THE JOURNAL OF B~oxmcnr. CHEMISTRY Vol. 249, No. 3, Issue of February 10, PP. 789-796, 1974

Printed in U.S.A.

Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme

Reductase Activity in Cultured Human Fibroblasts A

COMPARISON OF CELLS FROM A NORMAL SUBJECT AND FROM A PATIENT WITH HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA*

(Received for publication, August 13, 1973)

MICHAEL S. BROWN, SUZANNA E. DANA, AND JOSEPH L. GOLDSTEINI

From the Divisions of Gastroenterology-Liver and Medical Genetics, Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, Texas 75.235

SUMMARY

The activity of 3-hydroxy-d-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol bio- synthesis, is suppressed in normal fibroblasts cultured in medium containing whole serum. The factor responsible for this suppression was localized to the low density and very low density lipoproteins, but was not found in high density lipoproteins. Whole serum from a patient with abetalipoproteinemia, which is deficient in apolipoprotein B (the protein common to low density and very low density lipoproteins), did not suppress the activity of 3-hydroxy-d- methylglutaryl coenzyme A reductase. Since cholesterol itself was able to suppress enzyme activity when added to cells in a non-lipoprotein form, the data indicate that low density and very low density lipoproteins reduce enzyme activity by delivering cholesterol to the cell. The choles- terol, in turn, acts to inhibit the synthesis of new d-hydroxy- 3-methylglutaryl coenzyme A re’ductase molecules. Cul- tured cells from a homozygote with the disorder familial hypercholesterolemia, which showed a nearly complete failure of suppression of 3-hydroxy-3-methylglutaryl coen- zyme A reductase activity by low density or very low density lipoproteins, synthesized enzyme molecules at a rate nearly 60 times greater than that of normal cells. But when cho- lesterol was administered to these mutant cells in a non- lipoprotein form, it was able to exert a full inhibitory effect on 3-hydroxy-3-methylglutaryl coenzyme A reductase ac- tivity, suggesting that the primary defect in these cells re- sides in the transfer of cholesterol from extracellular low density lipoproteins to its presumed site of action on or within the cell. In further studies of the regulation of 3-hy- droxy-3-methylglutaryl coenzyme A reductase, enzyme activity in normal and mutant fibroblasts was shown to be stimulated by several factors in human serum, one of which is insulin.

* This research was supported by grants from the American Heart Association (72629), the National Institutes of Health (GM 19258, CA 08501, HLlCi024, and 5 TO1 AM 05490), and the Dallas Diabetes Association.

$ Recipient of a Research Career Development Award 1-Kr- GM-70, 277-01 from the United States Public Health Service.

The rate of cholesterol synthesis in cultured human fibroblasts is proportional to the activity of the rate-limiting enzyme in the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase)’ (l-3). In pre- vious studies we have demonstrated that in such cells the activity of this enzyme is regulated in part by the content of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) in the culture medium (l-3). We have also shown recently that this regulatory system is defective in fibroblasts cultured from the skin of subjects with the autosomal dominant disorder familial hypercholesterolemia (2). In cells of subjects with the homozygous form of this disorder, the failure of HMG-CoA re- ductase activity to decline in the presence of lipoproteins results in a marked overproduction of cholesterol, a defect that may be causally related to the accumulation of cholesterol that occurs in the serum and tissues of these patients. In contrast to this de- fect in cultured cells, the serum lipoproteins isolated from homo- zygotes were as effective as serum lipoproteins from normal sub- jects in suppressing HMG-CoA reductase activity of normal cells (2).

In the present studies we have compared the regulation of HMG-CoA reductase under different growth conditions in cells from normal and hypercholesterolemic subjects. The data show that: (a) the inhibitory factor in lipoproteins is cholesterol, (b) of the lipoprotein-bound forms of cholesterol in serum, only that which is bound to the apolipoprotein 13 in LDL and VLDL is inhibitory, (c) cholesterol delivered to normal cells in a non- lipoprotein form is able to suppress HMG-CoA reductase ac- tivity, (d) in contrast to the lack of effect of LDL-cholesterol on hypercholesterolemic cells,2 non-lipoprotein cholesterol exerts a full inhibitory effect on these cells, (e) the inhibitory effect of LDL on normal cells is due to an inhibition of synthesis of HMG- CoA reductase, (f) the high levels of activity in cells from homo- zygotes are due to overproduction of the enzyme, and (g) in addition to inhibitory factors human serum contains several

1 The abbreviations used are: HMG-CoA, 3-hgdroxy-3.methyl- glutaryl coenzyme A; LDL, low density lipoprotkins; VLDL, v&y low density lipoproteins; HDL, high densitv linonroteins. - _ _

2 For the sake of convenience, the term “hypercholesterolemic cells” is used to designate cell lines derived from subjects with the homozygous form of familial hypercholesterolemia.

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factors that stimulate HMG-CoA reductase which is insulin.

EXPERIMENTAL PROCEDURE

activity, one of

~l~ateria&-nL-3-Hydroxy-3-methyl[3-14C]glutaric acid (7.67 mCi per mmole) and nL-[5-aH]mevalonic acid (dibenzylethyl- enediamine salt) (6.7 Ci per mmole) were purchased from the New England Nuclear Corp. m-[2-W]Mevalonic acid lactone (17.5 mCi per mmole) was obtained from Amersham-Searle Corp. Cycloheximide, Tricine, n-glucose 6-phosphate (mono- sodium salt), TPN, and uL-mevalonic acid lactone were pur- chased from Sigma Chemical Co. 3-Hydroxy-3-methylglutaryl coenzyme A was obtained from P-L Biochemicals Inc. Coen- zyme A and glucose 6.phosphate dehydrogenase (350 units per mg) were purchased from Boehringer Mannheim. Cholesterol was obtained from Applied Science Labs, Inc. Insulin (porcine, single component, U-100) was a gift from the Eli Lilly Co. Topical thrombin (bovine origin) was purchased from Parke, Davis and Co. Crystalline bovine plasma albumin was ob- tained from Armour Pharmaceutical Co. Human serum albumin was purchased from Schwarz-Mann. Dithiothreitol (A grade) came from Calbiochem. Kyro EOB (Procter and Gamble Co.) was a gift from Dr. Robert Dowben. Silica Gel G (without gypsum) thin layer chromatography sheets with plastic backs were obtained from Brinkmann Industries. Fetal bovine serum was purchased from Flow Laboratories. Millipore filters (types SXHA, 0.45 pm, and Twin-go, 0.22 pm) were obtained from Millipore Corp. Eagle’s minimum essential medium (Catalogue No. F-11), penicillin (10,000 units per ml)-streptomycin (10,000 ng per ml) solution, trypsin-EDTA solution (1 X), nonessential amino acids solution (100 X), and Puck’s saline A (1 X) were purchased from GIBCO. Tissue culture dishes (60 X 15 mm) and flasks (75 cm2, 250 ml) were obtained from Falcon Plastics. The abetalipoproteinemic serum, collected from Patient R.Kl. (4), was the gift of Doctors H. Klaeveman and J. D. Gardner, National Institutes of Health, Bethesda, Md.

Cells-The normal human fibroblasts used in these studies were established from‘explants of foreskins of healthy newborns. Previous studies showed that the regulation of HMG-CoA re- ductase activity in these cells is similar to that of fibroblasts derived from adults and children with normal plasma lipid levels (2). The hypercholesterolemic fibroblast cell line was obtained from J.P., a 12.year-old subject with the homozygous form of familial hypercholesterolemia (2). All cells were grown in monolayer for about 5 to 20 generations prior to use. Cell lines were maintained in a humidified CO2 incubator at 37” in 75.cm2 flasks containing 10 ml of Eagle’s minimum essential medium supplemented with penicillin (100 units per ml) ; streptomycin (100 pg per ml) ; 0.05 M Tricine, pH 7.4; either 6 or 24 mM NaHC03; 1% (v/v) nonessential amino acids; and 10% (V/V)

fetal calf serum. For all experiments, cells from the stock flasks were dissociated with 0.05% trypsin-0.05% EDTA and were seeded (Day 1) at a concentration of approximately 2.5 X 105 cells per dish into dishes (60 X 15 mm) containing 3 ml of the above growth medium with 10% fetal calf serum. On Day 3 the medium was replaced with fresh growth medium containing 10% fetal calf serum. On Day 6 when the cells were confluent (average cell density, 6 to 9 X 106 cells per dish), the medium was removed, the cellular monolayer was washed with 2 ml of Puck’s saline A, and additions were made as indicated in the legends. All materials, including medium, human serum, and lipoprotein fractions, were sterilized by Millipore filtration be- fore addition to culture dishes.

E&u&-The medium from each flask was discarded and the cells were scraped with a rubber policeman into 1 ml of chilled buffer containing 50 mM Tris-Cl, pH 7.4, and 0.15 M NaCl (Buffer A). After centrifugation (900 X g, 3 min, 24”), the cell pellet was resuspended in 1 ml of Buffer A and washed once more in the same manner. Each pellet was frozen once in liquid nitrogen and kept at -196” until use. Cell extracts were pre- pared by dissolving the thawed pellet of fibroblasts in 0.1 ml of buffer containing 50 mM potassium phosphate, pH 7.4; 5 mM dithiothreitol; 5 mM EDTA; 0.2 M KCl; and 0.25% Kyro EOB.* After incubation for 10 min at 37”, the suspension was centrifuged for 1 min at 12,000 rpm in a Beckman Microfuge, and aliquots of the supernatant were assayed for HMG-CoA reductase activity and protein content. Under these conditions, more than 90% of the total cellular HMG-CoA reductase activity was found in the clear supernatant solution, and the enzyme activity was higher than that obtained by sonication, freeze-thawing, or Dounce homogenization.

HMG-CoA Reductase Assay-HMG-CoA reductase activity was measured by the method previously reported (1, 2). Ali- quots of the cell extract (20 to 100 pg of protein) were incubated for 120 min at 37” in a final volume of 0.2 ml containing 0.1 M potassium phosphate, pH 7.5; 20 mM glucose g-phosphate; 2.5 mM TPN; 0.7 unit of glucose g-phosphate dehydrogenase; 5 mM dithiothreitol; and 3 X 10e5 M DL-[3-14C]HMG-CoA (7.67 mCi per mmole). The DL-[3-14C]HMG-CoA was prepared from the DL-[~-‘~C]HMG acid as previously described (6). The reaction was stopped by addition of 20 ~1 of 5 N HCl. Three micromoles of [5-3H]mevalonolactone (21.8 mCi per mole) was added as an internal standard, and the mixture was incubated at 37” for 15 min and then extracted twice with 10 ml of diethyl ether in the presence of approximately 0.5 g of sodium sulfate. The ether extracts were evaporated to dryness and the mevalonolactone was isolated by thin layer chromatography using silica gel and counted as previously described, the efficiency of the counter being 26% for aH and 68% for 14C (6). Recovery of mevalono- lactone averaged about 50%. The amount of extract was ad- justed so that mevalonate formation was always linear with time and protein concentration (1). The mean variation in HMG- CoA reductase activity between duplicate dishes was less than l t5%. Unless otherwise stated each experimental point repre- sents the HMG-CoA reductase activity of cells from one culture dish.

Lipoproteins-LDL (d 1.019 to 1.063 g per ml), HDL (d 1.063 to 1.215 g per ml), and lipoprotein-deficient serum (d > 1.215 g per ml) were prepared from single 500.ml units of blood collected in 0.1% EDTA from healthy subjects who had been fasted for 15 hours. VLDL (d < 1.006 g per ml) was isolated from 100 ml of blood obtained from a patient with endogenous hy- pertriglyceridemia associated with partial lipodystrophy (fast. ing plasma lipid levels: cholesterol, 180 mg per dl; triglyceride 790 mg per dl). Lipoproteins were fractionated by sequential flotation in a Beckman preparative ultracentrifuge at 214,000 X g,, and 4-10” for 16 to 24 hours according to standard technique5 (7) using solid KBr for density adjustment (8). Isolated frac tions were dialyzed at least 36 hours at 4” against three changef of at least 50 volumes of buffer containing 0.15 M NaCl and 0.: mM EDTA, pH 7.4 (Buffer B). Following dialysis the lipopro. tein-deficient fraction was incubated at 24” for 10 min in the presence of thrombin (20 units per ml). The clot was removec

3 Kyro EOB is a synthetic nonionic detergent that solubilizer plasma membranes of cultured cells but not endoplasmic reticulun (5).

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with an applicator stick and the resulting serum was then clari- fied by centrifugation at 30,000 x g for l-to 2 hours at 4”. After Millipore filtration, all fractions were stored at 4” and maintained full biological activity for at least 2 months. Each isolated lipoprotein fraction migrated as a homogenous peak on lipopro- tein electrophoresis (9). The cholesterol content of sera and lipoprotein fractions was measured using a modification of the method of Zak (10).

Blood from a patient with abetalipoproteinemia and from a normal subject were collected in EDTA (1 mg per ml) and prior to use the plasma samples were coagulated with thrombin and centrifuged as described above. The cholesterol content of the unfractionated abetalipoproteinemic plasma was 55 mg per dl; lipoprotein electrophoresis of this specimen showed a complete absence of fi and pre-P-bands.

RESULTS

The addition of VLDL and LDL to normal human fibroblasts previously grown in the presence of lipoprotein-deficient serum resulted in a concentration-dependent decrease in HMG-CoA reductase activity (Fig. IA). In contrast, these lipoproteins had no effect on the enzyme activity of fibroblasts from a patient with homosygous familial hypercholesterolemia. LDL was ineffec- tive even when added at a cholesterol concentration of 2000 pg per ml, a level that was more than 600 times that required to achieve a 50% reduction of HMG-CoA reductase activity of normal cells (Fig. IB). HDL caused no significant reduction in the enzyme activity of either normal or hypercholesterolemic cells (Fig. 1). Since the two major inhibitory factors of serum, LDL and VLDL, both contain apolipoprotein B and since this protein is not present in HDL (ll), these data suggest that apo-

r B. Hypercholrsterolemic cells

0’ ’ ’ 1 I //’ ’ ’ ’ ’ /l-J- 0 10 20 30 40 0 IO 20 30 40 2000

CHOLESTEROL CONCENTRATION (pg/ml)

Fro. 1. Effect of lipoprotein fractions on HMG-CoA reductase activity of normal (A) and hypercholesterolemic (B) fibroblasts grown in the presence of lipoprotein-deficient serum. Cells were grown in dishes containing 10% fetal calf serum as described under “Experimental Procedure.” On Day 6 the medium was replaced with 3 ml of fresh medium containing human lipoprotein- deficient serum (6 mg of protein per ml). After 24 hours, 75 ~1 of Buffer B containing varying amounts of HDL (0), LDL (A), VLDL ( l ) , or no lipoproteins (0) were added to give the indicated cholesterol concentrations. After 6 hours extracts were prepared and assayed for HMG-CoA reductase activity as described under “Experimental Procedure.” Since the experiments with VLDL and HDL were performed on a different day than those with LDL, the results are expressed as a percentage of the HMG-CoA reduc- tase activity in control cells (0) on the appropriate day. These control activities (picomoles per min per mg of protein) were: BDL and VLDL experiment, 53.1 (normal cells) and 199 (hyper- cholesterolemic cells); LDL experiment, 97.9 (normal cells) and 213 (hypercholesterolemic cells).

lipoprotein B is involved in the previously described serum- mediated depression in HMG-CoA reductase activity (1, 3).

To test this hypothesis serum was obtained from a patient with abetalipoproteinemia, an inherited disorder in which apolipopro- tein B is undetectable in serum (12). When such serum was added to normal cells, it did not depress HMG-CoA reductase activity. In contrast, normal serum added to equal cholesterol concentrations achieved nearly complete suppression (Fig. 2). Since the cholesterol in abetalipoproteinemia serum is found primarily in the HDL fraction (12), these data further confirm the inability of HDL to reduce enzyme activity and suggest that the sole feedback inhibitors in human serum are associated with the lipoproteins containing apolipoprotein B, that is VLDL and

LDL. To determine whether cholesterol delivered to the cell in a

form other than that associated with apolipoprotein B is able to reduce HMG-CoA reductase activity, cholesterol dissolved in ethanol was added to cells in the presence of lipoprotein-deficient serum. Fig. 3 shows that at similar concentrations such non- lipoprotein cholesterol was nearly as effective as LDL-cholesterol in reducing enzyme activity of normal cells. A striking finding was that this non-lipoprotein cholesterol was able to reduce HMG-CoA reductase activity of cells from patients with ho- mozygous familial hypercholesterolemia, even though LDL- cholesterol was ineffective in these cells. When normal and hypercholesterolemic cells were grown for 24 hours in the presence of nonlipoprotein cholesterol, HMG-CoA reductase activity was reduced in both cell lines by more than 90% (Fig. 4).

Considered together, the data in Figs. 1, 2, 3, and 4 suggest that the physiological feedback suppressor of HMG-CoA reduc- tase activity of normal fibroblasts is cholesterol itself and that this cholesterol can exert its effect when it is bound to LDL, VLDL, and non-lipoproteins of plasma but not when bound to HDL. Moreover, these data suggest that the previously re- ported defect in regulation of HMG-CoA reductase activity in

Eo’ ’ I I I I I I 1 0 10 20 30 40

CHOLESTEROL CONCENTRATION (pg/ml)

FIG. 2. Failure of abetalipoproteinemic serum to suppress HMG-CoA reductase activity of normal fibroblasts. Cells were grown in dishes containing 10% fetal calf serum as described under “Experimental Procedure.” On Day 6 the medium was replaced with 3 ml of fresh medium containing human lipoprotein- deficient serum (4.3 mg of protein per ml). After 24 hours, 0.2 ml of 0.9% NaCl containing varying amounts of either unfractionated normal human serum (0) or unfractionated serum from a patient with abetalipoproteinemia (0) was added to give the indicated cholesterol concentrations. After 6 hours extracts were prepared and assayed for HMG-CoA reductase activity as described under “Experimental Procedure.”

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g 211 y.yF., g

0 10 20 30 40 50 CHOLESTEROL CONCENTRATION @g/ml)

FIG. 3 (left). Effect of varying concentrations of lipoprotein cholesterol and non-lipoprotein cholesterol on HMG-CoA reduc- tase activity of normal and hypercholesterolemic fibroblasts. Cells were grown in dishes containing 10% fetal calf serum as described under “Experimental Procedure.” On Day 6 the me- dium was replaced with 3 ml of fresh medium containing human lipoprotein-deficient serum (8.6 mg of protein per ml). After 24 hours the following additions were made to give the indicated cholesterol concentrations: 25 ~1 of ethanol containing varying amounts of cholesterol (0, A) ; 60 ~1 of Buffer B containing vary- ing amounts of LDL (0 ) ; and 60 ~1 of Buffer B containing varying amounts of LDL plus 25 ~1 of ethanol without cholesterol (A). After 6 hours extracts were prepared and assayed for HMG-CoA reductase activity as described under “Experimental Procedure.” Results are expressed as percentage of the activity in cells to which only ethanol or Buffer B was added. These control activ- ities (picomoles per min per mg of protein) were: 0,20.0; 0, 19.3;

cells from subjects with homozygous familial hypercholesterol- emia (2) may involve a specific inability of cholesterol to interact with the cell only when it is bound to LDL and VLDL.

To determine whether cholesterol exerts its effect within cells by direct allosteric inhibition of HMG-CoA reductase, the sterol was added to cell-free extracts in the form of whole serum, LDL, HDL, and in a suspension containing ethanol and lipoprotein- deficient serum. None of these solutions produced any direct inhibitory effect (Table I).

That the serum-mediated depression in HMG-CoA reductase activity was not due to the presence of an intracellular enzyme inhibitor was shown further by the mixing experiments in Table II. Extracts with depressed enzyme activity obtained from normal cells grown in fetal calf serum were mixed with extracts from cells grown in lipoprotein-deficient serum that had elevated enzyme activity. The amount of mevalonate produced by the mixture was equal to the sum of mevalonate formed by each of the two extracts when assayed separately (Table II, Experi- ment A). Additional evidence against direct effecters of HMG- CoA reductase activity in either normal or hypercholesterolemic cells was obtained from a similar experiment in which extracts from suppressed normal cells had no direct effect on the ab- normally elevated enzyme activity of hypercholesterolemic cells and vice versa (Table II, Experiment B).

Evidence that the level of HMG-CoA reductase activity in fibroblasts is related to the rate of synthesis of the enzyme was obtained by experiments in which the turnover rate of the en- zyme was measured under different conditions of growth (Fig. 5). After the administration of cycloheximide to normal cells grown in the presence of fetal calf serum, HMG-CoA reductase activity declined with first order kinetics and a half-life of 2.7 hours (Fig. 5A). I f one assumes that cycloheximide has no sig-

HOURS

A, 115; A, 150. Circles (0, 0) refer to normal cells; triangles (A, A) refer to hypercholesterolemic cells.

FIG. 4 (right). Time course of suppression of HMG-CoA re- ductase activity by non-lipoprotein cholesterol in normal and hypercholesterolemic fibroblasts. Cells were grown in dishes containing 10% fetal calf serum as described under “Experimental Procedure,” except that on Day 3 the medium was replaced with 3 ml of fresh medium containing human lipoprotein-deficient se- rum (6 mg of protein per ml). After 24 hours, 25 ~1 of ethanol containing either no cholesterol (0, A) or 180 pg of cholesterol (0, A) were added. After the indicated time extracts were pre- pared and assayed for HMG-CoA reductase activity as described under “Experimental Procedure.” In the inset enzyme activity in the presence of cholesterol is plotted as a percentage of the activitv in the absence of cholesterol at each time point. Circles (0, 0; (>) refer to normal cells; triangles (A, A, A) refer to hy- percholesterolemic cells. All values represent the mean of du- plicate dishes.

nificant effect on enzyme degradation, then the rate of decline of enzyme activity from the steady state level is a measure of the turnover rate of the enzyme in that steady state (13). It can thus be calculated that the synthesis rate of HMG-CoA reductase in the normal cells grown in the presence of fetal call serum was 0.27 unit per hour.4 When cycloheximide was ad. ministered to normal cells with elevated enzyme levels due tc growth in the presence of lipoprotein-deficient serum, the half. time of decay was similar to that in the fetal calf serum-growr cells (2.9 hours), but the calculated synthesis rate was markedly elevated at 5.9 units per hour. These data indicate that tht activity of HMG-CoA reductase is elevated in the absence 01 serum lipoproteins because the synthesis of the enzyme i: markedly accelerated whereas its fractional degradation rate if unchanged. If lipoproteins act like cycloheximide in inhibiting enzyme synthesis, then a maximal dose of LDL should produce 2 decline in enzyme activity at a rate similar to that produced by cycloheximide. Such an effect is shown in Fig. 5B, in which the HMG-CoA reductase activity of normal cells declined with firs1 order kinetics and a half-life of 3.0 hours after administration 01 LDL.

By similar analyses of enzyme turnover in the presence 0’ cycloheximide, the half-life of HMG-CoA reductase in the cell1 of the patient with hypercholesterolemia grown in fetal cal serum was observed to be 3.0 hours (Fig. 5A), a value not sig nificantly different from that calculated for normal cells. How ever, the calculated synthesis rate was 23.7 units per hour, : rate that is nearly 60 times greater than that of normal cell:

4 For the purposes of this discussion, 1 unit of HMG-CoA reduc tase activity is defined as that amount of enzyme protein tha catalyzes the formation of 1 pmole of mevalonate per min unde the assay conditions described under “Experimental Procedure.’

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TABLE I TABLE II

Effect of whole serum, lipoprotein fractians, and non-lipoprotein Effect of mixing extracts prepared from cells with differing HMG-

CoA reductase activities cholesterolonactivityofHMG-CoAreductaseincell-free extracts

Experiment A. Normal cells grown to confluence were incubated 18 hours in medium without serum after which extracts from three flasks were prepared as described under “Experimental Pro- cedure” and pooled in a final volume of 0.6 ml of Buffer A. Fifty- microliter aliquots of the extracts (250 rg of protein) were incu- bated at 37” for 15 min with 20 ~1 of Buffer B containing the indicated amounts of lipoprotein cholesterol, after which the components of the HMG-CoA reductase assay mixture were added to give a final volume of 0.2 ml and the reaction was started by the addition of [3-l%]HMG CoA. After incubation at 37” for a further 120 min, the amount of mevalonate formed was determined as described under “Experimental Procedure.” Experimerlt U. Normal cells grown to confluence were incubated 24 hours in medium containing human lipoprotein-deficient serum (6 mg of protein per ml) after which extracts from 18 dishes were prepared as described under “Experimental Procedure” and pooled in a final volume of 0.9 ml of Buffer A. Fifty-microliter aliquots of the extracts (100 rg of protein) were added to 0.2 ml of the stand- ard HMG-CoA reductase assay mixture containing the indicated serum fractions at a concentration of 4 mg of protein per ml. The mixtures were preincubated for 15 min at 37” after which the reaction was started by the addition of [3-‘“C]HMG-CoA. After a further 120 min at 37” the assays were terminated and the amount of mevalonate formed was determined as described under “Experi- mental Procedure.” In experiments in which cholesterol was tested, the sterol was first dissolved in ethanol (3.1 or 6.2 mg of cholesterol per ml) and then 40 ~1 were added to 0.5 ml of a solu- tion containing human lipoprotein-deficient serum (50 mg per ml in Buffer B), after which 20 ~1 of this suspension were added to the

Cells were grown in dishes containing lOy, fetal calf serum as described under “Experimental Procedure.” In Experiment A, the medium was replaced on Day 6 with 3 ml of fresh medium containing either 10% fetal calf serum or human lipoprotein- deficient serum (6 mg of protein per ml). After 24 hours cells were harvested, the washed cell pellets from eight dishes were pooled in a final volume of 0.4 ml of Buffer A, and the extract was clarified by centrifugation at 12,000 rpm as described under “Ex- perimental Procedure.” Twenty-five-microliter aliquots of the resulting supernatants were assayed for HMG-CoA reductase for 120 min either alone or together as indicated. In Experiment B, cells grown in 10% fetal calf serum were harvested on Day 6 and the supernatants were prepared and assayed as described in Ex- periment A. Each value represents the mean of duplicate deter- minations.

CdlS Condition of growth Amount of rlevalonate xtract adda formed

pg protein moles/Z hrs

Experiment A Normal Normal

Normal Experiment B

Normal (1). Hypercholes-

terolemic (2). (1) + (2).

Fetal calf serum (1) Lipoprotein-deficient

serum (2) (1) + (2)

73 38.9

64 401

Fetal calf serum

73 + 64

80

Fetal calf serum 25 Fetal calf serum 80 + 25

447

11.0

309 330

- assay mixture.

Addition to assay

Chol- esterol

mcentra. tion in assay

MeValO- nate

formed

Experiment A None...................................... 0 1060

LDL LDL LDL

HDL. HDL HDL. .

Experiment B

250 500

1000

1000 970 950

80 940 160 930 320 910

None......................................

Wholeserum..............................

Lipoprotein-deficient serum . Lipoprotein-deficient serum + 0.8yo ethanol Lipoprotein-deficient serum + 0.8y0 ethanol

+ cholesterol.. . Lipoprotein-deficient serum + 0.8% ethanol

+ cholesterol.. .

0 453

120 448

<l 463 <l 454

25 436

50 451

grown under identical conditions. The demonstration of an elevated synthesis rate of HMG-CoA reductase in hypercholes- terolemic cells grown in the presence of lipoproteins provides direct evidence that the elevated activity of HMG-CoA reduc-

tase in these cells is due to a failure of LDL to inhibit enzyme synthesis.

Since the control of enzyme synthesis in cultured cells may vary with different stages of growth (14), the regulation of HMG-CoA reductase activity was examined in nonconfluent and confluent cells (Fig. 6). When normal cells were noncon- fluent and in log phase growth, the removal of serum from the medium resulted in an increase in enzyme activity of approxi- mately 50-fold over a 24-hour period to a level of 50 pmoles per min per mg (Fig. 6C). In contrast, confluent cells in station- ary phase of growth responded with only about a 2.5-fold induction and attained a level of only 3.5 pmoles per min per mg when serum was removed (Fig. 6D). When fetal calf serum was replaced with lipoprotein-deficient serum, the nor- mal cells in both nonconfluent and confluent states were able to reach levels of enzyme activity above 50 pmoles per min per mg (Fig. 6, A and B). In normal confluent cells the activity of HMG-CoA reductase after 24 hours growth in lipoprotein- deficient serum was 15-fold higher than the activity after growth for 24 hours in medium devoid of serum. It is thus apparent that the lipoprotein-deficient serum contains factors that cause a marked increase in enzyme activity in confluent cells (Fig. 6, B and D).

Similar evidence for the presence of stimulatory factors of HMG-CoA reductase activity in lipoprotein-deficient serum was provided by the experiments using confluent cells of the subject with familial hypercholesterolemia (Fig. 6, B and D). The removal of serum from these cells resulted in a ‘I-fold decline in enzyme activity over a 24-hour period from the abnormally high levels maintained in fetal calf serum (Fig. 6D). This decline was completely prevented by the addition of human lipoprotein-deficient serum (Fig. 6B). In nonconfluent hy-

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TURNOVER RATE OF HMG Cop. REDUCTASE

200 r A. CYCLOHEXIMIOE A

8. LDL

‘Q./ Normal cdl6

ho lipoproteins) 0

HOURS AFTER ADDITION

FIG. 5. Decline of HMG-CoA reductase activity after adminis- tration of cycloheximide (A) or LDL (B) to normal and hyper- cholesterolemic fibroblasts. Cells were grown in dishes containing 10% fetal calf serum as describedunder “Experimental Procedure.” On Day 6 the medium was replaced with fresh medium containing either 10% fetal calf serum ( l , A) or human lipoprotein-deficient serum (5 mg of protein per ml) (0, 0). After 24 hours either 0.1 ml of 0.9% NaCl containing 420 Mg of cycloheximide (A) or 25 ~1 of Buffer B containing 360 pg of LDL-cholesterol (B) was added. At the indicated times extracts were prepared and HMG-CoA reductase activity was measured as described under “Experimen- tal Procedure.” Each point represents the average of duplicate dishes. All lines were fitted to the points by the method of least squares. Circles (0, l ) and squares (0) refer to normal cells. Triangles (A) refer to hypercholesterolemic cells.

percholesterolemic cells, the lipoprotein-deficient serum did

not appear to be required to maintain the high levels of en-

zyme activity (Fig. 6, A and C). Taken together, the data in Fig. 6 suggest that lipoprotein-deficient serum contains one or more factors that are required for the maintenance of high levels of HMG-CoA reductase activity in confluent cells. Noncon- fluent cells appear to be able to maintain high levels of enzyme activity in the absence of any serum factor or factors.

To determine whether insulin played any role in the stim- ulatory effect of lipoprotein-deficient serum, insulin, and lipo- protein-deficient serum were added to normal confluent cells in the presence and absence of fetal calf serum (Fig. 7). In the absence of fetal calf serum. both insulin (Fig. 7A) and lipopro- tein-deficient serum (Fig. 7B) were able to increase HMG-CoA reductase activity. The insulin effect was first apparent at a concentration in the physiological range of between 10~lo and lo-* M (Fig. 7A). Although lipoprotein-deficient serum was able to produce higher levels of enzyme activity than was insulin alone, the addition of insulin to maximally effective levels of lipoprotein-deficient serum caused a slight but definite increase in HMG-CoA reductase activity (Fig. 7B). In the presence of fetal calf serum, neither insulin nor lipoprotein-deficient serum was able to increase enzyme activity (Fig. 7, A and B), indi- cating that in whole serum the inhibitory effect of lipoproteins overcomes the stimulatory effects of any other serum factors. In other experiments not shown it was shown that noncon- fluent cells appeared to have less of a requirement for insulin, a finding similar to that observed for lipoprotein-deficient serum (Fig. 6).

In addition to insulin, albumin and HDL were two other

FIG. 6. Changes in HMG-CoA reductase activity of nonconflu- ent and confluent cells from normal and hypercholesterolemic sub- jects after removal of fetal calf serum and after replacement of fetal calf serum with lipoprotein-deficient human serum. On Day 1, cells were seeded at a concentration of 2.5 X lo6 per dish and grown in medium containing 10% fetal calf serum. For Experiments A and C, on Day 2 the medium was replaced with 3 ml of fresh medium containing either no serum (Experiment C) or human lipoprotein-deficient serum (6 mg per ml) (Experiment A). After the indicated intervals extracts were prepared and HMG- CoA reductase activity was assayed as described under “Experi- mental Procedure.” For Experimenk B and D, the cells were grown for 5 days in medium containing 10% fetal calf serum with fresh medium and fetal calf serum added on Day 3. On Day 6, the medium was replaced with 3 ml of fresh serum (6 mg per ml) (Experiment B). After the indicated time intervals extracts were prepared and HMG-CoA reductase activity was assayed as de- scribed under “Experimental Procedure.” Squares (0, n ) refer to normal cells. Circles (0, 0) refer to hypercholesterolemic cells. In Experiments A and C, the cells were nonconfluent and were in log phase growth; the soluble protein content averaged 54 fig per dish. In Experiments B and D, the cells appeared con- fluent; the soluble protein content averaged 174 rg per dish.

serum factors that appeared to play a permissive role in main- tenance of HMG-CoA reductase activity in confluent cells grown for a prolonged time in the absence of serum (Table III). After 48 hours without serum but in the presence of insulin, the level of enzyme activity was the same in normal and hyper- cholesterolemic cells. After growth in the absence of serum the addition of whole serum caused a decrease in the activity in normal cells. In contrast, the whole serum stimulated the activity in hypercholesterolemic cells, again indicating that in normal cells the inhibitory effect of lipoproteins predominates, but in hypercholesterolemic cells, which are resistant to lipo- protein suppression, the stimulatory effect of the non-lipopro- tein components is unmasked. Under the conditions of this experiment, lipoprotein-deficient serum, albumin, and HDL each allowed the maintenance of higher levels of HMG-CoA reductase activity in both normal and hypercholesterolemic cells. That similar levels of enzyme activity were observed

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in normal and hypercholesterolemic cells under all conditions in which inhibitory lipoproteins were absent is consistent with previous data suggesting that the only genetic abnormality in these cells involves a failure of regulation of HMG-CoA reduc- tase activity by LDL (2).

To determine whether the regulation of HMG-CoA reductase activity by LDL was present at all phases of cell growth, nor- mal and hypercholesterolemic cells were studied after growth at different cell densities (Table IV). In normal cells LDL inhibited HMG-CoA reductase activity by more than 90% regardless of the phase of growth. In hypercholesterolemic

10-12 x9 148 ice 0 INSULIN (mob/l) LIP”oPRO:ElN-D’EFIC:T

SERUM (mg/ml)

FIG. 7. Effect of insulin (A) and lipoprotein-deficient human serum (B) on HMG-CoA reductase activity of normal fibroblasts in the presence and absence of fetal calf serum. Cells were grown in dishes containing lOo]o fetal calf serum as described under “Ex- perimental Conditions.” On Day 6 the medium was replaced with 3 ml of fresh medium containing one of the following additions: none (O), 10e6M insulin (A), or 10% fetal calf serum (0). At the same time varying concentrations of insulin (A) or lipoprotein- deficient human serum (B) were added as indicated. After 24 hours extracts were prepared and assayed for HMG-CoA reductase activity as described under “Experimental Procedure.”

TABLE III Effect of serum factors on HMG-CoA reductase activity of normal

and hypercholesterolemic cells grown to conjluence

Cells were grown in dishes containing 10% fetal calf serum as described under “Experimental Procedure.” On Day 6 the me- dium was replaced with 3 ml of fresh medium containing no serum. After 24 hours the medium was replaced with fresh medium con- taining 10e6~ insulin and the indicated additions. After a further 24 hours, exctracts were prepared and assayed for HMG-CoA reductase activity as described under “Experimental Procedure.” The final concentrations of the added materials in the medium were: whole human serum, 8 mg of protein per ml; human lipo- protein-deficient serum, 6 mg of protein per ml; human serum albumin, 4 mg per ml; and human HDL, 50 Mg of cholesterol per ml. Each value represents the mean of duplicate determinations.

Addition

HMG-CoA reductase activity

Normal cells Hypercho- lesterolemic cells

None............................. Whole serum. Lipoprotein-deficient serum Albumin. HDL

pmoles/min/mg protein

10.2 9.1 4.0 47.5

29.5 38.5 15.5 25.0 18.0 32.3

cells LDL showed no significant inhibitory effect at any growth phase.

DISCUSSION

The studies presented in this paper suggest that the activity of HMG-CoA reductase in cultured human fibroblasts is gov- erned by factors in serum that suppress and stimulate syn- thesis of the enzyme. When normal cells are grown in the presence of unfractionated serum, the inhibitory effect of lipo- proteins predominates and consequently HMG-CoA reductase activity is low. Direct evidence that this serum-mediated sup- pression is due specifically to the presence of LDL and VLDL was obtained by: (a) the experiment in which the addition of LDL and VLDL but not HDL suppressed HMG-CoA reduc- tase activity of normal cells grown in lipoprotein-deficient serum and (b) the experiment in which unfractionated serum from a patient with abetalipoproteinemia was unable to suppress en- zyme activity of normal cells. These data indicate that apoli- poprotein B, which is a component of LDL and VLDL but not of HDL (ll), is specifically involved in the suppression of the enzyme by serum. LDL and VLDL appear to have the ability to suppress enzyme activity because, in contrast to HDL, they can deliver cholesterol to the cell in a form which permits the cholesterol itself to act. Cholesterol can decrease the enzyme activity in the absence of the protein component of LDL and VLDL, however, since when added to normal cells in the pres- ence of lipoprotein-deficient serum the sterol was as effective a suppressor as LDL-cholesterol. Considered together, these data indicate that cholesterol itself is the true feedback sup- pressor of HMG-CoA reductase activity in fibroblasts, but in order to act it must be delivered to the cell in a form other than that bound to HDL. These conclusions regarding the role of lipoproteins and cholesterol in the regulation of HMG-CoA reductase activity in normal human fibroblasts are in agree- ment with the scheme proposed by Bailey as a result of his studies

TABLE IV

Eflect of LDL on HMG-CoA reductase activity in normal and

hypercholesterolemic cells grown at different densities

On Day 1 cells were seeded at the indicated initial concentration and were grown in medium containing 10% fetal calf serum. On Day 3 the medium was replaced with 3 ml of fresh medium con- taining 10% fetal calf serum. On Day 5 the medium was removed and the cellular monolayer was washed with 2 ml of Puck’s saline A, after which 3 ml of fresh medium containing lipoprotein-defi- cient serum (6 mg per ml) with or without LDL (50 pg of choles- terol per ml) were added as indicated. After 24 hours extracts were prepared and HMG-CoA reductase activity was assayed as described under “Experimental Procedure.” Each value repre- sents the mean of duplicate dishes.

Cell density I

HMG-CoA reductase activity

Initial I

Final

No./dish

Experiment A: normal cells 0.68 x 106 0.9 x 106 1.25 x 106 4.7 x 106 2.50 x lo6 7.9 x 106

Experiment B : hypercholestero- lemic cells

0.68 x 106 1.1 x 106 1.25 x 106 3.2 X lo6 2.50 X 106 6.6 x 106

-LDL I

+LDL

pmoles/min/mg protein

99.1 4.2 41.2 2.7 30.7 2.0

105.6 144.0 96.5 91.0 97.4 71.4

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of cholesterol metabolism in mouse lymphoblasts and L cells

(15). LDL-cholesterol appears to reduce HMG-CoA reductase ac-

tivity of normal fibroblasts by inhibiting the synthesis of new enzyme molecules. This inhibition produces a rapid fall in enzyme activity because the enzyme has a very rapid rate of degradation with a half-life of approximately 3 hours, as shown by the exponential decline in enzyme activity after administra- tion of cycloheximide. The short half-life of the enzyme in human fibroblasts is remarkably similar to that reported for HMG-CoA reductase in intact rat liver (16). That LDL- cholesterol exerted its effect by reducing the number of enzyme molecules rather than by direct inhibition of the enzyme could also be inferred from the large number of negative experiments in which LDL-cholesterol, non-lipoprotein cholesterol, and ex- tracts from cells with suppressed activity failed to influence HMG-CoA reductase activity when added directly to the en- zyme.

The importance of LDL-cholesterol as a physiological reg- ulator of HMG-CoA reductase activity is emphasized by the fact that a genetic defect in this system exists in the fibroblasts of patients with familial hypercholesterolemia. Cultured cells from a homozygote with this disorder failed to show suppression of HMG-CoA reductase activity when grown in the presence of LDL even at concentrations 600 times greater than that which inhibited normal cells. In the presence of serum, the hyper- cholesterolemic cells synthesized HMG-CoA reductase enzyme molecules at a rate nearly 60 times greater than that of normal cells, an observation that further confirms the inability of lipo- proteins to inhibit enzyme synthesis in these cells. These data, considered together with our previously reported evidence that the HMG-CoA reductase from normal and mutant cells has identical kinetic properties (2), suggests that the mutation in the hypercholesterolemic cells involves a gene product other than HMG-CoA reductase. This putative gene product ap- pears to be critical for the normal regulation of enzyme syn- thesis by LDL. In this regard, it was striking to note that cholesterol in a non-lipoprotein form could suppress HMG-CoA reductase activity in these mutant cells. This finding can be interpreted to indicate that the hypercholesterolemic cells possess all the factors necessary for normal inhibition of enzyme syn- thesis provided that cholesterol can be delivered to its presumed receptor site. Thus, the primary genetic defect in the hyper- cholesterolemic cells may be localized to one of the processes which mediate the transfer of cholesterol from extracellular LDL to its site of action on or within the cell.

In addition to LDL and VLDL, which inhibit HMG-CoA

reductase activity of normal cells, human serum also contains factors that stimulate enzyme activity. It is unclear from the present data whether these stimulatory factors act directly to induce the synthesis of HMG-CoA reductase or whether they play a permissive role by facilitating cell growth and hence general protein synthesis. As pointed out by Schimke, en- zymes with rapid rates of degradation (such as HMG-CoA reduc- tase) may show a rise in activity relative to total cellular protein content in response to an agent which stimulates the synthesis of all cellular proteins at the same rate (13). Insulin and other factors in the lipoprotein-deficient fraction of serum that cause an increase in the specific activity of HMG-CoA reductase may be acting in such a nonspecific fashion. Some evidence that this may be the case is provided by the experiments indicating that these serum-stimulatory factors are more effective in con- fluent cultures than in actively growing cells. Irrespective of the mechanism of action of these stimulatory factors, recognition of their effects in cultured fibroblasts is necessary for develop- ment of a reproducible system for the study of regulation of HMG-CoA reductase activity.

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REFERENCES

BROWN, M. S., DANA, S. E., AND GOLDSTEIN, J. L. (1973) Proc. Nat. Acad. Sci. U. 8. A. 70, 2162-2166

GOLDSTEIN, J. L., AND BROWN, M. S. (1973) Proc. Nat. Acad. hi. U. S. A., 70, 2804-2808

BROWN, M. S., GOLDSTEIN, J. L., AND SIPERSTEIN, M. D. (1973) Fed. Proc., in press

BASSEN, F. A., AND KORNZWEIO, A. L. (1950) Blood 6.381-387 BIRCHBICHLER, P. J., AND PRYME, I. F. (1973) Eur. J. Biochem.

33, 368-373 BROWN, M. S., DANA, S. E., DIETSCHY, J. M., AND SIPERSTEIN,

M. D. (1973) J. Biol. Chem. 243,4731-4738 HAVEL, R. J., EDER, H. A., AND BRAGDON, J. H. (1955) J. Clin.

Invest. 34, 1345-1353 RADDING, C. M., AND STEINBERG, D. (1960) J. Clin. Invest. 39,

1560-1569 NOBLE, R. P. (1968) J. Lipid Res. 9,693-700 ZAK, B. (1957) Amer. J. CZin. Pathol. 2’7, 583-588 ALAUPOVIC, P., LEE, D. M., AND MCCONATHY, W. J. (1972)

Biochim. Biophys. Acta 260, 689-707 FREDRICKSON, D. S., GOTTO, A. M., AND LEVY, R. I. (1972) in

2’he Metabolic Basis of Inherited Disease (STANBURY, J. B.: WYNGAARDEN, J. B., AND FREDRICKSON, D. S., eds) 3rd Ed pp. 493-530, McGraw-Hill Book Co., New York

SCHIMKE, R. T. (1969) Curr. Top. Cell. Regul. 1, 77-124 HERSHKO, A., MAMONT, P., SHIELDS, R., AND TOMPKINS, G. M

(1971) Nature New BioZ. 232, 206-211 BAILEY, J. M. (1973) Atherogenesis: Initiating Factors. Cibc

Foundation Symposium II, pp. 63-92, Elsevier Scientific Publishing Co., Amsterdam

EDWARDS, P. A., AND GOULD, R. G. (1972) J. BioZ. Chem. 247, 1620-1524

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Michael S. Brown, Suzanna E. Dana and Joseph L. GoldsteinHYPERCHOLESTEROLEMIA

SUBJECT AND FROM A PATIENT WITH HOMOZYGOUS FAMILIALCultured Human Fibroblasts: COMPARISON OF CELLS FROM A NORMAL

Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Activity in

1974, 249:789-796.J. Biol. Chem. 

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