea [ 351 resulted in the disappearance of about 84% of the cholesterol from the DNA (Table I). Only a small part (10% or less) of the original cholesterol was detected in the histone fractions, and the rest was found associated with the acidic protein fraction. Changes in free and esterified ctyomatin-bound cholesterol during the circadian rhythm. Although the total cholesterol present in chromatin remained constant during the circadian cycle, the proportions of free and esterified cholesterol changed significantly (Fig. 1). Shortly before the midpoint of the dark phase (D-6)* there was a rapid increase in the free cholesterol and a corresponding decrease in the esterified fraction. 3-4 h later the free cholesterol concentration started to decrease rapidly and reached a level slightly

* D-6 denotes t’he 6th h of the dark phase. Rats were kept on a standard lighting cycle of 12 h of light and 12 h of dark. In one animal room the 12-h light phase started at 6:00 a.m. and in a second it started at 4:00 p.m. Consequently the terminology defining the time the animal was killed relative to the number of hours after the beginning of a light (L) or dark (D) phase is used throughout.

64 TABLE

I

DISTRIBUTION DNA, protein, The chromatin

OF CHOLESTEROL

IN CHROMATIN

FRACTIONS

and cholesterol were determined for each fraction as described in Experimental fractions from livers of four animals were pooled in each experiment.

: DNA

Experiment

Fraction

Protein

ratio

Cholesterol

1

whole chromatin DNA + acidic protein histones

1.34 0.52

100 89 11

2

whole chromatin DNA + 10% acidic protein histones + 90% acidic protein

1.40 0.10 ~-

100 16 84

in protein

Procedure.

fraction

(5%)

below the basal level between D-10 and D-12. Corresponding increases occurred in the esterified cholesterol resulting in no significant changes in the total amount of cholesterol per mg DNA. During the 16 h between L-l and D-4 there were no significant changes in the free or esterified cholesterol concentrations. The mean value of 1.05 for free cholesterol during the period from D-6 to D-10 was significantly greater than the mean value of 0.75 from L-4 to D-5 (P< 0.001,?2 = 38). It is well established that 3-hydroxy-3-methylglutaryl-CoA reductase activity increases from about L-12 to D-6, followed by a steep decline until about D-12, and from L-l until about L-11 it remains at a relatively constant basal level (Fig. 1) [1,3,6]. In the present experiments, the rapid rise in chromatinbound free cholesterol was observed to occur shortly before the rapid decrease in activity of the reductase; the later rapid decrease in free chromatin cholesterol occurred shortly before the stabilization of the reductase activity at the basal level. Concentrations of cholesterol in some subcellular fractions of rat liver. To obtain more information on possible contamination of chromatin by membrane fragments we isolated a number of hepatic subcellular fractions and determined the total cholesterol per mg protein and the free to esterified cholesterol ratios to compare with results for chromatin. Total cholesterol, expressed as pg/mg protein, and free to esterified cholesterol ratios for these subcellular fractions are shown in Table II. These ratios did not change significantly in the microsomal or nuclear fractions during the circadian rhythm but did change in chromatin. Only about 10% of the total nuclear cholesterol was found in the chromatin fraction (Table II). The remainder was very largely in the nuclear membranes. Much, and probably most, of the nuclear membrane cholesterol is in the outer membrane which in our studies was removed by treatment with Triton X-100 as described by Blobel and Potter [ 331. They reported that treatment of rat liver nuclei with 0.5-2s Triton X-100 removed the outer membrane, leaving the nuclei otherwise intact as judged by electron microscopy and DNA and RNA analyses. Cholesterol per mg DNA in whole nuclei was 12.0 Erg compared with that in chromatin of only 1.3 pg. Consequently, the free to esterified cholesterol ratio for nuclei reflects that of the nuclear mem-

65 TABLE

II

CHOLESTEROL CONTENT AND FREE TO ESTERIFIED CELLULAR FRACTIONS OF RAT LIVER

CHOLESTEROL

RATIOS

IN SOME

SUB-

Rat liver subceIIular fractions were isolated and analyzed for p’rotein. total free and esterified cholesterol as described in Experimental Procedure. Values are f S.E. The number of determinations is given in parentheses. Three animals were used in each experiment. Fraction

pg cholesterol per mg protein

pg cholesterol per mg DNA

Free to esterified cholesterol ratio

Nuclei (7) Chromatin (15) (basal) Chromatin (10) (post-midnight) Microsomes (6) Cytosol(5)

5.02 0.78 0.78 24.22 0.66

12.00 ?: 1.20 1.30 + 0.14 1.30 + 0.14

7.4 1.45 3.7 13.6 1.06

+ i t + +

0.48 0.09 0.09 1.85 0.08

braill. The high free to esterified cholesterol ratios of the microsomal and nuclear fractions are typical of membranes [ 50-521 and contrast with the free to esterified cholesterol ratios of about 1 for chromatin and cytosol from basal rats. L3H] Cholesterol studies. When [ 3H] cholesterol was administered to rats it was found in a series of exploratory experiments that labelled cholesterol appeared rapidly in liver subcellular fractions including chromatin. In a typical experiment with animals killed 2 h after administration of trace amounts of [ 3H] cholesterol in corn oil by stomach tube, the specific activities of cholesterol from some subcellular fractions (relative to whole liver cholesterol as 1.0) were: cytosol = 0.90, nuclei = 0.82, microsomes = 0.27, chromatin = 0.14. Discussion Previous investigations on lipids in nuclei and in chromatin have largely been restricted to phospholipids [26,27,29--311. However, Rose and Frenster [28] reported finding 25 pg of phospholipids and 3 pg of cholesterol per mg DNA in calf thymus chromatin. It is not clear whether the considerably smaller values which we found in rat liver chromatin were due to the use of different species and tissues or to different methods of preparation of chromatin. The protein : DNA ratios were 1.8 in the Rose and Frenster [ 281 study and 1.3 in ours; in addition, the free to esterified cholesterol ratio was 10 in the Rose and Frenster [28] study and in the range 0.8-4 in ours, depending on the time of day the animals were killed. The molar cholesterol : phospholipid ratio also differed; it was about 0.1 in the Rose and Frenster [28] study compared with 1.1 in our studies. It is very difficult to establish that a preparation of chromatin is completely free of contaminating membrane fragments or other proteins or lipids. The evidence accumulated in the present investigation indicated no significant contamination and may be summarized as follows: (a) Examination of the nuclei after removal of the outer membrane by optical and electron microscopy gave no evidence of contamination by nuclear outer membrane or by other mem-

66

branes. (b) The protein : DNA ratio of 1.3 of the chromatin preparation indicates relatively little if any extraneous protein. (c) The molar ratio of free to esterified cholesterol in chromatin varied from 0.8 to 3.7 depending on the circadian rhythm whereas that in other membranes did not vary and was much higher for nuclear membrane, microsomal, and mitochondrial outer membranes. (d) The amount of phospholipid found in rat liver chromatin relative to cholesterol was quite different from that found in membranes. The molar ratio of phospholipids : cholesterol was 1.1 + 0.1 compared to about 0.1 [12,50521 for intracellular membranes and 0.76 [ 521 for plasma membranes. It seems highly unlikely that plasma membrane fragments could contaminate the chromatin preparations, and other membranes differ so markedly in this ratio that significant contamination by them also appears to be ruled out. The evidence above supports the hypothesis that the cholesterol found bound to rat liver chromatin was not due simply to random membrane contamination but was an integral part of chromatin. Treatment of chromatin to remove basic proteins resulted in a DNA . acidic protein complex that contained nearly the same amount of cholesterol as the original chromatin. Similar observations have been reported for binding of steroid hormones to chromatin [20----251. The binding of cholesterol to chromatin may be considered to be hydrophobic since the cholesterol was extractable by certain organic solvents, and no apparent dissociation of cholesterol from chromatin occurred on treatment with high salt concentrations (2.0 M NaCl) . Several investigators have concluded that the changes in activity of 3hydroxy-3-methylglutaryl-CoA reducatse during the circadian rhythm result from an increased rate of synthesis of the enzyme from about L-12 until D-6, followed by little or no synthesis from D-6 to about D-12 when a relatively slow, constant rate is maintained [7,6]. The period when the free to esterified cholesterol ratio in chromatin was high corresponds very closely to the period when synthesis of the enzyme is repressed. Although there was no apparent change in the free to esterified cholesterol ratio at the time the rate of enzyme synthesis increased, there was a striking increase in the ratio shortly before the sudden turning off of enzyme synthesis. These findings suggest that the cholesterol associated with chromatin may play a role in the control of the circadian rhythm in cholesterol synthesis; e.g. changes in the amount of chromatinbound free (or esterified) cholesterol may influence the synthesis or inactivation of 3-hydroxy-3-methylglutaryl-CoA redllctase through the genetic mechanism. The actual amount of cholesterol bound to chromatin coresponds to one molecule to about 1000 nucleotide units. Steroid hormones are bound to chromatin at considerably lower ratios (one molecule to about 3 million nucleotide units [53] ) and are generally considered to exert their effects through the genetic mechanism. Acknowledgements We wish to thank Adeiine Shrewsbury for the 3-hydroxy-3-methylglutar~ l-CoA reductase assays, Dr Ray Clayton for the gas-liquid chromatography

67

analysis of chromatin extracts, graphs. This work was supported

and Dr John Frenster for the electron microby N.I.H. Grants HL08476 and HL05360.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

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