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THE JOURNAL. OF BIOLOGICAL. CHEMISTR YVol. 254, No. 12, Issue of June 25, pp. 5144-5149, 1979Prmted in U.S. A.

Active and Inactive Forms of 3-Hydroxy-3-methylglutaryl Coenzyme AReductase in the Liver of the RatCOMPARISON 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. DietschyFrom the Denartments of Internal Medicine and Molecular Genetics, The University of Texas Health Scienc e Center atDallas, Dalias, T exas 7i235

Hepatic 3-hydroxy-3-methylglutaryl coenzyme A re-ductase, the rate-controlling enzyme in cholesterol syn-thesis, exists in a phosphorylated (inactive) and de-phosphorylated (active) form. The current studies wereundertaken to determine whether long term regulationof reductase in rat liver is achieved through changes inthe proportion of enzyme in the two form s. Rates ofcholesterol synthesis were varied 50-fold by diurnallight cycling, fasting, stress, and feeding of cholesteroland cholestyramine. Portions of each liver were usedto measure 1) rate of cholesterol synthes is in tissueslices, and 2) reductase act ivi ty in microsomes pre-pared in four different ways: after isolation in absenceand presence of 50 mM sodium fluoride and after prein-cubation in absence and presence of Escherichia colialkaline phosphatase. Sodium fluoride was added tothe homogenization buffer because it inhibits dephos-phorylation of the enzyme during the microsomal iso-lation. E. coli alkaline phosphatase was used to maxi-mally activate the enzyme by removing a phosphategroup.

In livers of animals at midpoint of the dark cycle,reductase act ivi ty was I-fold higher in microsomes pre-pared in absence o f fluoride than in microsomes pre-pared in presence of fluoride. Alkaline phosphataseactivated reductase of fluoride-treated microsomes 13-fold , but it produced only a 1.4-fold activation of micro-somes isolated in absence of fluoride. Using enzymeact ivi ty after phosphatase treatment as a measure oftotal reductase, we calculated that 75 to 90% of reduc-tase under all physiological conditions was in a phos-phorylated (inactive) form at the time the homogenateswere prepared. The proportion of phosphorylated en-zyme remained constant under conditions in whichtotal reductase act ivi ty and the rate of cholesterol syn-thesis varied as much as 50-fold. Thus, long term alter-ations in cholesterol synthesis in rat liver are due notto changes in the state of phosphorylation of reductase,but rather to changes in the total amount of enzymeprotein.

Under physiologic conditions, the microsomal enzyme 3-hydroxy-3-methy lglutary l coenzyme A reductase appears tocontrol the rate of cholesterol biosynthesis in rat liver and ina number of other mammalian tissues as well (l-3). HMG-

* This research was supported by United States Pub lic H ealthService Research Grants HL 20948, HL 09610, AM 1638 6, and AM19329. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked aduertisement in accordan ce with 18 U.S.C. Section 1734solely to indicate this fact.

CoA reductase act ivi ty and the rate of cholesterol synthes isare both enhanced by treatments that remove cholesterolfrom the liver such as the administration of Triton-WR 1339or the feeding of the bile acid-binding resin cholestyramine(l-3). On the other hand, enzyme act ivi ty and the rate ofcholesterol synthesis in liver are reduced in parallel when ratsare fed cholesterol or when they are subjected to prolongedfasting (l-3). In addition, the act ivi ty of hepatic HMG-CoAreductase is subject to a marked diurnal cycle, which isaccompanied by parallel changes in the rate of cholesterolsynthesis from acetate (l-3).The mechanisms for the changes in hepatic HMG-CoAreductase act ivi ty have received much attention. Early studiesof the decay of enzyme acti vity following the administrationof cycloheximide to rats indicated that the enzyme has a rapidturnover with a half- life of approximately 4 h (4). Changes inenzyme act ivi ty during the diurnal cycle were interpreted asbeing due to changes in the rate of enzyme synthesis (4).Using an immunochemical approach to measure the amountof HMG-CoA reductase protein and its rate of synthesis,Higgins et al. also concluded that changes in enzyme acti vityduring the diurnal cycle and following cholestyramine feedingwere due to alterations in the rate o f synthesis of HMG-CoAreductase protein (5, 6). On the other hand, these workersfound that suppression of enzyme act ivi ty b y cholesterol feed-ing was more complex. Within 6 h after cholesterol feeding,enzyme acti vity was relati vely low as compared with theamount of immunochemically detectable enzyme protein.However, after 12 h both the amount of enzyme protein andenzyme act ivi ty were equally reduced. These data suggestedthat cholesterol feeding lowered HMG-CoA reductase acti vityby two mechanisms, an immediate inactivation of preformedenzyme and a longer term reduction of enzyme synthes is (5,6).

A series of in vitro studies has supported the notion that inaddition to its regulation by changes in enzyme synthetic ratesthe act ivi ty of HMG-CoA reductase may be regulated byposttranslational enzyme modification. Beg et al. reportedthat hepatic HMG-CoA reductase was inactivated in vitrowhen microsomes were incubated with cytoso l in the presenceof ATP and magnesium (7). Similar ATP-dependent inacti-vation of HMG-CoA reductase was reported to be catalyzedby a cytoso lic enzyme in human fibroblasts (8). Nordstrom etal. made the important observation that HMG-CoA reductasethat had been inact ivated in the ATP-dependent reactioncould be reactivated by incubation with a cytoso lic enzymefrom rat liver in the presence of EDTA (9). This react ivationwas inhibited by sodium fluoride, an inhibitor of phosphatases,

The abbreviation used is: HMG-CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase.

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Active and Inact ive Forms of HMG-CoA Reductase 5145suggesting that the reactivation involved a dephosphorylationof a previously phosphorylated enzyme (9). Gibson and co-workers have reported that the reactivation of inactive HMG-CoA reductase can also be achieved by incubation with apartially purified preparation of hepatic phosphoprotein phos-phatase (10, 11). Recent ly, Beg et al. demonstrated that theinactivation of hepatic HMG-CoA reductase in the presenceof ATP/magnesium is accompanied by the incorporation of32P from [y-P]ATP into a microsomal protein that could beprecipitated by an antibody to HMG-CoA reductase (12).

In the course of their studies, Nordstrom et al. made thesurprising observation that activation of rat liver HMG-CoAreductase appears to occur during the isolation of microsomesby the usual techniques employed in most laboratories (9).These workers showed that if the tissue homogenization andmicrosome isolation were carried out in the presence of fluo-ride, so as to inhibit the putative hepatic phosphoproteinphosphatase, the resultant microsomal HMG-CoA reductaseact ivi ty was only one-seventh of the amount that was foundwhen the microsomes were isolated in the absence o f fluoride.Moreover, the inactive enzyme isolated in the presence offluoride could be reactivated by incubation with the cytosolicactivation factor in the absence of fluoride (9). These investi-gators suggested that under ordinary conditions more than85% of the HMG-CoA reductase in rat liver was in the phos-phorylated (inactive) form and that the enzyme became acti-vated artifactual ly during isolation of the microsomes (9).

The above studies raise the question as to whether thephysiologic factors known to regulate HMG-CoA reductase inrat liver operate predominantly by changing the amount o fenzyme protein or by changing the state of phosphorylationof the enzyme. Accordingly, in the present study we haveperformed a series of manipulations designed to alter the ratesof hepatic cholesterol synthesis in rats (3, 13, 14). We meas-ured the absolute rate of carbon flux into cholestero l in liverslice s and made homogen ates of the same rat livers in theabsenc e and presence of fluoride. The microsom es were thenpreincubated with a preparation of E. coli alkaline phospha-tase that was shown to convert inactive HMG-CoA reductaseto its active form. The latter preincuba tion allowed an esti-mate of the total amount of HMG-CoA reductase (active plusinactive) in the tissue.

EXPERIMENTAL PROCEDURESMaterials-nL-3-Hyd roxy-3-methy l-[3-4C]glutaric ac id (49 mCi/mmol) and sodium [1-%]octanoate (1.0 mCi/mg) were purchased

from New England Nuclear. Glucose-6-phosphate dehydrogenase(350 units/mg) was purchased from Boehringer Mannheim. E. colialkal ine phosphatase suspended in 2.6 M ammonium sulfate (34 to 56units/mg of protein) was obtained from Worthington Bioch emica ls,Inc. (catalog no. 06124). Just prior to use, the enzyme suspe nsioncontaining 20 mg of protein was centrifuged (12,060 x g, 45 min, 4C),the supernatant was discarded, and the pellet (con taining the alkalinephospha tase activity) was resuspended in 1 ml of Buffer A (20 m Mimidazole/chloride, pH 7.4, and 5 rnM dithiothreitol).

Anima l Preparations-Female, Sprague-Dawley-derived ratswere pu rchased in the weight range of 140 to 170 g from the CharlesRiver Breeding Laboratories and placed in gang cage s in light cyclingrooms with alternating 12-h periods of light and darkness (14, 15).The anima ls were allowed to adapt to the light cy cling for 2 to 3weeks during which time they were fed ad libitum Ralston PurinaRat Chow diet. After this period of adaptation, the anima ls wereallocated to eight different experimental groups designated A throughH in Table II and Fig. 2, and these groups were treated as follows. A,control, mid-dark; anima ls were fed ad libitum and killed at the mid-dark phase of the light cycle. B, control, mid-light; anima ls were fedad Zibitum and killed at the mid-light phase of the light c ycle. C andD, fasted, mid-dark; food was removed from the cages either 12 h or48 h prior to the time the rats were killed at the mid-dark point of the

light cycle. E, fasted, stresse d, mid-dark; anima ls were placed inindividual restraining cages and fasted for 48 h prior to the time theywere killed at the mid-dark point of the light cycle. F, CHO-fed, mid-dark; anima ls were fed a 1% cholestero l, 10% corn oil diet (w/w) for12 h before they were killed at the mid-dark point of the light cycle.G and H, CS-fed, mid-dark and mid-light; anima ls were fed a 2%cholestyramine diet (w/w) for 72 h before they were killed at eitherthe mid-dark or the mid-light phase of the light cycle. Imm ediatelyafter killing the various groups of anima ls by decap itation, the liverswere removed and chilled in cold 0.9% NaCl so lution. Aliquots of eachliver were then taken for the preparation of microsom es and tissuesl ices as described below.

Preparation of Liver Microsomes -Aliquots of each liver (approx-imately 1 g) were weighed and placed into 4:l (v/w) cold homogeni-zation medium containing 0.3 M sucrose, 10 m M 2-mercaptoethanol,10 InM sodium EDTA (pH 7.4), and either 50 m M sodium chloride or50 mM sodium fluoride. The livers were homogenized at 4C in aDounce homogenizer with 10 strokes of a loose fitting pestle followedby 5 strokes of a tight fitting pestle. Each hom ogenate was centrifugedfor 15 min at 12,000 x g at 4C and the supernatant fraction wasthen centrifuged for 60 min at 100,000 x g in a Beckman-Sp incoultracentrifuge at 4C. Unless otherwise indicated, the resulting mi-crosom al pellets were immediately frozen and were stored at -190C.Prior to assay of HMG-CoA reductase activity, each pellet (5 to 10mg of protein) was resuspended in 2 to 2.5 ml of Buffer A.

Assay of Microsomal HMG-CoA Reductase Activity-The stan-dard assay for HMG-CoA reductase activity cons isted of two sequen-tial steps: 1) a preincubation period du ring wh ich microsom es wereincubated in the absen ce or presence of E. coli alkaline phosph atase,and 2) a subsequ ent incubation period during which the activity ofHMG-CoA reductase was measured. The preincubation mixture con-tained the following concen trations of compon ents in a volume of 90d: 20 InM imidazole/chloride (pH 7.4), 5 m M dithiothreitol, 20 to 150pg of microsom al protein, and 10 units of E. coli alkaline phospha tasewhere indicated. The tubes were incubated at 37C for 60 min, afterwhich 100 ~1 of solution containing 0.2 M potassium phosphate (pH7.4), 40 mM glucos e 6-phosphate, 5 InM TPN, 0.7 unit of glucose-6-phospha te dehydrogenase, 20 mM sodium EDTA, and 10 m M dithio-threitol were added to the preincubation mixture. The HMG-CoAreductase assay was then initiated with the addition of DL-[%%I-HMG-CoA (23,000 cpm/nmol) to a final concentration of 176 PM(final assay volume, 200 ~1). After incubation for 30 min at 37C, the[%]mevalonate formed was converted into the lactone, isolated bythin layer chromatography (16), and counted using an internal stan-dard of [:H]mevalonate to correct for incomp lete recovery (17). In allfigures and tables, each value represents the average of duplicateassays. HMG-CoA reductase activity is expressed as the picom oles of[Clmevalonate formed per min per mg of microsom al protein (pmol.min . mg o f protein-).

Assay of Rates of Cholesterol Synthesis-Portions of each liverwere cut into ribbons approximately 2 to 3 mm thick. Liver slic esO.&mm thick were then prepared on a tissue slicer, and 3O@mgaliquots were placed in 25-ml Erlenmeyer flasks fitted with center-wells and containing 5 ml of oxygenated Krebs bicarbonate bufferand [1-%]octanoate at a concen tration of 1.0 mrvr (3, 18). Generally,6 flasks were run from each an imal; 2 flasks were used for zero timecorrections of mas s and radioactivity in the ketone de terminationswhile the remaining 4 flasks were incu bated for 90 min at 37C in ametab olic shaker set at 160 oscillatio ns per min. At the comp letion ofthe incubation period one pair of flasks that was incubated at 37Cwas used to determine the rates of incorporation of radiolabeledoctanoate into CO* and cholestero l as previously described (3, 18). Inboth situations the total radioactivity found in each product wasdivided by the spe cific activity of the radiolabeled precursor to yieldrates of incorporation that were expressed as either the micromo lesor nanom oles of [I-Cloctanoate incorporated into the two productsper h per g wet weight of liver (pm01 or nmol. h- . g-). The remainingpair of flasks was utilized to determine the rate of synthesis of ketonebodies and their spe cific a ctivities. The production rates of acetoac-etate and /3-hydroxybutyrate were combine d and are reported as themicromo les of total ketone synthesized per h per g of liver. The totalradioactivity incorporated into acetoacetate and P-hydroxybutyratealso was measured (19, 20). The observed value for the spe cificactivity of the ketones was then calculated and was compared to thetheoretical value that would be expected if no intracellular dilution ofthe spe cific activity of the acetyl-CoA pool occurred. The latter valueshould equ al half of the spe cific activity of the [I-%]octanoate added


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5146 Active and Inactive Forms of HMG-CoA Reductaseto the incubation media. In Table s I and II, the term relative ketonespe cific activity equa ls the absolute ketone spec ific activity dividedby the theoretical spe cific activity times 100. The rates of incorpora-tion of [1-%]octanoate into cholestero l and CO2 also were correctedfor intracellular dilution by endogeno us acetyl-CoA units. In addition,data on the incorporation of [l-%]octanoate were used to calcula teincorporation rates in terms o f acetyl-CoA units, i.e. C2 units, ratherthan nanom oles of octanoate. Hence, the corrected CL flux from [l-%]octanoate into cholestero l equa ls the rate of incorporation of [l-%]octanoate into cholestero l times 4 times 1.5 times 100 divided bythe relative ketone spe cific activity. The factor 4 converts the incor-poration rates of. octanoate to incorporation rates of CZ units. Theadditional factor of 1.5 corrects for the loss of 33% of the radioactivityas [C!]C02 during the conversion of [l-4C]acetyl-CoA to cholestero l.A similar calcula tion was carried out to determine the CP flux intoCO*, except that the factor of 1.5 is not required (18).

Calculations-Where appropriate the data obtained in the rats ineach experimental group were combine d and are given as mean values+ 1 S.E. In Fig. 2 microsom al HMG-CoA reductase activity wascorrelated with the rate of cholesterol synthes is by fitting the datafor these two variables obtained in individual anima ls to linear regres-sion curves. The regression curves have the usual form of y = a + bxwhere a is the intercept on the vertical axis and b is the slope of theregression curve.

For purposes of compa rison, rates of HMG-CoA reductase activitywere converted to rates of Cz flux into cholesterol. On average, 20 mgof microsom al protein were recovered from the homogen ization of 1g wet weight of liver. Th us, the rates of enzyme activity, in pmol.mini . mg of protein-, were multiplied by (20 mg of protein. g-) (60min. h-) (3 C!lunits.p mol of mevalonatee) (1 x lo-) to yield therates of Cz flux into cholestero l with the units of nmol. he. g-l, thesame units u sed to describe the rate of incorporation of acetyl-CoA

units from [1-Cloctanoate into cholestero l in the experiments usingliver slices.

RESULTSThe major purpose of these studies was to investigate the

poss ibility that phosphorylation-dephosphorylation reactionsinvolving HMG-CoA reductase might play a role in the longterm regulation of cholestero l synthes is in the liver underdifferent physiological circumstances. To this end absoluterates of cholestero l synthes is were measured in liver slice susing [I-%]octanoate as the precursor. Corrections for dilu-tion of the spe cific activity of the intracellular acetyl-CoA poolwere made by using the measured spe cific activity of thenewly synthesized ketone b odies. The rate of acetyl-CoA (i.e.C,) flux into cholestero l determined in this manner was thencompared to the activity of HMG-CoA reductase found in thesame livers after preparation of microsom es in the presenceand absen ce of 50 mM sodium fluoride and after activation ofenzyme activity by preincubation with E. coli alkaline phos-phatase.

Initial studies were undertaken to measure the relativeactivities of HMG-CoA reductase in microsom es prepared inthe presence and absen ce of 50 InM sodium fluoride (Table I).Groups of rats were killed at either the mid-dark or mid-lightphase of the light cycle. As expected from previous work, therate of cholestero l synthes is was 3.2-fold higher at the mid-dark point of the light cycle than at the mid-light time while

TABLE IHMG-CoA reductase activity in liver microsom es prepared in the presence and absen ce of 50 m M sodium fluoride

The rats used in this study were adapted to light c ycling for 2 absen ce (0) of 50 mM sodium fluoride in the homoge nization buffer.weeks and were then killed at either the mid-dark or mid-light point The data in column s 3 to 6 were d erived from the liver slice s whileof the cycle. One portion of each liver was used to prepare liver slic es those in colum ns 7 and 8 were obtained with the microsom e prepa-for assay of the rate of cholestero l synthes is while two other aliqu ots rations. Each value represents the mean t 1 SE. for results obtainedwere used to prepare hepa tic microsom es in the presence (+) or in three anima ls in each experimental group.

Ketone bodies CI flux into HMG-CoA reductase activitvExperimental group 1. Animalweight 2. Liver weight 3. Relativespecific activ- 4. Synthesisrate 5. con 6. Cholesterol 7. (+) Fluoride 8. (0) Fluorideity

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 + 106B. 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 IIComparison 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 colum ns 7 through 10 werecholesterol synthesis were varied over a large range by using anima ls measured in microsom es prepared in the presence (+) or absenc e (0)subjected to eight different treatments; these experimental groups are of 50 mM sodium fluoride and after preincubation in the presencedesignated A through H. Columns 1 and 2 give the body weights and (+) or absen ce (0) of E. coli alkaline phospha tase as described underliver weights, respectively, at the time the anima ls were killed. The Experimental Procedures. Each represents the mean + 1 SE. fordata on ketone synthes is and the rates of CZ flux into CO* and results obtained in three to seven anima ls in each experimental group.cholesterol given in column s 3 through 6 were obtained in liver slice s.

Ketone bodies C., flux into HMG-CoA redu ctase activitvExperimental group 1. Anima l 2. Liverweight weight 3. Relativespecif ic 4. Synthesisrate 5. co1

6. Cho,es - 7. (+) Flue- 8. (0) Flue- 9. (0) Flue- 10. (+) Flu-two1 ride (0) ride (0) ride (+) oride (+)activi tv Phosphatase Phosphatase Phosphatase Phosphatase

g R % them-e t- pmunol$.id g pd~i.g n?d,hi~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 62B, control, mid-light 215 + 3 9.0 f 0.3 69 f 2 8.4 + 0.2 11.9 -c 0.6 305 + 89C, 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 & 20D, 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+ 17E, fasted, stressed, mid- 182 f 6 5.7 + 0.3 56 -+ 4 17.8 f 0.6 11.7 + 0.7 500 r+ 143

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 + 7h)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 66H. 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

87 e 1332 f 8

122 f 2811 f 235 f 3

8-+1244 + 28185 + 53

613 + 82 862 f 156 1152 k 162138 + 26 225 t 35 227 -c 97562 + 64 758 f 196 691 f 79

53f 17 82 f 31 55& 19267 + 48 488 + 102 392 f 65

70f 13 105 f 21 76 f 102096 k 168 2833 + 191 2 216 +- 473

844 f 107 1 437 + 306 1393 + 138


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Active and Inactive Forms of HMG- CoA Reductase 5147the rates of CO, production and ketone synthes is and theketone relative specif ic activi ties were the same. MicrosomalHMG-CoA reductase acti vity manifested a nearly identical3.2- to 3.7-fold relative difference between the two phases ofthe light cycle regardless of whether the microsomes wereprepared in the presence or absence of fluoride. However, theabsolute act ivi ty in microsomes prepared in the presence offluoride was only about 18% of that found in microsomesprepared in the absence o f this ion. This d ifference wasessential ly the same whether the microsomes were preparedfrom animals killed at the mid-dark or mid-light phase of thelight cycle . These results confii those reported by Nord-strom et al. (9) and demonstrate that the HMG-CoA reductaseact ivi ty found in a particular liver is profoundly influenced bythe conditions under which the microsomes are prepared.

A second group of studies was next undertaken to establishthe conditions giving maximal activation of HMG-CoA reduc-tase by treatment of the microsomes with a phosphatase.Previously published work has shown that alkaline phospha-tase prepared from E. coli is eff ect ive in dephosphorylating avariety of phosphorylated mammalian proteins including bo-vine heart glycogen synthase D, mixed phosphohistones, andrabbit skeletal muscle phosphorylase kinase (21). In prelimi-nary studies we found that this phosphatase preparation alsowas eff ect ive in activating hepatic microsomal HMG-CoAreductase. This activation was inhibited by 50 mM sodiumphosphate, indicating that it was probably due to a phospha-tase act ivi ty (21). The time course for this activation processis shown in Fig. 1. In this study, microsomes from the liver ofa control rat were prepared in the presence of fluoride. Ali-quots of this preparation of microsomes having relatively lowlevels of HMG-CoA reductase act ivi ty were then preincubatedfor either 30 or 60 min with 1 to 10 units of the E. coli alkalinephosphatase in the absence of fluoride. When the microsomalHMG-CoA reductase was subsequently assayed, there was amarked increase in act ivi ty that was dependent upon both theduration of the preincubation period and the amount of phos-phatase in the preincubation medium (Fig. 1). When themicrosomes were preincubated for 60 min with 10 units o fphosphatase, maximal activation of HMG-CoA reductase ac-tivi ty was achieved. These conditions, therefore, were chosento activate the microsomal reductase act ivi ty in all subsequentexperiments.

The results of the principal group of experiments are sum-marized in Table II . In these experiments rats were subjectedto a variety o f physiological manipulations known to alterrates of hepatic cholesterol synthesis. Microsomes were thenprepared from the livers of these animals in the presence (+)and absence (0) of 50 mM sodium fluoride. An aliquot of eachpreparation of microsomes was preincubated in the presence(+) and absence (0) of the E. coli alkaline phosphatase. Thus,four diffe rent values for HMG-CoA reductase act ivi ty weredetermined in each animal and these values, in turn, werecompared to the overall rates of acetyl-CoA (C,) incorporationinto cholesterol in tissue slices prepared from the same livers.

As is evident in experimental groups A and B, light cyclingcaused a 3.4-fold change in the C%flux into cholesterol withoutany significant alteration in the rates of CO2 production orketone synthesis. Similar relative differences between themid-light and mid-dark animals were seen in assays of HMG-CoA reductase act ivi ty in microsomes prepared in the pres-ence of fluoride (2.7-fold, column 7), in the absence of fluoride(4.4-fold, column 8), or after activat ion of either of theseenzyme preparations with phosphatase (3.8. and 5.0-fold , col-umns 9 and 10, respect ively) . At both the peak and val ley ofthe diurnal cycle the absolute activi ties of HMG-CoA reduc-

I I , I I0 2 4 6 8 10 IALKALINE PHOS PHATA SE (U.tube-1

FIG. 1. Activation of rat liver microsomal HMG-CoA reduc tase bypreincubation with E. coli alkaline phospha tase. Microsomes (45 pgof protein) prepared from a rat liver that was homogenized in thepresence of 50 mM sodium fluoride were preincubated at 37C foreither 30 min or 60 min with the indicated amount of E. coli alkalinephospha tase under the standard cond itions. HMG-CoA reductaseactivity was then determined as described under Experimental Pro-cedures.

tase were approximately 7- and 4-fold higher, respectively, inthe microsomes prepared in the absence of fluoride than inthose prepared in the presence of this ion (column 7 versus 8).With the microsomes prepared in the absence of fluoride,treatment with phosphatase resulted in only a small incrementin enzyme act ivi ty (column 8 uersus 9). With the microsomesprepared in the presence of fluoride, phosphatase treatmentraised the level of HMG-CoA reductase by 13- and 7-fold inthe mid-dark and mid-light animals, respect ively (column 7uersus 10). As a result, after phosphatase treatment the levelof HMG-CoA reductase act ivi ty was similar whether themicrosomes had been prepared with or without fluoride in thehomogenization medium (column 9 versus IO).

As anticipated from previous work, fasting for 12 or 48 h(experimental groups C and D) caused a significant increasein the rate of ketone body synthes is and a decrease in theketone relative specific activ ity, reflecting increased levels offa tt y acid oxidation and acetyl-CoA production. In the animalsfasted for 48 h the rate o f C, flux into cholesterol was sup-pressed to 4% of the level found in the appropriate controlanima ls (experimental group D uersus A), and HMG-CoAreductase act ivi ty was similar ly suppressed to 13%, 9%, lo%,and 5% of the appropriate control values in the microsomesprepared in the four different ways (columns 7, 8, 9, and 10,respect ively) . In each experimental group, there was onceagain a 5-fold higher level of HMG-CoA reductase act ivi ty inmicrosomes prepared in the absence of fluoride. This differ-ence was eliminated when the enzymes were activated withphosphatase. Qualitat ively similar relative changes in Cz fluxinto cholesterol and in microsomal HMG-CoA reductase ac-ti vi ty were found when the rates of cholesterol synthesis weresuppressed by cholesterol feeding rather than by fasting (ex-perimental group F).

In two final experimental groups of animals, the rates ofcholesterol synthesis were enhanced either by stressing theanimals (experimental group E) or by short term cholestyra-mine feeding (experimental groups G and H). Again, qua lita-tively similar relative changes were seen in HMG-CoA reduc-tase acti vity, i.e. the enzyme activ ity was reduced by 4- to 7-fold in the microsomes prepared in the presence of fluoride,and it was activated by alkaline phosphatase.The data of Table II are plotted in Fig. 2 to show therelation between the HMG-CoA reductase acti vity (ordinate)and the CZ flux into cholesterol (abscissa) under each of the


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5148 Active and Inactive Forms of HMG-CoA Reductase

D A Fasted, M,d-dark (48hrlE . Fr,s+ed, Stressed,

M,d-dark (48 hr)F . CHO-Fed, f&d-dark

(0) PHOSPHATASE

0 500 1000 1500 2000C, FLUX INTO CHOLESTEROL (nmol.hr-.g-)

FIG. 2. Relation between HMG-CoA reductase ac tivity and cho-lesterol synthesis under four different conditions of assay of HMG-CoA reductase activity. These data were derived from the data ofTable II . The data on cholesterol synthesis were obtained in liverslices. HMG-CoA reductase acti vity was measured in microsomesfour diffe rent conditions of enzyme treatment (panels A to D).Whether the microsomes were isolated in the absence orpresence of fluoride or whether or not they were subjected toprior incubation with alkaline phosphatase, the fina l measuredact ivi ty was proportional to the Ca flux into cholesterol underall of the physiological conditions tested. In all cases theintercepts on the x and y axes were not signif icantly differentfrom zero, and there was a high degree of correlation betweenthe HMG-CoA reductase act ivi ty and the Cp flux into choles-terol (r = 0.86 to 0.89). There was, however, a lo-fold differ-ence between the slopes of the lines obtained with the micro-somes prepared in the presence of fluoride (panel A) andthose activated with phosphatase (panels C and D). Micro-somes prepared in the conventional manner, i.e. without flu-oride ion or phosphatase treatment (panel B), manifested anintermediate proportionality constant, and there was greaterscatter in the data.

DISCUSSIONIn the current experiments, the rates of hepatic cholesterol

synthesis were varied over a nearly 50-fold range in eightexperimental groups of animals by a var iety of physiologicalmanipulations. In all experimental groups three similar find-ings were observed. First , under all physiological circum-stances, the act ivi ty of HMG-CoA reductase was 4- to &foldhigher in microsomes prepared in the absence of fluoride thanin those prepared in the presence of fluoride. Second, in allexperimental groups, preincubation of the microsomes withE. coli alkaline phosphatase preferentia lly activated the en-zyme that had been isolated in the presence of fluoride andhence abolished the difference between the reductase activ i-ties in microsomes prepared in the absence and presence ofthis ion. Third, under all conditions changes in the rate of C2flux into cholesterol were reflected by proportional changes inthe relative levels of HMG-CoA reductase act ivi ty regardlessof whether the microsomes were prepared in the presence orabsence of fluoride and regardless of whether or not they wereincubated with E. coli alkaline phosphatase.

Two lines of evidence indicate that under the conditions ofthe current experiments changes in the Cs flux into cholesterolas measured in liver slices were brought about by changes inthe amount of HMG-CoA reductase protein and not by

prepared in the presence (+) or absence (0) of 50 m M sodium fluorideand after preincubation in the presence (+) or absence (0) of E. col ialkaline phosphatase. The linear regression curves were fitted to dataobtained in 36 individual animals although not all of the points areshown because of overlap, particularly at the lower rates of synthesis.changes in the state of phosphorylation of the enzyme. Thefir st line of evidence comes from the experiments in which E.coli alkaline phosphatase was used to convert phosphorylated(inactive) HMG-CoA reductase into its active form. As shownin panels C and D of Fig. 2, total HMG-CoA reductase act ivi ty,as measured in the phosphatase-treated microsomes, wasstrongly correlated with the Cz flux into cholesterol. I f enzymeact ivi ty had been regulated by changes in the state o f phos-phorylation, the total amount of enzyme as measured afterdephosphorylation should have been constant after the var-ious physiologic manipulations.

The second line of evidence comes from the finding thatunder each of the physiological conditions studied, HMG-CoAreductase act ivi ty in microsomes isolated in the absence offluoride was 4- to &fold higher than the act ivi ty in microsomesisolated in the presence of fluoride (Fig. 2, panels A and B).Although slight differences in these ratios were seen in thevarious experimental groups, the differences were not suf fi -cient to account for the marked differences in cholesterolsynthesis in the various groups.

The simplest interpretation of the current data is that 75 to90% of the HMG-CoA reductase in the liver cell is in aphosphorylated form under all physiologic conditions andhence is inactive. When the livers are homogenized and themicrosomes are isolated in the absence of fluoride, dephos-phorylation of the enzyme occurs, and the latent enzymebecomes activated. If fluoride is included in the homogeniza-tion mixture, this dephosphorylation is prevented and themeasured activ ity of the enzyme reflects the acti vity that waspresent initially in the tissue.

The reason for having more than 75% of HMG-CoA reduc-tase in an inactive (phosphorylated) form at all times in liveris not clear. The inactive enzyme might serve as a reservoir toallow rapid increases in HMG-CoA reductase by the mecha-nism of dephosphorylation when hepatocytes are faced withsudden extraordinary demands for cholesterol. It should benoted that all of the experiments in the current paper repre-sented relat ively long term physiological manipulations. It ispossible that the enzyme act ivi ty may be altered by phospho-rylation and dephosphorylation transiently during some shortterm control processes.

In experiments not shown, we have observed that HMG-CoA reductase act ivi ty in the corpus luteum of the rabbit


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active and inactive forms in the liver rat

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