THE JOURNAL. OF BIOLOGICAL.

CHEMISTRY
Vol. 254, No. 12, Issue of June 25, pp. 5144-5149, 1979
Prmted in U.S. A.

Active and Inactive Forms of 3-Hydroxy-3-methylglutaryl Coenzyme A

Reductase in the Liver of the Rat
COMPARISON WITH THE RATE OF CHOLESTEROL SYNTHESIS IN DIFFERENT PHYSIOLOGICAL STATES*

(Received for publication, November 17, 1978)

Michael S. Brown, Joseph L. Goldstein, and John M. Dietschy

From the Denartments of Internal Medicine and Molecular Genetics, The University of Texas Health Science Center at
Dallas, Dalias, Texas 7i235

Hepatic 3-hydroxy-3-methylglutaryl coenzyme A re- CoA reductase’ activity and the rate of cholesterol synthesis
ductase, the rate-controlling enzyme in cholesterol syn- are both enhanced by treatments that remove cholesterol
thesis, exists in a phosphorylated (inactive) and de- from the liver such as the administration of Triton-WR 1339
phosphorylated (active) form. The current studies were or the feeding of the bile acid-binding resin cholestyramine
undertaken to determine whether long term regulation (l-3). On the other hand, enzyme activity and the rate of
of reductase in rat liver is achieved through changes in cholesterol synthesis in liver are reduced in parallel when rats
the proportion of enzyme in the two forms. Rates of

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are fed cholesterol or when they are subjected to prolonged
cholesterol synthesis were varied 50-fold by diurnal fasting (l-3). In addition, the activity of hepatic HMG-CoA
light cycling, fasting, stress, and feeding of cholesterol reductase is subject to a marked diurnal cycle, which is
and cholestyramine. Portions of each liver were used accompanied by parallel changes in the rate of cholesterol
to measure 1) rate of cholesterol synthesis in tissue
synthesis from acetate (l-3).
slices, and 2) reductase activity in microsomes pre-
The mechanisms for the changes in hepatic HMG-CoA
pared in four different ways: after isolation in absence
and presence of 50 mM sodium fluoride and after prein- reductase activity have received much attention. Early studies
cubation in absence and presence of Escherichia coli of the decay of enzyme activity following the administration
alkaline phosphatase. Sodium fluoride was added to of cycloheximide to rats indicated that the enzyme has a rapid
the homogenization buffer because it inhibits dephos- turnover with a half-life of approximately 4 h (4). Changes in
phorylation of the enzyme during the microsomal iso- enzyme activity during the diurnal cycle were interpreted as
lation. E. coli alkaline phosphatase was used to maxi- being due to changes in the rate of enzyme synthesis (4).
mally activate the enzyme by removing a phosphate Using an immunochemical approach to measure the amount
group. of HMG-CoA reductase protein and its rate of synthesis,
In livers of animals at midpoint of the dark cycle, Higgins et al. also concluded that changes in enzyme activity
reductase activity was ‘I-fold higher in microsomes pre- during the diurnal cycle and following cholestyramine feeding
pared in absence of fluoride than in microsomes pre- were due to alterations in the rate of synthesis of HMG-CoA
pared in presence of fluoride. Alkaline phosphatase reductase protein (5, 6). On the other hand, these workers
activated reductase of fluoride-treated microsomes 13- found that suppression of enzyme activity by cholesterol feed-
fold, but it produced only a 1.4-fold activation of micro- ing was more complex. Within 6 h after cholesterol feeding,
somes isolated in absence of fluoride. Using enzyme enzyme activity was relatively low as compared with the
activity after phosphatase treatment as a measure of amount of immunochemically detectable enzyme protein.
total reductase, we calculated that 75 to 90% of reduc- However, after 12 h both the amount of enzyme protein and
tase under all physiological conditions was in a phos- enzyme activity were equally reduced. These data suggested
phorylated (inactive) form at the time the homogenates that cholesterol feeding lowered HMG-CoA reductase activity
were prepared. The proportion of phosphorylated en-
by two mechanisms, an immediate inactivation of preformed
zyme remained constant under conditions in which
enzyme and a longer term reduction of enzyme synthesis (5,
total reductase activity and the rate of cholesterol syn-
thesis varied as much as 50-fold. Thus, long term alter- 6).
ations in cholesterol synthesis in rat liver are due not A series of in vitro studies has supported the notion that in
to changes in the state of phosphorylation of reductase, addition to its regulation by changes in enzyme synthetic rates
but rather to changes in the total amount of enzyme the activity of HMG-CoA reductase may be regulated by
protein. posttranslational enzyme modification. Beg et al. reported
that hepatic HMG-CoA reductase was inactivated in vitro
when microsomes were incubated with cytosol in the presence
of ATP and magnesium (7). Similar ATP-dependent inacti-
Under physiologic conditions, the microsomal enzyme 3- vation of HMG-CoA reductase was reported to be catalyzed
hydroxy-3-methylglutaryl coenzyme A reductase appears to by a cytosolic enzyme in human fibroblasts (8). Nordstrom et
control the rate of cholesterol biosynthesis in rat liver and in al. made the important observation that HMG-CoA reductase
a number of other mammalian tissues as well (l-3). HMG- that had been inactivated in the ATP-dependent reaction
* This research was supported by United States Public Health could be reactivated by incubation with a cytosolic enzyme
Service Research Grants HL 20948, HL 09610, AM 16386, and AM from rat liver in the presence of EDTA (9). This reactivation
19329. The costs of publication of this article were defrayed in part by was inhibited by sodium fluoride, an inhibitor of phosphatases,
the payment of page charges. This article must therefore be hereby
marked “aduertisement” in accordance with 18 U.S.C. Section 1734 ’ The abbreviation used is: HMG-CoA reductase, 3-hydroxy-3-
solely to indicate this fact. methylglutaryl coenzyme A reductase.

5144
 Active and Inactive Forms of HMG-CoA Reductase 5145

suggesting that the reactivation involved a dephosphorylation light cycle. E, fasted, stressed, mid-dark; animals were placed in
of a previously phosphorylated enzyme (9). Gibson and co- individual restraining cages and fasted for 48 h prior to the time they
were killed at the mid-dark point of the light cycle. F, CHO-fed, mid-
workers have reported that the reactivation of inactive HMG-
dark; animals were fed a 1% cholesterol, 10% corn oil diet (w/w) for
CoA reductase can also be achieved by incubation with a 12 h before they were killed at the mid-dark point of the light cycle.
partially purified preparation of hepatic phosphoprotein phos- G and H, CS-fed, mid-dark and mid-light; animals were fed a 2%
phatase (10, 11). Recently, Beg et al. demonstrated that the cholestyramine diet (w/w) for 72 h before they were killed at either
inactivation of hepatic HMG-CoA reductase in the presence the mid-dark or the mid-light phase of the light cycle. Immediately
of ATP/magnesium is accompanied by the incorporation of after killing the various groups of animals by decapitation, the livers
were removed and chilled in cold 0.9% NaCl solution. Aliquots of each
32P from [y-“‘P]ATP into a microsomal protein that could be
liver were then taken for the preparation of microsomes and tissue
precipitated by an antibody to HMG-CoA reductase (12). slices as described below.
In the course of their studies, Nordstrom et al. made the Preparation of Liver Microsomes-Aliquots of each liver (approx-
surprising observation that activation of rat liver HMG-CoA imately 1 g) were weighed and placed into 4:l (v/w) cold homogeni-
reductase appears to occur during the isolation of microsomes zation medium containing 0.3 M sucrose, 10 mM 2-mercaptoethanol,
by the usual techniques employed in most laboratories (9). 10 InM sodium EDTA (pH 7.4), and either 50 mM sodium chloride or
50 mM sodium fluoride. The livers were homogenized at 4°C in a
These workers showed that if the tissue homogenization and
Dounce homogenizer with 10 strokes of a loose fitting pestle followed
microsome isolation were carried out in the presence of fluo- by 5 strokes of a tight fitting pestle. Each homogenate was centrifuged
ride, so as to inhibit the putative hepatic phosphoprotein for 15 min at 12,000 x g at 4°C and the supernatant fraction was
phosphatase, the resultant microsomal HMG-CoA reductase then centrifuged for 60 min at 100,000 x g in a Beckman-Spinco
activity was only one-seventh of the amount that was found ultracentrifuge at 4°C. Unless otherwise indicated, the resulting mi-
when the microsomes were isolated in the absence of fluoride. crosomal pellets were immediately frozen and were stored at -190°C.
Prior to assay of HMG-CoA reductase activity, each pellet (5 to 10
Moreover, the inactive enzyme isolated in the presence of
mg of protein) was resuspended in 2 to 2.5 ml of Buffer A.
fluoride could be reactivated by incubation with the cytosolic

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Assay of Microsomal HMG-CoA Reductase Activity-The stan-
activation factor in the absence of fluoride (9). These investi- dard assay for HMG-CoA reductase activity consisted of two sequen-
gators suggested that under ordinary conditions more than tial steps: 1) a preincubation period during which microsomes were
85% of the HMG-CoA reductase in rat liver was in the phos- incubated in the absence or presence of E. coli alkaline phosphatase,
phorylated (inactive) form and that the enzyme became acti- and 2) a subsequent incubation period during which the activity of
vated artifactually during isolation of the microsomes (9). HMG-CoA reductase was measured. The preincubation mixture con-
tained the following concentrations of components in a volume of 90
The above studies raise the question as to whether the d: 20 InM imidazole/chloride (pH 7.4), 5 mM dithiothreitol, 20 to 150
physiologic factors known to regulate HMG-CoA reductase in pg of microsomal protein, and 10 units of E’. coli alkaline phosphatase
rat liver operate predominantly by changing the amount of where indicated. The tubes were incubated at 37’C for 60 min, after
enzyme protein or by changing the state of phosphorylation which 100 ~1 of solution containing 0.2 M potassium phosphate (pH
of the enzyme. Accordingly, in the present study we have 7.4), 40 mM glucose 6-phosphate, 5 InM TPN, 0.7 unit of glucose-6-
performed a series of manipulations designed to alter the rates phosphate dehydrogenase, 20 mM sodium EDTA, and 10 mM dithio-
threitol were added to the preincubation mixture. The HMG-CoA
of hepatic cholesterol synthesis in rats (3, 13, 14). We meas- reductase assay was then initiated with the addition of DL-[%‘%I-
ured the absolute rate of carbon flux into cholesterol in liver HMG-CoA (23,000 cpm/nmol) to a final concentration of 176 PM
slices and made homogenates of the same rat livers in the (final assay volume, 200 ~1). After incubation for 30 min at 37”C, the
absence and presence of fluoride. The microsomes were then [‘%]mevalonate formed was converted into the lactone, isolated by
preincubated with a preparation of E. coli alkaline phospha- thin layer chromatography (16), and counted using an internal stan-
tase that was shown to convert inactive HMG-CoA reductase dard of [:‘H]mevalonate to correct for incomplete recovery (17). In all
figures and tables, each value represents the average of duplicate
to its active form. The latter preincubation allowed an esti-
assays. HMG-CoA reductase activity is expressed as the picomoles of
mate of the total amount of HMG-CoA reductase (active plus [“‘Clmevalonate formed per min per mg of microsomal protein (pmol.
inactive) in the tissue. min’ . mg of protein-‘).
Assay of Rates of Cholesterol Synthesis-Portions of each liver
EXPERIMENTAL PROCEDURES were cut into ribbons approximately 2 to 3 mm thick. Liver slices
O.&mm thick were then prepared on a tissue slicer, and 3O@mg
Materials-nL-3-Hydroxy-3-methyl-[3-’4C]glutaric acid (49 mCi/ aliquots were placed in 25-ml Erlenmeyer flasks fitted with center-
mmol) and sodium [1-‘%]octanoate (1.0 mCi/mg) were purchased wells and containing 5 ml of oxygenated Krebs’ bicarbonate buffer
from New England Nuclear. Glucose-6-phosphate dehydrogenase and [1-‘%]octanoate at a concentration of 1.0 mrvr (3, 18). Generally,
(350 units/mg) was purchased from Boehringer Mannheim. E. coli 6 flasks were run from each animal; 2 flasks were used for zero time
alkaline phosphatase suspended in 2.6 M ammonium sulfate (34 to 56 corrections of mass and radioactivity in the ketone determinations
units/mg of protein) was obtained from Worthington Biochemicals, while the remaining 4 flasks were incubated for 90 min at 37°C in a
Inc. (catalog no. 06124). Just prior to use, the enzyme suspension metabolic shaker set at 160 oscillations per min. At the completion of
containing 20 mg of protein was centrifuged (12,060 x g, 45 min, 4”C), the incubation period one pair of flasks that was incubated at 37’C
the supernatant was discarded, and the pellet (containing the alkaline was used to determine the rates of incorporation of radiolabeled
phosphatase activity) was resuspended in 1 ml of Buffer A (20 mM octanoate into CO* and cholesterol as previously described (3, 18). In
imidazole/chloride, pH 7.4, and 5 rnM dithiothreitol). both situations the total radioactivity found in each product was
Animal Preparations-Female, Sprague-Dawley-derived rats divided by the specific activity of the radiolabeled precursor to yield
were purchased in the weight range of 140 to 170 g from the Charles rates of incorporation that were expressed as either the micromoles
River Breeding Laboratories and placed in gang cages in light cycling or nanomoles of [I-“‘Cloctanoate incorporated into the two products
rooms with alternating 12-h periods of light and darkness (14, 15). per h per g wet weight of liver (pm01 or nmol. h-’ . g-‘). The remaining
The animals were allowed to adapt to the light cycling for 2 to 3 pair of flasks was utilized to determine the rate of synthesis of ketone
weeks during which time they were fed ad libitum Ralston Purina bodies and their specific activities. The production rates of acetoac-
Rat Chow diet. After this period of adaptation, the animals were etate and /3-hydroxybutyrate were combined and are reported as the
allocated to eight different experimental groups designated A through micromoles of total ketone synthesized per h per g of liver. The total
H in Table II and Fig. 2, and these groups were treated as follows. A, radioactivity incorporated into acetoacetate and P-hydroxybutyrate
control, mid-dark; animals were fed ad libitum and killed at the mid- also was measured (19, 20). The observed value for the specific
dark phase of the light cycle. B, control, mid-light; animals were fed activity of the ketones was then calculated and was compared to the
ad Zibitum and killed at the mid-light phase of the light cycle. C and theoretical value that would be expected if no intracellular dilution of
D, fasted, mid-dark; food was removed from the cages either 12 h or the specific activity of the acetyl-CoA pool occurred. The latter value
48 h prior to the time the rats were killed at the mid-dark point of the should equal half of the specific activity of the [I-‘%]octanoate added
5146 Active and Inactive Forms of HMG-CoA Reductase

to the incubation media. In Tables I and II, the term “relative ketone units from [1-‘Cloctanoate into cholesterol in the experiments using
specific activity” equals the absolute ketone specific activity divided liver slices.
by the theoretical specific activity times 100. The rates of incorpora-
tion of [1-‘%]octanoate into cholesterol and CO2 also were corrected RESULTS
for intracellular dilution by endogenous acetyl-CoA units. In addition,
data on the incorporation of [l-‘%]octanoate were used to calculate The major purpose of these studies was to investigate the
incorporation rates in terms of acetyl-CoA units, i.e. C2 units, rather possibility that phosphorylation-dephosphorylation reactions
than nanomoles of octanoate. Hence, the corrected CL flux from [l-
involving HMG-CoA reductase might play a role in the long
‘%]octanoate into cholesterol equals the rate of incorporation of [l-
‘%]octanoate into cholesterol times 4 times 1.5 times 100 divided by term regulation of cholesterol synthesis in the liver under
the relative ketone specific activity. The factor 4 converts the incor- different physiological circumstances. To this end absolute
poration rates of. octanoate to incorporation rates of CZ units. The rates of cholesterol synthesis were measured in liver slices
additional factor of 1.5 corrects for the loss of 33% of the radioactivity using [I-‘%]octanoate as the precursor. Corrections for dilu-
as [“‘C!]C02 during the conversion of [l-‘4C]acetyl-CoA to cholesterol. tion of the specific activity of the intracellular acetyl-CoA pool
A similar calculation was carried out to determine the CP flux into
were made by using the measured specific activity of the
CO*, except that the factor of 1.5 is not required (18).
Calculations-Where appropriate the data obtained in the rats in newly synthesized ketone bodies. The rate of acetyl-CoA (i.e.
each experimental group were combined and are given as mean values C,) flux into cholesterol determined in this manner was then
+ 1 S.E. In Fig. 2 microsomal HMG-CoA reductase activity was compared to the activity of HMG-CoA reductase found in the
correlated with the rate of cholesterol synthesis by fitting the data same livers after preparation of microsomes in the presence
for these two variables obtained in individual animals to linear regres- and absence of 50 mM sodium fluoride and after activation of
sion curves. The regression curves have the usual form of y = a + bx
enzyme activity by preincubation with E. coli alkaline phos-
where a is the intercept on the vertical axis and b is the slope of the
regression curve. phatase.
For purposes of comparison, rates of HMG-CoA reductase activity Initial studies were undertaken to measure the relative

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were converted to rates of Cz flux into cholesterol. On average, 20 mg activities of HMG-CoA reductase in microsomes prepared in
of microsomal protein were recovered from the homogenization of 1 the presence and absence of 50 InM sodium fluoride (Table I).
g wet weight of liver. Thus, the rates of enzyme activity, in pmol. Groups of rats were killed at either the mid-dark or mid-light
mini’ . mg of protein-‘, were multiplied by (20 mg of protein. g-‘) (60
phase of the light cycle. As expected from previous work, the
min. h-‘) (3 C!lunits.pmol of mevalonatee’) (1 x lo-“) to yield the
rates of Cz flux into cholesterol with the units of nmol. he’. g-l, the rate of cholesterol synthesis was 3.2-fold higher at the mid-
same units used to describe the rate of incorporation of acetyl-CoA dark point of the light cycle than at the mid-light time while

TABLE I
HMG-CoA reductase activity in liver microsomes prepared in the presence and absence of 50 mM sodium fluoride
The rats used in this study were adapted to light cycling for 2 absence (0) of 50 mM sodium fluoride in the homogenization buffer.
weeks and were then killed at either the mid-dark or mid-light point The data in columns 3 to 6 were derived from the liver slices while
of the cycle. One portion of each liver was used to prepare liver slices those in columns 7 and 8 were obtained with the microsome prepa-
for assay of the rate of cholesterol synthesis while two other aliquots rations. Each value represents the mean t 1 SE. for results obtained
were used to prepare hepatic microsomes in the presence (+) or in three animals in each experimental group.
Ketone bodies CI flux into HMG-CoA reductase activitv
1. Animal
Experimental group weight 2. Liver weight 3. Relative 4. Synthesis
specific activ- rate 5. con 6. Cholesterol 7. (+) Fluoride 8. (0) Fluoride
ity
g 67 % theoretical pmol.h-‘.g-’ /mol.h-‘.g-’ nmol.h-‘.g-’ pmo1.min-‘.mgprotein~’
A, mid-dark 196 -t 4 7.2 + 0.3 69 f 3 9.8 k 0.5 12.4 + 0.6 967 -c 25 128 + 20 762 + 106
B. mid-light 199 rf: 6 7.4 f 0.2 71 f 2 9.0 -c 0.3 13.0 + 0.4 303 + 30 40 + 12 206 + 5

TABLE II
Comparison of rates of hepatic cholesterol synthesis and HMG-CoA reductase activity in differentphysiological states
As described under “Experimental Procedures,” the rates of hepatic HMG-CoA reductase activities shown in columns 7 through 10 were
cholesterol synthesis were varied over a large range by using animals measured in microsomes prepared in the presence (+) or absence (0)
subjected to eight different treatments; these experimental groups are of 50 mM sodium fluoride and after preincubation in the presence
designated A through H. Columns 1 and 2 give the body weights and (+) or absence (0) of E. coli alkaline phosphatase as described under
liver weights, respectively, at the time the animals were killed. The “Experimental Procedures.” Each represents the mean + 1 SE. for
data on ketone synthesis and the rates of CZ flux into CO* and results obtained in three to seven animals in each experimental group.
cholesterol given in columns 3 through 6 were obtained in liver slices.
Ketone bodies C., flux into HMG-CoA reductase activitv
1. Animal 2. Liver 6. Cho,es- 7. (+) Flue- 8. (0) Flue- 9. (0) Flue- 10. (+) Flu-
Experimental group weight weight 3. Relative 4. Synthesis
specific 5. co1 two1 ride (0) ride (0) ride (+) oride (+)
activitv rate Phosphatase Phosphatase Phosphatase Phosphatase
% them-et- pmunol$‘. pd~i’. n?d,hi’~
g R id g g g
pnd.min-’ m g protein-’

A, control, mid-dark 211 + 7 7.7 f 0.4 69 -t 2 10.1 f 0.9 13.6 f 0.5 1023 f 62 87 e 13 613 + 82 862 f 156 1152 k 162
B, control, mid-light 215 + 3 9.0 f 0.3 69 f 2 8.4 + 0.2 11.9 -c 0.6 305 + 89 32 f 8 138 + 26 225 t 35 227 -c 97
C, fasted, mid-dark (12 h) 163 f 3 5.1 + 0.1 59 -c 1 19.4 f 0.4 8.3 + 0.8 624 & 20 122 f 28 562 + 64 758 f 196 691 f 79
D, fasted, mid-dark (48 h) 171 f 9 5.2 + 0.3 52 f 3 19.7 f 0.8 10.2 -c 0.5 36+ 17 11 f 2 53f 17 82 f 31 55& 19
E, fasted, stressed, mid- 182 f 6 5.7 + 0.3 56 -+ 4 17.8 f 0.6 11.7 + 0.7 500 r+ 143 35 f 3 267 + 48 488 + 102 392 f 65
dark (48 h)
F, CHO-fed, mid-dark (12 215 f 8 8.3 + 0.5 67 k 2 10.3 -c 0.4 14.2 f 0.3 50 + 7 8-+1 70f 13 105 f 21 76 f 10
h)
G, CS-fed, mid-dark (72 h) 288 f 7 10.2 * 1.0 80 + 2 8.3 f 0.5 11.3 + 0.5 1685 f 66 244 + 28 2096 k 168 2833 + 191 2216 +- 473
H. CS-fed. mid-light (72 h) 260 + 5 9.8 + 0.2 77 + 4 6.6 + 0.3 11.9 -t 0.8 1427 f 154 185 + 53 844 f 107 1437 + 306 1393 + 138
 Active and Inactive Forms of HMG- CoA Reductase 5147

the rates of CO, production and ketone synthesis and the

ketone relative specific activities were the same. Microsomal
HMG-CoA reductase activity manifested a nearly identical
3.2- to 3.7-fold relative difference between the two phases of
the light cycle regardless of whether the microsomes were
prepared in the presence or absence of fluoride. However, the
absolute activity in microsomes prepared in the presence of
fluoride was only about 18% of that found in microsomes
prepared in the absence of this ion. This difference was
essentially the same whether the microsomes were prepared
from animals killed at the mid-dark or mid-light phase of the
light cycle. These results confii those reported by Nord- I I , I I I
strom et al. (9) and demonstrate that the HMG-CoA reductase ‘0 2 4 6 8 10
ALKALINE PHOSPHATASE (U.tube-‘1
activity found in a particular liver is profoundly influenced by
the conditions under which the microsomes are prepared. FIG. 1. Activation of rat liver microsomal HMG-CoA reductase by
preincubation with E. coli alkaline phosphatase. Microsomes (45 pg
A second group of studies was next undertaken to establish of protein) prepared from a rat liver that was homogenized in the
the conditions giving maximal activation of HMG-CoA reduc- presence of 50 mM sodium fluoride were preincubated at 37°C for
tase by treatment of the microsomes with a phosphatase. either 30 min or 60 min with the indicated amount of E. coli alkaline
Previously published work has shown that alkaline phospha- phosphatase under the standard conditions. HMG-CoA reductase
tase prepared from E. coli is effective in dephosphorylating a activity was then determined as described under “Experimental Pro-
variety of phosphorylated mammalian proteins including bo- cedures.”
vine heart glycogen synthase D, mixed phosphohistones, and

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rabbit skeletal muscle phosphorylase kinase (21). In prelimi- tase were approximately 7- and 4-fold higher, respectively, in
nary studies we found that this phosphatase preparation also the microsomes prepared in the absence of fluoride than in
was effective in activating hepatic microsomal HMG-CoA those prepared in the presence of this ion (column 7 versus 8).
reductase. This activation was inhibited by 50 mM sodium With the microsomes prepared in the absence of fluoride,
phosphate, indicating that it was probably due to a phospha- treatment with phosphatase resulted in only a small increment
tase activity (21). The time course for this activation process in enzyme activity (column 8 uersus 9). With the microsomes
is shown in Fig. 1. In this study, microsomes from the liver of prepared in the presence of fluoride, phosphatase treatment
a control rat were prepared in the presence of fluoride. Ali- raised the level of HMG-CoA reductase by 13- and 7-fold in
quots of this preparation of microsomes having relatively low the mid-dark and mid-light animals, respectively (column 7
levels of HMG-CoA reductase activity were then preincubated uersus 10). As a result, after phosphatase treatment the level
for either 30 or 60 min with 1 to 10 units of the E. coli alkaline of HMG-CoA reductase activity was similar whether the
phosphatase in the absence of fluoride. When the microsomal microsomes had been prepared with or without fluoride in the
HMG-CoA reductase was subsequently assayed, there was a homogenization medium (column 9 versus IO).
marked increase in activity that was dependent upon both the As anticipated from previous work, fasting for 12 or 48 h
duration of the preincubation period and the amount of phos- (experimental groups C and D) caused a significant increase
phatase in the preincubation medium (Fig. 1). When the in the rate of ketone body synthesis and a decrease in the
microsomes were preincubated for 60 min with 10 units of ketone relative specific activity, reflecting increased levels of
phosphatase, maximal activation of HMG-CoA reductase ac- fatty acid oxidation and acetyl-CoA production. In the animals
tivity was achieved. These conditions, therefore, were chosen fasted for 48 h the rate of C, flux into cholesterol was sup-
to activate the microsomal reductase activity in all subsequent pressed to 4% of the level found in the appropriate control
experiments. animals (experimental group D uersus A), and HMG-CoA
The results of the principal group of experiments are sum- reductase activity was similarly suppressed to 13%, 9%, lo%,
marized in Table II. In these experiments rats were subjected and 5% of the appropriate control values in the microsomes
to a variety of physiological manipulations known to alter prepared in the four different ways (columns 7, 8, 9, and 10,
rates of hepatic cholesterol synthesis. Microsomes were then respectively). In each experimental group, there was once
prepared from the livers of these animals in the presence (+) again a 5-fold higher level of HMG-CoA reductase activity in
and absence (0) of 50 mM sodium fluoride. An aliquot of each microsomes prepared in the absence of fluoride. This differ-
preparation of microsomes was preincubated in the presence ence was eliminated when the enzymes were activated with
(+) and absence (0) of the E. coli alkaline phosphatase. Thus, phosphatase. Qualitatively similar relative changes in Cz flux
four different values for HMG-CoA reductase activity were into cholesterol and in microsomal HMG-CoA reductase ac-
determined in each animal and these values, in turn, were tivity were found when the rates of cholesterol synthesis were
compared to the overall rates of acetyl-CoA (C,) incorporation suppressed by cholesterol feeding rather than by fasting (ex-
into cholesterol in tissue slices prepared from the same livers. perimental group F).
As is evident in experimental groups A and B, light cycling In two final experimental groups of animals, the rates of
caused a 3.4-fold change in the C%flux into cholesterol without cholesterol synthesis were enhanced either by stressing the
any significant alteration in the rates of CO2 production or animals (experimental group E) or by short term cholestyra-
ketone synthesis. Similar relative differences between the mine feeding (experimental groups G and H). Again, qualita-
mid-light and mid-dark animals were seen in assays of HMG- tively similar relative changes were seen in HMG-CoA reduc-
CoA reductase activity in microsomes prepared in the pres- tase activity, i.e. the enzyme activity was reduced by 4- to 7-
ence of fluoride (2.7-fold, column 7), in the absence of fluoride fold in the microsomes prepared in the presence of fluoride,
(4.4-fold, column 8), or after activation of either of these and it was activated by alkaline phosphatase.
enzyme preparations with phosphatase (3.8. and 5.0-fold, col- The data of Table II are plotted in Fig. 2 to show the
umns 9 and 10, respectively). At both the peak and valley of relation between the HMG-CoA reductase activity (ordinate)
the diurnal cycle the absolute activities of HMG-CoA reduc- and the CZ flux into cholesterol (abscissa) under each of the
5148 Active and Inactive Forms of HMG-CoA Reductase

(0) PHOSPHATASE

D A Fasted, M,d-dark (48hrl

E . Fr,s+ed, Stressed,
M,d-dark (48 hr)
F . CHO-Fed, f&d-dark

0 500 1000 1500 2000

C,
FIG. 2. Relation between HMG-CoA reductase activity and cho-
lesterol synthesis under four different conditions of assay of HMG-
CoA reductase activity. These data were derived from the data of
Table II. The data on cholesterol synthesis were obtained in liver
FLUX INTO CHOLESTEROL (nmol.hr-‘.g-‘)
prepared in the presence (+) or absence (0) of 50 mM sodium fluoride
and after preincubation in the presence (+) or absence (0) of E. coli
alkaline phosphatase. The linear regression curves were fitted to data
obtained in 36 individual animals although not all of the points are

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slices. HMG-CoA reductase activity was measured in microsomes shown because of overlap, particularly at the lower rates of synthesis.

four different conditions of enzyme treatment (panels A to D). changes in the state of phosphorylation of the enzyme. The
Whether the microsomes were isolated in the absence or first line of evidence comes from the experiments in which E.
presence of fluoride or whether or not they were subjected to coli alkaline phosphatase was used to convert phosphorylated
prior incubation with alkaline phosphatase, the final measured (inactive) HMG-CoA reductase into its active form. As shown
activity was proportional to the Ca flux into cholesterol under in panels C and D of Fig. 2, total HMG-CoA reductase activity,
all of the physiological conditions tested. In all cases the as measured in the phosphatase-treated microsomes, was
intercepts on the x and y axes were not significantly different strongly correlated with the Cz flux into cholesterol. If enzyme
from zero, and there was a high degree of correlation between activity had been regulated by changes in the state of phos-
the HMG-CoA reductase activity and the Cp flux into choles- phorylation, the total amount of enzyme as measured after
terol (r = 0.86 to 0.89). There was, however, a lo-fold differ- dephosphorylation should have been constant after the var-
ence between the slopes of the lines obtained with the micro- ious physiologic manipulations.
somes prepared in the presence of fluoride (panel A) and The second line of evidence comes from the finding that
those activated with phosphatase (panels C and D). Micro- under each of the physiological conditions studied, HMG-CoA
somes prepared in the conventional manner, i.e. without flu- reductase activity in microsomes isolated in the absence of
oride ion or phosphatase treatment (panel B), manifested an fluoride was 4- to &fold higher than the activity in microsomes
intermediate proportionality constant, and there was greater isolated in the presence of fluoride (Fig. 2, panels A and B).
scatter in the data. Although slight differences in these ratios were seen in the
various experimental groups, the differences were not suffi-
DISCUSSION
cient to account for the marked differences in cholesterol
synthesis in the various groups.
In the current experiments, the rates of hepatic cholesterol The simplest interpretation of the current data is that 75 to
synthesis were varied over a nearly 50-fold range in eight 90% of the HMG-CoA reductase in the liver cell is in a
experimental groups of animals by a variety of physiological phosphorylated form under all physiologic conditions and
manipulations. In all experimental groups three similar find- hence is inactive. When the livers are homogenized and the
ings were observed. First, under all physiological circum- microsomes are isolated in the absence of fluoride, dephos-
stances, the activity of HMG-CoA reductase was 4- to &fold phorylation of the enzyme occurs, and the latent enzyme
higher in microsomes prepared in the absence of fluoride than becomes activated. If fluoride is included in the homogeniza-
in those prepared in the presence of fluoride. Second, in all tion mixture, this dephosphorylation is prevented and the
experimental groups, preincubation of the microsomes with measured activity of the enzyme reflects the activity that was
E. coli alkaline phosphatase preferentially activated the en- present initially in the tissue.
zyme that had been isolated in the presence of fluoride and The reason for having more than 75% of HMG-CoA reduc-
hence abolished the difference between the reductase activi- tase in an inactive (phosphorylated) form at all times in liver
ties in microsomes prepared in the absence and presence of is not clear. The inactive enzyme might serve as a reservoir to
this ion. Third, under all conditions changes in the rate of C2 allow rapid increases in HMG-CoA reductase by the mecha-
flux into cholesterol were reflected by proportional changes in nism of dephosphorylation when hepatocytes are faced with
the relative levels of HMG-CoA reductase activity regardless sudden extraordinary demands for cholesterol. It should be
of whether the microsomes were prepared in the presence or noted that all of the experiments in the current paper repre-
absence of fluoride and regardless of whether or not they were sented relatively long term physiological manipulations. It is
incubated with E. coli alkaline phosphatase. possible that the enzyme activity may be altered by phospho-
Two lines of evidence indicate that under the conditions of rylation and dephosphorylation transiently during some short
the current experiments changes in the Cs flux into cholesterol term control processes.
as measured in liver slices were brought about by changes in In experiments not shown, we have observed that HMG-
the amount of HMG-CoA reductase protein and not by CoA reductase activity in the corpus luteum of the rabbit
 Active and Inactive Forms of HMG-CoA Reductase 5149

ovary (22) also appears to be predominantly in a phosphoryl- takes place rapidly while the animal is being killed and the
ated and inactive form as judged by the same criteria used in liver is being removed.
the current paper.” As with the rat liver enzyme, the propor- From a technical point of view, the present results indicate
tion of rabbit corpus luteal enzyme in the phosphorylated that the relative levels of HMG-CoA reductase as measured
form was similar whether the enzyme activity was low (during in microsomes prepared with or without fluoride correlate
the first few days after ovulation) or high (during the peak of with relative rates of cholesterol synthesis after long term
activity that occurs during pregnancy) (22). On the other variations of cholesterol synthesis in rat liver. The correlations
hand, in cultured human fibroblasts all of the HMG-CoA are somewhat better when the enzyme is assayed after acti-
reductase appears to be in the active form under all conditions, vation with phosphatase. Nevertheless, previous physiological
i.e. there is no reduction in activity when extracts are prepared conclusions about relative levels of HMG-CoA reductase ac-
in the presence of fluoride and no increase when the micro- tivity based on measurements of HMG-CoA reductase in
somes are treated with alkaline phosphatase.” Thus, tissues microsomes prepared in the absence of fluoride, as has been
such as liver and steroid-secreting organs, which may have conventionally done, remain essentially correct.
sudden demands for large amounts of cholesterol, may use the
phosphorylation-dephosphorylation mechanism to maintain Acknowledgments-We thank Gloria Y. Brunschede and Joyce
a reservoir of inactive HMG-CoA reductase that can be Eckles for their expert technical assistance during these studies.
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