in vitro and in vivo inhibition of hepatic cholesterol synthesis by 3-hydroxy-3-methylglutaric acid

BIOCHIMICA ET BIOPHYSICA ACTA 439

BBA 56009

IN VITRO AND IN VII’0 INHIBITION OF HEPATIC CHOLESTEROL

SYNTHESIS BY 3-HYDROXY-3-METHYLGLUTARIC ACID

2. H. BEG* AND P. J. LUPIEN**

Centre de Recherche SW les Maladies Lipidiques, D@artement de Biochimie, H&da1 Universitaive, Facultg de Me’dicine, Universite’ Laval, Qadbec I o (Canada) (Received March 1st 1971) (Revised manuscript received June 7th 1971)

SUMMARY

I. Hepatic cholesterol synthesis was compared in rat liver slices and homogenates with and without added 3-hydroxy-3-methylglutaric acid (HMG) by measuring the rates or incorporation of [I-“Clacetate into cholesterol. Addition of HMG in vitro produced dose-dependent decrease in the rate of sterol synthesis. Addition of cholesterol to liver slices failed to block the rate of cholesterogenesis. HMG inhibition was not masked by cholesterol, when added together. HMG had no effect on the rate of oxidation of [I-14C]acetate to CO, by the Krebs cycle.

2. The rate of incorporation of [I-Wlacetate into cholesterol by liver slices of cholesterol-fed rats was also assayed with and without HMG. The rate of cholesterogenesis was significantly suppressed by HMG in comparison to control values.

3. Measurement of rates of incorporation of [I-14C]acetate, the [3-14C]HMG-CoA and [V4C]mevalonic acid into cholesterol in the presence of HMG, revealed that the site of HMG inhibition is at the enzymatic step mediated by HMG-CoA reductase (mevalonate : NADP oxidoreductase (acylating CoA), EC I .I. I .34).

4. HMG was also inhibitory to cholesterol synthesis when injected to intact animals. Greater inhibition of liver cholesterogenesis was evident with HMG administration to rats fed cholesterol in the diet than with rats fed only cholesterol. These findings indicate an additive inhibitory effect of HMG. The possible role of HMG as a regulatory molecule for the physiological regulation of cholesterol biosynthesis is discussed.

INTRODUCTION

There is now good evidence that the de novo rate of cholesterol synthesis from acetate in liver is regulated at the site of reaction catalyzed by 3-hydroxy-g-methyl-

* Post-doctoral fellow, on leave from the Department of Chemistry, Faculty of Science, Aligarh Muslim University, Aligarh, India. * * Professor and Director of the Centre de Recherche sur les Maladies Lipidiques. Abbreviations: HMG and HMG-CoA, 3-hydroyx-3-methylglutaric acid and its CoA ester.

Biochim. Biophys. Acta, 260 (1972) 439-448

440 z. H. REG. I'. J. Ll'I'IliN

glutaryl (HMG)-CoA reductase (mevalonate:NADP oxidoreductase (acylating C‘(J~~), EC I.I.I.34)'-4. An important aspect of this evidence is the fact that both cholesterol synthesis and HMG-CoA reductase activity from acetate are depressed b!. dietar! cholesterol and starvation5+‘. The mechanism(s) by which the HMG-CoA reductase activitv is altered are still not completelv understood. At the same time there exist discrepancies regarding the nature of the regulatory molecule9. Postulated mechanisms include allosteric inhibition by cholesterol at the site of conversion of HM(;-CO.\ to mevalonic acid”.

Bile salts have also been shown to inhibit cholesterol synthesis in rat liver homogenates in ~jitrol~. However, there are data indicating that the concept of direct inhibition of cholesterol synthesis in the liver by bile salts is not correct12-15. Moreover, attempts to demonstrate that the activity of HMG-CoA reductase is controlled by bile acids ilz &JO have not been conclusivelfl.

From all the information available, very little can be said about the mechanism responsible for decreased reductase activity brought about by cholesterol feeding. Cholesterol synthesis is not inhibited by addition of cholesterol to homogenates or liver slices”. More recently it has been shown that solubilized cholesterol failed to inhibit the highly purified HMG-CoA reductase enzyme from the rat liver microsomal fractionIT. These findings suggest that the actual regulatory molecule for hepatic cholesterogenesis may not be cholesterol itself but some other metabolite.

Earlier reports have indicated that HMG, which is formed in the liver of mam- mals from HMG-CoA by the hydrolytic action of enzyme HMG-CoA hydrolase (EC

3.I.2.5)'8, is a competitive inhibitor for HMG-CoA reductase in the bacterial system]“.

HMG has also been shown to have some hypolipidemic characteristics when given to rats20-21

The data presented in this manuscript strongly supports the view that HMG may be the true physiological metabolite responsible for the control of cholesterol

synthesis. In order to assess the role of HMG in the control of hepatic cholesterogenesis,

various types of i?a vitro and in zlivo investigations have been done. The possible effect of HMG on the Krebs cycle has also been examined.

MATERIALS AND METHODS

Animals

Male hooded rats weighing 150-200 g, were used. Dietary regimens consist of Purina Laboratory Chow, supplemented as indicated. All animals were given water ad libitum.

Preparation of tissue specimens and analytical methods For measuring the radioactive cholesterol and X0, production in liver slices,

normal rats were decapitated, and livers immersed in an ice-cold solution of Krebs-- Ringer phosphate buffer. Liver slices (approx. 0.5 mm thick) were cut using a tissue slicer (Arthur Thomas and Co., Pa.). The slices (250-300 mg) were suspended in Warburg flasks in IO ml of Krebs-Ringer phosphate buffer (pH 7.4) containing sodium/r-X] acetate (spec. act. z mC/mmole). Whenever HMG inhibition was assayed, different concentrations of neutralized solution of HMG (pH 7.4) were added, Concentrated HCl

INHIBITION OF CHOLESTEROL SYNTHESIS 441

(0.25 ml) was pipetted into the side arm of the flasks and a multifold Whatman NO. I

filter paper (3 cm x 5 cm), previously moistened with hyamine hydroxide IO-X, was introduced in the center well of the flask. The flasks were then flushed with O,-CO, (95 : 5, v/v), closed with a glass stopper, and the mixture was incubated in a Dubnoff metabolic shaker for 2 h at 37”. The determination of 14C0, released during the incubation period was carried out as described earlier 22 In short, the reaction is ter- . minated by the addition of HCl contained in the side arm of the flask, the reaction vessels are then shaken for 15 min at 37’ to assure the complete trapping of labeled CO, released and each filter paper is introduced into a scintillation counting vial containing 15 ml of PPO-POPOP solution in toluene (42 ml of liquifluor-1000 ml of toluene). The contents of the Warburg flasks were transfered to a 5o-ml Erlenmeyer equipped with a condenser tube, the flasks were rinsed twice with 5 ml of alcohol. 15 pellets of KOH and carrier cholesterol (4 mg) were added, the suspension was heated overnight at 70’ and the cholesterol was extracted with light petroleum (b.p. 30-60”). Cholesterol was isolated, purified and counted as its digitonide23. Digitonin pads were dissolved in I ml of hyamine hydroxide IO-X in a counting vial, the volume was made up to 15 ml with PPO-POPOP solution (45 ml of liquifluor-rooo ml of toluene). All the radioactive samples were counted twice either in a Mark I of Mark II liquid scintillation counter (Nuclear Chicago) for IO min.

For the 60oxg supernatant fraction, livers of normal rats were excised and flushed with 0.25 M ice-cold sucrose solution. Individual livers were weighed and homogenized in 2.5 vol. of 0.04 M potassium phosphate buffer (pH 7.2), containing 0.028 M nicotinamide, 0.126 M sucrose and 0.0007 M MgCl,. The homogenization was done in a Potter-Elvehjem homogenizer by hand using 6 up-and-down strokes of a loose-fitting Teflon pestle. The resulting homogenates were centrifuged at 600 xg for IO min and the resultant supernatant solutions of each rat were pooled.

To obtain IOOOO xg supernatant fraction, livers from normal rats were chilled thoroughly in ice-cold phosphate-nicotinamide buffer, blotted and weighed, All sub- sequent operations were carried out at o-4”. The livers were homogenized by IO up- and-down strokes with a motor-driven, loose-fitting Teflon pestle at low speed24. The homogenates were centrifuged at 700 xg for IO min, and the supernatant liquid centrifuged at IOOOO xg for 15 min, the supernatant fractions were removed with a spinal needle and syringe to avoid disturbing either the lipid layer or the sediment. The supernatant fractions from individual rats were pooled and used for incubations.

Cholesterol determination

Total cholesterol levels in serum and liver homogenates were determined by the methodof FERROANDHAM~~.

Source of compounds Sodium [I-14C]acetate, DL-[2-14C]mevalonic acid and 3-hydroxy-3-methyl

[3-14C]glutaric acid were purchased from New England Nuclear (Boston, Mass.). HMG (anhydrous, m.p. IIO’), cholesterol, coenzyme A, NADP, NAD and glucose 6-phosphate were obtained from Sigma Chemical Company (St. Louis) and ATP from Mann Research Laboratories (New York). DL [3-14C]HMG-CoA was synthesized by the proce- dure of HILZ et aLzs. HMG[3-14C]anhydride was prepared and crystallized twice with benzene (m.p. 1o1-1o2~). Then the anhydride was allowed to react with a solution of

Biochim. Biophys. Acta, 260 (1972) 43g-448

442 z. H. I’&(;, I’. J, I.I’PII<S

TABLE I

EFFECTOFADDING HMGinvitroON THEINCORPORATIONOF [I-'4C!ACETA1.E1~'~O(.HOLI'SI.UROL ,X1)

14C02PRODUCTIONBYNORMALRATLIVERSLICES

Liver slices prepared from normal animals tvere incubated in Krcbs-Ringer phosphatr metlinn~ in the presence of IO,& of [+%]acetate with or without HMG. Radioactive cholesterol and lJ(‘O, evolution was measured as described in MATERIALS AND METHODS. Values given are mean 1. S. I<. for 4 rats per group. For each animal four incubations were made, two each for control and 1112C;- added flasks. ____ - HMG added Cholesterol OO Inhibztion ‘“CO, Oc, lnhibztzon (pmoles) (disint. /win $PY wag (disint. lmin PPV wzg

endogenous cholesterol) liver) ___ .___ None 37640 & I4240 0 1590 + 109 Insignificant

50 17640 i 5040 +t* 2464 + Io5 None 31800 + 12360 0 2565 * 2X4

75 I1380 4: 2000 64* ~284 * 126 None 30459 * 3306 0 2018 + 169 100 11640 $- 1257 61.X 2I3I i I33

(P < 0001) None 46943 & 9705 0 2891 & 209

150 I7697 * 2Iso ;; < 0.001)

2976 zt I99

None 27663 + 404.5 0 3904 + 236

250 9636 * I366 ;; < 0.001)

3792 + 188

* Differences not significant.

coenzyme A until the nitroprusside test for free sulfhydryl groups was negativeZ7. [3-14C]HMG-CoA solution was stored frozen, when in use it was kept at o-4”.

RESULTS

In vitro studies The results (Table I) show that the addition of different concentrations of HMG

produced a marked decrease in [I-14C]acetate incorporation into cholesterol in liver

TABLE II

EFFECT OF ADDING HMG, CHOLESTEROL AND HMG PLUS CHOLESTEROL in Vitf’O ON THE INCOR- PORATION OF [I-W]ACETATE INTO CHOLESTEROL AND “Co, PRODUCTION BY NORMAL RAT LIVER

SLICES

Cholesterol dissolved in propylene glycol (6 pmoles), HMG (250 pmoles), or HMG (250 ,umoles) @us cholesterol (6 pmoles) were added to the Krebs-Ringer medium in which liver slices from normal rats were incubated with 6 ,uC (z mg) of [I-Wlacetate. For further experimental details, see MATERIALS AND METHODS. Data are presented as mean 5 S.E. for 6 rats.

~~

Addition Cholesterol 0/O Inhibition “CO, S;Inhibition (disint.lmin per mg (disint.lmin per mg endogenous cholesterol) liver)

None 4883 f 813 0 975 * 64 Insignificant

HMG (250 ,umoles) 1057 & 298 78 946 % 5I (P < 0.001)

Cholesterol 5846 * 935 0 755 I-t 85 (6 ,umoles) HMG (z50,umoles) 1362 & 326 plus cholesterol (6 ymoles)

76.7 770 + 62 (P < 0.001)



TABLE III

EFFECT OF ADDING HMG ilz vitro ON THE INCORPORATION OF[I-W]ACETATEINTOCHOLESTEROL

BY LIVER SLICES FROM RATS FED CHOLESTEROL

Cholesterol was fed at 5% level in the diet for 7 days and then the animals were hilled by decapita- tion. Liver slices prepared from normal and cholesterol-fed animals were incubated in Krebs- Ringer phosphate medium in the presence of 6 yC (z mg) of [I-i*C]acetate with (250 pmoles) or without HMG. Experimental details were the same as those described in MATERIALS AND METHODS. Values reported are mean + S.E. for 6 rats.

Groups Cholesterol (disint. /min per mg endogenous cholesterol)

0/O Inhibition

Normal 8706 zt 467 0

Cholesterol-fed 2948 + 328 ;i: < 0.001)

Cholesterol-fed, 542 f 84 93.7 HMH added (250 pmoles) (P < 0.001)

TABLE IV

EFFECT OF ADDING HMG in vitro ON THE INCORPORATION OF [I-14C]~~~~~~~ INTO CHOLESTEROL BY NORMAL RAT LIVER SUPERNATANT FRACTION (600 X g)

I .3 ml of pooled 600 x g supernatant fractions were used for each incubation flask. Prior to incubation necessary cofactor@ and 6 ,uC of [I-Wlacetate were added to make a total volume of 2 ml. Reaction vessels with or without added HMG were incubated for 2 h (37’) in an atmosphere of 0,. The reaction was terminated with I2 pellets of KOH. Experimental details for counting radioactivity in the isolated cholesterol was the same as mentioned in MATERIALS AND METHODS. Values given are from pooled samples of 600 x g supernatant from =J rats.

HMG added (pmoles)

Cholesterol (disint. lmin per mg endogenous cholesterol)

%Inhibition

None

Gone

Gone IO

None 25 None so

5065 *I54 5586 1538 5278

757 4970

378 4876

378

0

72

81.6 0

92 0

92

slices. Up to a certain extent the depression in the rate of cholesterol synthesis was dose dependent. The 14C0, evolved from [I-14C]acetate, via the Krebs cycle with or without added HMG, was identical.

The addition of solubilized cholesterol (dissolved in propylene glycol) to the liver slices caused no inhibition of sterol synthesis. When HMG was added with the cholesterol the percentage of inhibition was almost the same as in the case of HMG alone (Table II). However, a decrease in %O, level was noted in the cholesterol-supplemented group in comparison to the control values.

As shown in Table III, cholesterol feeding caused a supression of [I-l%]acetate incorporation into liver cholesterol (66%). Addition of HMG to the liver slices of cholesterol-fed rats further depressed the rate of incorporation (93%).

In Table IV are shown the results obtained with 600 xg supernatant fraction. It will be observed that when HMG (25 or 50 pmoles) was introduced in the medium,


444 Z. H. BEG, I’. .[. I.I~I’IIiS

the rate of incorporation of /I-%]acetate into cholesterol was almost completely inhibited. Addition of z pmoles of HMG (2 ml of incubation medium) caused j7’;,& decrease in the rate of cholesterol synthesis, when compared to the control values.

Rates of incorporation of !I-%]acetate, [3-14C]HMG-CoA, and Lz-Y]mevalo- nit acid into cholesterol of 10000 >cg supernatant fractions of liver with and without added HMG are shown in Table V. It may be seen that the rates of incorporation of [I-Klacetate and [3-X]HMG-CoA into liver cholesterol of the supernate were significantly inhibited (74% and 58%, respectively). No significant difference was observed between the rates of incorporation of mevalonic ]z-14C]acid into cholesterol in HMG-added and control groups. Again, no effect was noticed on the citric acid cycle, since l*CO, production from both groups was the same.

TABLE 1:

EFFECT OF ADDIP~G HMG in vitro ON THE INCORPORATION or [I-14C]~~~~~~~, [3-IY]HMG CoX AND [z-I%]MEVALONIC ACID INTO CHOLESTEROL BY NORMAL RAT LIVER SUPERKATAKT FRACTION (~ooooxg)

Incubations were carried out with 2.5 ml of the pooled IOOOOX~ supernatant fraction containing either [I-i*C]acetate (6 ,nC, spec. act. 2 mC/mM), [3-W]HMG CoA (0.3 ,uC, spec. act. 1.02 mC/mM) or [2-I4C] mevalonic acid (0.5 PC, spec. act. 2.73 mC/mM). The cofactors include 4 pmoles ATP, Y pmoles glucose 6-phosphate, 2 pmoles NADP and 2 ,nmoles of NAD to make a total volume of 3 ml. The homogenates with (25 ,umoles) or without HMG were incubated for 2 h at 37’. For further experimental details see MATERIALS AND METHODS. Data presented are mean and S.D. for triplicate determinations in each case. The pooled supernatant was from 5 rats.

Tracev Cholesterol o,Inhlbition (disint. lmin pev mg endogenous cholrstevol)

Acetate 5201 & 264 0

Acetate-HMG (25 ,nmolcs) 1151 * 4x0 77.8 (P < 0.001)

HMG-CoA 2781 zk 345 HMG-CoA-HMG (25 pmoles) “95 & r+o ji

(P <. 0.001)

Mevalonic acid To3777 -k z-l79 0

Mevalonic acid-HMG (25 pmoles) 94567 i ‘0.52 x.x*

* Statistically insignificant.

CO, (disint.lmin pcv ur,lnhibition mg liver)

424 i 21 Insignificant

445 + IOI

TABLE VI

in viva INCORPORATION OF [I-Y]ACETATE INTO LIVER AKD SERUM CIIOLESTEROL OF NORMAL AKD HMG- TREATED RATS

Normal rats were given intraperitoneal injections of either 6 mg or I5 mg HMG per day per rat for IO days. Control animals received saline injections (I ml) for the same duration. 2 h before killing, all animals wereinjected with [I-W]acetate (IO & per Ioo g body weight). Cholesterol specific activities (disint./min per mg endogenous cholesterol) in liver homogenates and serum were determined as described under MATE- RIALS AND METHODS. Values given are mean k S.E. for 5 rats in each group.

Group Livev cholesterol q&Inhibition Serum cholesterol O,Inhibition (disint./min per mg (disint. /min per mg endogenous cholesterol) endogenous cholesterol)

Saline-injected (normal) 8782 i 1217 0 5673 f 923 0

HMG-injected (6 mg) 5327 f 732 39 3314 & 628 41.5 (P < 0.05) (P CL 0.05)

HMG-injected (15 mg) 4Ioo + 348 53 2590 l 4’6 54 (P < 0.001) (P ‘. 0.01)

Riochim. Biophys. Ada, 260 (1972) 43g-+#8


TABLE VII

in vivo INCORPORATION OF [1-r4C] ACETATE INTO LIVER AND SERUM CHOLESTEROL OF CHOLESTEROL AND HMG $hS CHOLESTEROL-TREATED RATS

18 rats were divided into three groups, i.e. a control group given normal Purina Chow, a cholesterol-fed group given Purina Chow supplemented with 5% cholesterol, and a HMG plus cholesterol-fed group given 5% cholesterol in diet and treated with HMG intraperitoneally (15 mg per day per rat). The animals of the first two groups received daily injections of saline (I ml). The rats were placed in individual cages and fed their respective diets for a period of 7 days. 2 h prior to killing, all animals were injected with [r-Wlacetate (IO PC per IOO g body weight). Liver and serum cholesterol specific activities were measured according to the techniques described in MATERIALS AND METHODS. Data are presented as mean i S.E. for 6 rats per group.

Group Liver cholesterol 0/O Inhibition Serum cholesterol “/b Inhibition (disint./min per mg (disint.lmin per mg endogenous cholesterol) endogenous cholesterol)

Normal 3176 & 612 0 2230 * 416 0

Cholesterol-fed 845 zt 78 {S

612 + 92 72.5 < 0.001) (P < 0.001)

Cholesterol-fed plus 439 53 * 479 f 80 78.5 HMG(15 mg)-treated

;Y < 0.001) (P < 0.001)

TABLE VIII

RATES OF INCORPORATION OF [I-W]ACETATE INTO CHOLESTEROL WITH AND WITHOUT UNLABELED ACETATE BY LIVER SLICES OF NORMAL AND HMG-TREATED RATS

Normal rats were injected with HMG (IO mg per day per rat) intraperitoneally for 7 days. Control animals received injections of saline (I ml) for the same period. Liver slices prepared from control and HMG-treated rats were incubated with [I-Xlacetate (6 ,uC each) with and without unlabeled acetate. Experimental conditions were the same as those described in MATERIALS AND METHODS. Data are presented as mean -J= S.E. for 3 rats per group. For each animal four incubations were made; two each for [I-“Clacetate, with unlabeled acetate (2 mg) and [I-W]acetate without unlabeled acetate added to the incubation flasks.

Group Tracer Cholesterol (disint./min per mg endogenous cholesterol

Of0 Inhibition

Controls [r-Wlacetate with unlabeled acetate 3286 i 533 0

HMG-treated [r-*%]acetate with unlabeled acetate ‘798 zt 302 45.2 (P < 0.02)

Controls [I-“Clacetate without unlabeled acetate 4140 + 602 0

HMG-treated [I-Wlacetate without unlabeled acetate 2380 & 269 42.3 (P < 0.01)

In vivo stzcdies

In Table VI are shown the results obtained from the experiments performed by injecting HMG to intact rats. It will be observed that the Io-day administration of HMG lowered the rate of [I-14C]acetate incorporation into hepatic and serum cholesterol. The inhibitory effect seemed to be more pronounced with the higher dose.

The data presented in Table VII demonstrated the in vivo effect of HMG plzls cholesterol on the rate of incorporation of [I-14C]acetate into cholesterol. It is clear that cholesterol feeding suppressed the rate of sterol synthesis (73%). Intraperitoneal administration of HMG with cholesterol caused further depression, when compared to the cholesterol-treated rats (48%, P < 0.02). Almost similar effects were obtained with the serum.

The results in Table VIII demonstrate that the depression in the rates of incorporation of [I-14C]acetate into cholesterol by liver slices of HMG-treated rats were


446 z. H. HK, I'. j. I.I.I'II:S

virtually identical in the presence and absence of unlabeled acetate, when coml)ared to their relative controls.

DISCUSSIOS

In 1958 DEKKER ct al. l8 discovered an essentially irreversible hydrolysis of HMG-CoA to HMG and coenzyme A in liver and a number of other animal iissues. There has been no clear cut demonstration of the conversion of HMG to HJIGCoA, although the possible existence of an ATP-dependent esterification has been suggested by the ifz ~iw incorporation of HMG into cholesterolZ9 and the incorporation of small amounts of [3-%jHMG into cholesterol by rat liver homogenates:‘“. DIT~XI t? al.:” concluded that deacylation of HMG-CoA to free HMG led to a metabolic “dead end”, as far as further metabolism was concerned. Although BURCH et al.“” ha\re demonstrated the activation of HMG to HMG-CoA via a coenzyme A transferase in rat kidne!- mitochondria, no conclusive evidence is yet available to substantiate the presence of a kinase or CoA-transferase system in liver, which is the chief site for cholesterol synthesis.

The present study provides evidence that HMG markedly depresses hepatic cholesterol synthesis from acetate in liver slices and homogenates of normal rats. Significant but less marked inhibition of rate of cholesterol synthesis was alsO noticed when HMG was administered to intact rats. The reports that cholesterol is not in-

hibitory to cholesterol synthesis in zitvo from acetates!ll were confirmed in these studies. The results also demonstrated that the inhibitory effect to HMG QW not masked when solubilized cholesterol was added with the HMG (Table II). Furthermore addition of HMG significantly inhibited the rate of cholesterol synthesis from acetate in liver slices of cholesterol-fed rats (i.r. with suppressed cholesterol synthesis ; Table

III). The rate of X0, evolution from [I-Klacetate, which estimates the rate of activity of the citric acid cycle, was almost identical for the control and HMG-treated tissues (Tables I, II and V). These findings indicate that energy production mechanisms of the liver cells are not affected by the addition of HMG. However, addition of cholesterol ipz llitvo lowered l*CO, production. No such effect was observed by cholester-

ol feeding33. The inhibition of rate of cholesterogenesis by HMG in liver homogenates was

much more striking than in liver slices. The effect appears to be dose-dependent (Table IV). The results of Table V show that the site of HMG inhibition of cholesterol biosynthesis from II-Klacetate ilz vitro is between HMG-CoA and mevalonic acid (1’~.

enzyme HMG-CoA reductase). The data thus support the observations of FIMOGNARI AND RODWELL~~J~ that

the incorporation of acetate into mevalonate was significantly inhibited by HMG in rat liver preparations. It was also concluded that the inhibitory effect of HMG can not be attributed to the incorporation of HMG into mevalonate, as purified ;3-X]HMC; was a poor precursor of mevalonate. It was suggested that the lower incorporation at higher concentration of [3-%]HMG may also reflect HMG inhibition of this incorpora-

tions”. A mammalian preparation catalysing the irreversible cleavage of HMGCo.4 to

acetoacetate and acetyl-CoA has been obtained by BAKKAWAT ct a1.35. Acetoacetyl- CoA and acetyl-CoA are also precursors for HMG-CoA synthesis, therefore enhanced


cleavage of HMG-CoA in the presence of HMG might dilute the [x-l*C]acetate pool at the step of HMG-CoA synthesis. However, the decrease in the rates of incorporation of [I-Wlacetate in the presence and absence of unlabeled acetate, into cholesterol with liver slices of HMG-treated animals was identical (Table VIII). This elimi- nates the possibility of [r-14C]acetate pool dilution in HMG-treated animals. The possible dilution of one of the other intermediaries in cholesterol synthesis by HMG should not be ruled out.

The nature of HMG inhibition at the reductive step leading to mevalonic acid formation may be the same as described for bacterial system elsewherelg. The rate of hepatic cholesterol biosynthesis was also inhibited in intact animals when HMG was administered intraperitoneally (Table VI). The results (Table VII) demonstrate that when HMG was given with the cholesterol supplemented diet the rate of inhibition of cholesterol biosynthesis was significantly (86% ; P < 0.001) increased, indicating therefore an additive effect. These data lend support to the hypothesis that in vivo the rate controlling effect of HMG on liver cholesterogenesis is independent of inhibition caused by cholesterol feeding. Furthermore, the mechanism of HMG inhibition both in vitro and in vivo seems to be different from that of cholesterol per se. The decrease in the radioactivity of isolated serum cholesterol (Tables VI and VII) confirms further the inhibition of hepatic cholesterogenesis from [r-14C]acetate in HMG-treated rats.

The aformentioned facts lead us to believe that HMG may be one of the important metabolites responsible for the physiological regulation of cholesterol synthesis. Since HMG is a natural metabolite and blocks the hepatic cholesterogenesis both in vitro and in vivo, the possibility of cholesterol or bile acids being the most important regulatory molecules of cholesterol biosynthesis at the hepatic level appear to us to be much less important than previously described.

In the light of our findings, and the fact that recently the first preparation of highly purified HMG-CoA reductase has been obtained from ratsIT, further attempts will be made to determine whether cholesterol metabolism is regulated by alterations in the activity of HMG-COA reductase as well as in the enzyme population, because any change of the amount of HMG-CoA reductase may be the major mechanism through which the activity of this enzyme is regulated.

Since the pathways of rat intestinal cholesterol biosynthesis appear to be similar to those of rat live?, it was therefore of interest to investigate the possible role of HMG in the control of intestinal cholesterogenesis, these results will be reported shortly.

ACKNOWLEDGMENTS

The authors are greatly indebted to Mr. J. G. Bertrand for all of the technical help which he provided. We are also grateful to the technical staff of the Centre de Recherche sur les Maladies Lipidiques for their technical contributions.

This work was supported by grants from Centre de Recherche en Nutrition de 1’UniversitC Lava1 and the Medical Research Council of Canada.

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in vitro and in vivo inhibition of hepatic cholesterol synthesis by 3-hydroxy-3-methylglutaric acid

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