' A portion of this work was presented at the 1981 meeting of the American Heart Association at Dallas, TX, and published in abstract form (1).

Transcription

1 Effect of removl of lipoproteins of different composition on heptic 3-hydroxy-3-methylglutryl coenzyme A reductse ctivity nd heptic very low density lipoprotein secretion' Pul E. A. Vn Zuiden, Sndr K. Erickson, nd Allen D. Cooper Division of Gstroenterology, Stnford University Medicl Center, Stnford, CA 9435 Abstrct The effect of remnnt lipoproteins on heptic 3- hydroxy-3-methylglutryl coenzyme A (HMG-CoA) reductse nd heptic very low density lipoprotein (VLDL) secretion ws studied in the perfused rt liver nd in vivo. As hd been observed previously, when the liver ws perfused with lipidfree medium, HMG-CoA reductse ctivity incresed bout twofold fter 15 min, nd this increse could be prevented by the ddition of chylomicron remnnts to the medium. However, suppression below bse line ctivity did not occur even with incresing mounts of remnnt cholesterol. When chylomicron remnnts prepred from triglyceride-rich prticles were included in the medium, reductse ctivity ws incresed even bove tht in the control perfusions despite the fct tht pproximtely the sme mount of cholesterol ws removed from these prticles s from stndrd prticles. In contrst, prticles tht were low in triglycerides nd rich in cholesterol not only prevented the rise in reductse ctivity but inhibited it significntly below bse line ctivity. Agin, the totl mount of cholesterol removed ws the sme s with the other types of prticles. These results suggested tht both the triglycerides nd cholesterol exerted n effect on HMG-CoA reductse. Consistent with this hypothesis, significnt correltion ws found between reductse ctivity nd the rtio of triglycerides to cholesterol removed, but not to either lone. To explore the role of triglycerides further, the effect of these lipoprotein prticles on VLDL secretion ws determined. VLDL secretion ws stimulted by both stndrd nd triglyceride-rich remnnts but not by triglyceride-poor remnnts. The degree of stimultion with stndrd chylomicron ws comprble to tht induced by infusion of comprble ftty cid lod s oleic cid bound to lbumin. In vivo similr effect of these lipoproteins on HMG-CoA reductse ctivity ws observed. Rts were injected with lipoprotein bolus contining 7 mg of cholesterol, nd reductse ctivity in the liver ws mesured 2 hr lter. Stndrd chylomicrons nd triglyceride-rich chylomicrons stimulted reductse to 157% nd 187% of control ctivity, respectively, wheres cholesterol-rich VLDL suppressed reductse ctivity to 3% of control ctivity.l These observtions support the hypothesis tht remnnt lipoproteins hve dul effect on heptic HMG-CoA reductse ctivity; the cholesterol in these lipoproteins suppresses heptic reductse ctivity while the triglycerides concommitntly delivered stimulte reductse ctivity t lest in prt becuse they stimulte heptic VLDL secretion. Therefore, the net response of heptic HMG-CoA reductse to prticulr dietry lipopro- tein will depend upon the blnce between the cholesterol nd triglycerides crried to the liver.-vn Zuiden, P. E. A., S. K. Erickson, nd A. D. Cooper. Effect of removl of lipoproteins of different composition on heptic 3-hydroxy-3- methylglutryl coenzyme A reductse ctivity nd heptic very low density lipoprotein secretion.]. Lipid Res : Supplementry key words cholesterol triglycerides It is well estblished tht heptic cholesterol synthesis is subject to inhibition by dietry cholesterol. It hs lso been demonstrted tht chylomicrons, the lipoproteins of intestinl origin, when injected into rts in vivo lso reduce the rte of cholesterol synthesis in liver in dosend time-dependent mnner. Mximl inhibition (bout 6%) is chieved 12 hr fter single bolus of 8 mg of lipoprotein cholesterol/loo g body weight (2). 3-Hydroxy-3-methylglutryl coenzyme A (HMG-CoA) reductse, the rte-limiting enzyme for cholesterol synthesis, is inhibited by bout 8% fter feeding cholesterol for hr. The hlf-life for the enzyme ctivity hs been estimted s 1-4 hr (3, 4). Therefore, one would expect tht inhibition of enzyme ctivity by 5% should occur within 1-4 hr fter dministrtion of bolus of lipoprotein cholesterol. To dte this hs not been demonstrted. When chylomicron remnnts were dded to liver perfusions the increse in HMG-CoA reductse normlly induced by perfusion ws prevented; however, HMG-CoA reductse ws not inhibited below the initil vlue (5). Thus, it ppered tht lthough HMG-CoA reductse could respond to bolus of cho- Abbrevitions: VLDL, very low density lipoproteins; HMG-CoA, 3-hydroxy-3-methylglutryl coenzyme A; TCA, trichlorocetic cid; MVA, mevlonolctone. ' A portion of this work ws presented t the 1981 meeting of the Americn Hert Assocition t Dlls, TX, nd published in bstrct form (1). 418 Journl of Lipid Reserch Volume 24, 1983

2 lesterol in lipoprotein, the degree of inhibition ws not s substntil s might hve been expected. Chylomicron remnnts lso contin lrge mounts of triglyceride which is hydrolyzed to free ftty cids fter uptke by the liver. Thus, in ddition to cholesterol, chylomicron remnnts my trnsport lrge mounts of ftty cids in the form of triglycerides to the liver. Goh nd Heimberg (6) hve estblished tht when ftty cids re delivered to the liver there is both n increse in heptic very low density lipoprotein (VLDL) triglyceride secretion nd n increse in HMG-CoA reductse ctivity. Therefore, it is possible tht two opposing regultory events occur with HMG-CoA reductse nd cholesterol synthesis when lipoproteins contining both cholesterol nd triglycerides re tken up by the liver. The degree of inhibition of reductse observed could thus be the net result of the inhibitory effect of cholesterol nd the stimultory effect of ftty cids. To test this hypothesis, the cute effects on heptic HMG-CoA reductse of three different types of lipoprotein prticles contining mrkedly different rtios of triglyceride nd cholesterol were studied in the perfused liver system nd in vivo. In ddition, the effects of these lipoproteins on heptic VLDL secretion by the perfused liver were investigted. Animls MATERIALS Mle Sprgue-Dwley rts were used in ll experiments. They were housed in windowless room illuminted between 7 AM nd 7 PM nd fed commercil rt chow for t lest 7 dys prior to use. Retired breeders, fed n rtherogenic diet (7) for 3 to 4 weeks, were used s plsm donors for cholesterol-rich very low density lipoproteins. Liver donors weighed g, lymph donors weighed 3-4 g, nd the rts used in in vivo experiments weighed g. Retired breeders were used for lipoprotein remnnt preprtion s described previously (8). Chemicls D,L 3-Hydroxy-3-methyl-[ 3-4C]glutryl coenzyme A (4-6 mci/mmol), R,S[5- H]mevlonic cid (dibenzyldiethylenedimine slt, 1-5 Ci/mmol) nd Aqusol were obtined from New Englnd Nucler; oleic cid, NADP, glucose-6-phosphte dehydrogense, bovine serum lbumin, glucose-6-phosphte (disodium slt), triolein (prcticl grde), nd L--phosphtidylcholine (soyben, purity pprox. 5%) were purchsed from Sigm. Sodium turocholte nd dithiothreitol were from Clbiochem. 3-Hydroxy-3-methylglutryl coen- zyme A ws from P.L. Biochemicls, nd the triglyceride ssy kit ws from Boehringer. Egle s bsl medium contining Egle s slts nd L-glutmine ws from Grnd Islnd Biochemicl Compny. All other regents were nlyticl grde. Lipoprotein preprtion METHODS Mesenteric lymph chylomicrons were prepred s described previously (9). A silstic ctheter ws inserted into the mesenteric lymph duct of 3-4 g rt. Twenty-four hr lter solution of one whole egg dispersed in 125 ml of norml sline ws continuously infused through n intrgstric ctheter t rte of 1.8 ml/hr. The lymph ws filtered through ten lyers of guze, lyered under.9% NCI, nd centrifuged in Beckmn SW 41 rotor t 1.1 X lo5 g for 45 min. The superntnt lyer ws seprted with tube slicer nd resuspended in smll volume of buffered norml sline. Chylomicrons rich in triglycerides were prepred by slight modifiction of the method of Bennett-Clrk (1). A sonicted emulsion contining 7 g of triolein, 4 g of L--phosphtidycholine,.1 g of sodium turocholte, nd.5 g of Tween 8 in 1 ml of wter ws infused intrgstriclly t rte of 1.8 ml/hr. The collected lymph ws processed for chylomicrons s described bove. Very low density lipoproteins rich in cholesterol were prepred s previously described (7). A group of 2-3 retired breeders ws fed diet contining 5% lrd, 1% cholesterol,.1 % propylthiourcil, nd 3% turocholic cid for period of t lest 3 weeks. The rts were killed between 13 nd 15 hr by ortic puncture nd the serum ws seprted from red blood cells by low speed centrifugtion. Serum VLDL ws collected by djusting the density to 1.19 g/ml nd centrifuging t 14, g for 16 hr in Beckmn SW 41 rotor. A tube slicer ws used to collect the top lyer contining the VLDL. Lipoprotein remnnts were prepred in eviscerted rts s previously described (8). Ntive (precursor) lipoproteins were injected t rte of.6 ml/min through femorl vein ctheter. The injected lipoproteins contined no more thn 8 mg of cholesterol in chylomicrons or cholesterol-rich VLDL nd 2 mg of cholesterol in triglyceride-rich chylomicrons nd were llowed to circulte for 3 hr. Cholesterol-rich VLDL ws llowed to circulte for 3 min. The nimls were exsnguinted by ortic puncture nd blood ws llowed to clot for 2 hr. Plsm ws seprted from red blood cells by low speed centrifugtion. Serum contin- Voji Zitideti, Errckso~, oud Coopr Lipoprotein composition, HMG-CoA reductse, nd VLDL secretion 419

3 ing chylomicron remnnts ws centrifuged t 2. X 1 O5 g for 12 min in Beckmn SW 41 rotor. The lipoprotein remnnts were seprted with tube slicer nd resuspended in.9% NCI. Serum contining VLDL remnnts ws centrifuged t 2 X lo5 g for 16 hr t density 1.19 g/ml nd lipoprotein remnnts were seprted nd resuspended. Triglyceride, cholesterol, nd cholesteryl ester contents were determined on ll lipoprotein preprtions. Lipoproteins tht were to be rdioiodinted were chromtogrphed on Bio-Gel A-5 M (BioRd Lbortories, Richmond, CA) to remove heme products, trces of lbumin, nd other impurities (11). The lipoproteins were resuspended in.15 M NCI,.1 % EDTA, ph 7.4, djusted to the pproprite density with KBR, nd recentrifuged. They were lbeled with IZ5I (sodium iodide, New Englnd Nucler, Boston, MA) s previously described (12) ccording to the method of McFrlne (13) s modified by Bilheimer, Eisenberg, nd Levy (14). Not more thn 2 ml of lipoproteins contining mg of protein were dilyzed ginst 1.O M glycine buffer, ph 1., overnight. One to 3 mci of crrier free N-Iz5I ws dded to the lipoprotein followed by rpid injection of IC1 solution (1: 1, mol:mol, iodine:protein rtio ssuming moleculr weight of 3, for the protein constituents). Unbound iodine ws removed by pssing the lbeled lipoproteins through smll column of 2% grose followed by dilysis t 4 C ginst the NCI, EDTA buffer with t lest four chnges of buffer. The distribution of rdioiodine ws determined by mesuring the percentge of I tht ws trichlorocetic cid (TCA)-precipitble (12). In vivo 125~-1beied lipoprotein disppernce studies lz5i-lbeled lipoproteins of high specific ctivity were injected into ctheter tht hd been previously inserted into the inferior ven cv through the femorl vein. The totl injected mss ws less thn.1 mg of protein. Control nd experimentl nimls were of equivlent weight (3 k 2 g). The ctheter ws rpidly flushed with.9% NCI. Blood smples (.1 ml) were drwn t 2-min intervls for 15 min then t 1- min intervls up to 45 min. Following the collection of ech blood smple, the ctheter ws wshed with.1 ml of.9% NCl. Red blood cells were removed by centrifugtion. A smll smple ws counted nd, fter ddition of crrier protein, 1% TCA precipittion ws performed on ech time point. The kinetic prmeters (slope, Y intercept, nd tllz) were clculted using the MLAB curve fitting progrm (15). Liver perfusion After pentobrbitl nesthesi, liver perfusion ws performed in situ by the method of Mortimer (1 6) s 42 Journl of Lipid Reserch Volume 24, 1983 described previously (5). Portl blood flow ws interrupted t most for few seconds during preprtion of the orgn. Oxygention ws ccomplished with silstic coil (Dow Corning Corp., Midlnd, MI) s described by Hmilton et l. (1 7). The entire perfusion system except the rtificil lung ws siliconized s described by Hmilton et l. (1 7). The plsm-free perfuste (4 ml, ph 7.4) contined 22% wshed humn red cells in Egle s bsl medium supplemented with 3 g of bovine serum lbumin nd 1 mg of glucose for ech 1 ml of medium. Hemolysis during the perfusion ws negligible. The perfuste circulted t 1.1 ml per min per g liver. Oxygention prior to nd during perfusion ws mintined using gs mixture contining 95% 2 nd 5% COz. Vibility of the liver ws judged t 5-1 min by color, O2 extrction, nd the bsence of perfuste loss. This ws monitored throughout the perfusion. Previously published dt from this lbortory hve shown excellent vibility for 3 hr of perfusion (5) nd unpublished evidence shows tht rtes of VLDL secretion nd bile flow were liner for 4-5 hr. Immeditely prior to estblishing recircultion, smll lobe of liver weighing between.2 nd.3 g ws ligted, excised, nd microsomes were prepred. Perfusions were crried out s outlined in Fig. 1. Lipoprotein remnnts were dded nd llowed to circulte for 15-2 min. This lipoprotein-contining perfuste ws then wshed out over 5-1 min with pproximtely 6-8 ml of lipoproteinfree perfuste. When residul lipoproteins were ll removed, recircultion ws re-estblished. The effectiveness of the wshout ws determined by the bsence of triglycerides in the perfuste. All three types of lipoprotein remnnts were tken up with comprble efficiency. In most instnces bout 5% of the dded mteril ws removed in 15-2 min. The rtes of removl were consistent with wht ws predicted by previous publictions from this lbortory (18, 19). The perfusion ws continued for n dditionl 2 hr nd hourly smples were tken for triglyceride determintion. At the end of the perfusion, the finl perfuste ws collected, its hemtocrit nd volume were recorded, nd the red cells were removed by low speed centrifugtion. The liver ws removed, weighed, chilled in.9% NCI, nd portion ws tken for preprtion of microsomes. A smple of the perfuste ws tken for triglyceride determintion nd the reminder ws used to estimte very low density lipoprotein secretion fter isoltion of VLDL by ultrcentrifugtion. In one group of experiments insted of lipoproteins, oleic cid complexed to lbumin ws dded to the perfuste t concentrtions of.25 mm,.5 mm, or 1 mm. During the entire perfusion period oleic cid ws dded continuously to mintin constnt concentrtion (2). Free ftty cid uptke ws equted with the mount infused (2.5, 5, nd 1 mg/g liver per 2 hr). The re-

4 Perfuste for Remnnt removl Perfuste for Lipoprotein < secretion determintion TIME(min I2 Removl of liver segment ( gm) Strt perfusion (1st recircultion) Estblish vibility Remnnts in recircultory perfuste Lipoprotein-free perfuste (wsh out) Lipoprotein-free perfuste (2nd recircultion) - Rpid excision of liver nd preprtion of microsomes Fig. 1. Protocol for liver perfusion studies. Prior to strting recirculting liver perfusion, smll lobe of the rt liver ws resected, microsomes were prepred, nd HMG-CoA reductse ctivity ws determined. The perfusion ws strted by connecting inferior ven cv nd portl veins to recirculting system s described (5) previously. After vibility ws estblished lipoprotein remnnts were dded nd llowed to circulte for 15-2 min. The remining remnnts were wshed out with n excess of lipid-free perfuste consisting of Egle's medium contining 3% ftty cid-poor bovine lbumin, nd 1 mg glucose/loo ml, ph 7.4. The efficiency of the wshout ws determined by mesuring perfuste triglyceride. When ll remnnts were removed, recircultion with lipid-free medium ws continued for n dditionl 2 hr. minder of the perfusion protocol ws performed s described. In vivo experiments The femorl vein of nesthetized rts weighing g ws cnnulted with polyethylene tubing (PE1, Cly-Adms). A lipoprotein bolus contining 7 mg of cholesterol nd vrious mounts of triglycerides ws injected through the cnnul into the experimentl rts. Control rts were infused with n equl volume of norml sline (ph 7.4) The nimls were killed 2 hr fter injection nd their livers were removed for nlysis. Preprtion of microsomes The liver smples were blotted dry, weighed, nd homogenized in 5 volumes of Buffer A (.1 M sucrose,.5 M KCI,.25 M KH2P4,.3 M EDTA, ph 7.4) by three strokes t moderte speed in glss-teflon Potter-Elvejhem homogenizer. The homogente ws centrifuged for 15 min t 1, g to sediment unbroken cells, nuclei, mitochondri, nd lysosomes. The superntnt ws centrifuged for 6 min t 15, g to sediment microsomes. The 15, g superntnt ws discrded nd the pellet ws resuspended in Buffer A. The 15, g centrifugtion ws repeted nd the finl pellet ws resuspended in Buffer A. The microsomes were ssyed for protein content nd HMG-CoA reductse ctivity. In the in vivo experiments, livers were divided nd microsomes were prepred in Buffer A nd in Buffer A contining 5 mm sodium fluoride. The finl resuspension ws done in Buffer A lone. Assy of HMG-CoA reductse ctivity HMG-CoA reductse ws ssyed essentilly s described previously (21), except tht totl ssy volume of.5 ml ws used contining mg of microsoml protein. Chemicl ssys Protein ws determined by the method of Lowry et l. (22) or by the biuret method (23) using bovine serum lbumin s reference stndrd. Phospholipid phosphorus ws determined colorimetriclly by the method of Brtlett (24). Triglycerides were ssyed enzymticlly by the method of Eggstein nd Kreutz (25) using the triglyceride ssy kit from Boehringer. Totl cholesterol ws determined by gs-liquid chromtogrphy s previously described (26). RESULTS Effect of lipoprotein composition on heptic HMG- CoA reductse in liver perfusions The livers of rts killed 3 hr (1 AM) fter the onset of the light phse were perfused with lipid-free perfuste. HMG-CoA reductse ctivity ws determined before nd fter 2.5 hr of perfusion. As hd been reported previously (5), enzyme ctivity incresed during the period of perfusion rising from.16 f.3 to.28 k.4 nmol MVA min-' mg protein-' (P <.5, n = 12). It hd been shown previously (5) tht this increse ws inhibited by the presence of cholesterol in the perfuste either in phospholipid dispersion or in chylomicron remnnts (5, 9). Despite ddition of incresing mounts of cholesterol in chylomicron remnnts, reductse ctivity remined ner the initil level (not shown). However, in the intct niml, cholesterol feeding suppresses heptic HMG-CoA reductse ctivity by greter thn 5% within few hours. Thus, it ws surprising tht cholesterol crried in chylomicron remnnts ws not effective in suppressing reductse ctivity below the initil vlue. To explin this observtion it ws hypothesized tht the mount of concommitntly dministered triglyceride might be fctor in determining the response of heptic HMG-CoA reductse to given mount of cholesterol in remnnt lipoproteins. To test this hypothesis, lipoprotein remnnts of vrious trig1yceride:cholesterol rtios were prepred. The Vn Zuiden, Erickson, nd Cooper Lipoprotein composition, HMG-CoA reductse, nd VLDL secretion 421

5 TABLE 1. Composition of precursor lipoprotein remnnts Totl Free Triglyceride Cholesterol Cholesterol Phospholipid Protein" R d? u,eight Precursor lipoproteins Chylomicrons Triglyceride-rich chylomicrons Cholesterol-rich VLDL Lipoprotein remnnts Chylomicrons N D ~ Triglyceride-rich chylomicrons ND Cholesterol-rich VLDL (' Lipoprotein protein content ws determined ccording to Lowry et l. (2) following Bio-Gel A5m chromtogrphy. ND, not determined. Chylomicrons were prepred from lymph obtined from lymph fistul rts tht were fed mixture of egg in 12 ml of.9%, NCl through n intrgstric ctheter (9). Triglyceride-rich chylomicrons were obtined similrly except tht these rts were fed mixture of triolein nd lecithin (IO). Cholesterol-rich VLDL ws obtined from rts fed stndrd therogenic diet (7). Lipoprotein remnnts were prepred in eviscerted rts s described in Methods. Results shown re the verge of determintions on two seprte preprtions of lipoproteins in which the complete chemicl composition ws done. They re consistent with the trig1yceride:cholesterol rtios tht were determined on every lipoprotein btch used. composition of the three types of remnnts studied is given in Tble 1. Cholesterol-rich VLDL remnnts were used becuse more extreme ltertion of the triglyceride:cholesterol rtio could be obtined with these prticles thn with ny chylomicron remnnt preprtion. The remnnts were perfused for 15-2 min, the perfuste ws removed, the liver ws wshed out with lipoprotein-free perfuste, nd then recircultion with lipoprotein-free perfuste ws estblished for 2 dditionl hours (see Fig. 1). The perfustes were collected to determine the mount of remnnts removed. The cholesterol removed per g of liver in these experiments verged.74 k.5 pmol for norml chylomicron remnnts,.88 t.18 pmol for triglyceride-rich chy- TABLE 2. lomicron remnnts, nd pmol for cholesterol-rich VLDL remnnts. These mounts were not significntly different. The three types of remnnts hd profoundly different effects on heptic HMG-CoA reductse ctivity (Tble 2). Remnnts prepred from the stndrd chylomicrons prevented the increse in HMG-CoA reductse ctivity tht occurs during lipid-free liver perfusions, but s noted before, they did not suppress reductse ctivity below the pre-perfusion level. In contrst, cholesterol-rich prticles not only prevented the rise in reductse ctivity, but they decresed reductse ctivity to level significntly below the initil vlue. The decrese of 25% over 2.5-hr period is con- Effect of lipoprotein remnnts on HMG-CoA reductse in the perfused liver HMG-CoA Reductse Pre- vs Post-perfusion Post- Post- Control vs Lipoprotein Number Pre-perfusion perfusion perfusion Post-perfusion iimu/ ~ v.4 x mg pro-' x mn-1 Control (lipoprotein-free) f.3.28 f.4 P i.5 Chylomicron remnnts iz.3.17 f.2 NSO P <.1 Trig1 yceride-rich chylomicron remnnts r f.6 P <.5 NS Cholesterol-rich VLDL remnnts r f.1 P <.2 P <.1 (1 NS, not sttisticlly significnt. Livers were perfused s described in Methods. At the brginning of the perfusion, lobe of the liver ws removed nd microsoml HMG-CoA reductse ctivity ws determined. Chylomicron remnnts, triglyceriderich chylomicron remnnts, nd cholesterol-rich VLDL remnnts were prepred s described. The mounts of cholesterol removed by the livers from these different lipoprotein remnnts were comprble. After 15-2 min, lipoprotein remnnts were wshed out nd the perfusion ws continued for 2 hr. Heptic HMG-CoA reductse ctivity ws then determined in freshly prepred microsomes. 422 Journl of Lipid Reserch Volume 24, 1989

6 C? 'C m e : P E m - E.2- ;.1 $ 1 I Y =.25-4 X r =.14 m ' : *. I, I 1 I I n W CHOLESTEROL REMOVED (Fmdes gm liver-') C I 2 1 I I I,, W OO ? e.31 FATTY ACID REMOVED (pnmdes gm liver-'). * /. ~ W j l I I I I, OO IO FATTY ACID REMOVED / CHOLESTEROL REMOVED Fig. 2. Reltionship between heptic HMG-CoA reductse ctivity nd lipid uptke from lipoprotein remnnts by perfused rt livers. The techniques of liver perfusion nd preprtion of remnnts were s described in the legend to Fig. 1 nd in Methods. The removl of remnnt cholesterol ws clculted s the difference between the mount dded nd the mount of cholesterol recovered in the wshout s B C sistent with the rte of suppression expected if synthesis of HMG-CoA reductse hd cesed. The triglyceriderich chylomicron remnnts, on the other hnd, filed to prevent the increse in reductse ctivity nd in fct ctully enhnced it bove the level chieved with control (lipid-free) perfusions, lthough this did not rech sttisticl significnce. It must be stressed tht these striking differences in reductse response were obtined fter removl by the liver of very similr mounts of cholesterol. When HMG-CoA reductse ctivity ws plotted s function of either the mount of cholesterol (Fig. 2) or the mount of triglyceride removed (Fig. 2b), the correltions were poor. However, if reductse ctivity ws plotted s function of the molr rtio of the mount of ftty cids removed to tht of cholesterol removed, distinct reltionship ws observed (Fig. 2c). Thus, the more cholesterol delivered per unit of ftty cid the more suppressive the prticle is likely to be. This demonstrtes the importnce of both lipids in the physiologic regultion of HMG-CoA reductse. The lipid composition of the prticles remining in the perfuste fter the initil perfusion period ws the sme s the prticles dded to the perfuste. This is consistent with previous observtions suggesting tht the prticles re removed s unit (9) nd tht neither the triglycerides nor the cholesterol were removed preferentilly by the liver. Becuse cholesterol removl from the different remnnt types ws comprble, chnges in the rtio of trig1ycerides:cholesterol determined the response of HMG-CoA reductse ctivity in confirmtion of our initil hypothesis. The effect of remnnt lipoprotein composition on heptic VLDL secretion by the perfused liver It is well-estblished tht free ftty cid influx is principl determinnt of the rte of VLDL secretion by the liver (18). VLDL secretion must result in cholesterol efflux. Thus, ftty cid infusion might be expected to stimulte the ctivity of HMG-CoA reductse to compenste for this loss. This sequence of events ws in fct shown to occur by Goh nd Heimberg (6). We hypothesized tht the effects on HMG-CoA reductse observed in this study due to ltering the lipoprotein triglyceride:cholesterol rtio might be explined by chnges induced in VLDL secretion s consequence of different ftty cid lods. Accordingly, the effects of comprble volume. Ech point represents seprte experiment. Tble I gives the reltive weight composition of the lipoprotein remnnts used. A: HMG-CoA reductse ctivity s function of cholesterol removed. B: HMG-CoA reductse ctivity s function of triglyceride removed. C: HMG-CoA reductse ctivity s function of the rtio of the mount of ftty cid:cholesterol removed. Dt from the sme 29 perfusions were used to clculte the regressions lthough in some instnces the points did not fit on the xis used for the illustrtion. Vriii Zuiden, Ericksori, nud Cooper Lipoprotein composition, HMG-CoA reductse, nd VLDL secretion 423

7 NO CHYLOMKRON TO -RICH CHOL- RICH LIPO- REM 114) CHYLO- VLDL REM PROTEIN MICRON (6) (14) REM 19) *p <.m **p <.1 Fig. 3. Effect of lipoprotein remnnts on heptic very low density lipoprotein triglyceride secretion rte fter comprble cholesterol uptke by the perfused rt liver. VLDL triglyceride secretion in the lst 2 hr of recircultion with lipid-free medium (see protocol, Fig. 1) ws determined by enzymtic ssy. Lipoprotein remnnt removl ws clculted s described in Fig. 2. The cholesterol removed per g liver verged.74 f.5 pmol for stndrd chylomicron remnnts,.88 f.18 rmol for triglyceride-rich chylomicron remnnts, nd.96 f.16 pmol for cholesterol-rich VLDL remnnts. These mounts were not significntly different. *, Different from control P <.5; **, different from control P <.1. mounts of infused free ftty cids on this prmeter were lso studied. Chylomicron remnnts stimulted the rte of VLDL secretion s compred to tht observed with lipid-free perfusion (Fig. 3). The degree of stimultion ws proportionl to the mount of triglyceride / REM AN.EIC ACID 4 7 2t I I 1 I I FATTY ACID REMOVED ( pmole. gm liver-' Fig. 4. The effect of Ftty cids, either s chylomicron remnnt triglycerides or oleic cid, on heptic VLDL secretion in perfused rt livers. Oleic cid cornplexed to bovine seruni lbumin ws infused continuously throughout the entire perfusion period. VLDL. secretion nd coniposition were determined on the lst 2 hr of the recircultion perfuste. Dt on VLDL composition of the high nd low oleic cid infusion rtes re given in Tble 3. VLDL secretion following removl of norml chylomicron remnnts ws plotted s function of the mount of ftty cid contined in remnnts s triglycerides removed during the perfusion crried out s described in the legend to Fig. 1 nd in Methods. I"' removed nd the mgnitude of the stimultion ws similr to tht induced by comprble mount of free ftty cid (Fig. 4). Triglyceride-rich chylomicron remnnts lso stimulted VLDL secretion (Fig. 3), but the mgnitude of the stimultion ws not s gret s would hve been expected from the mount of ftty cid removed. As predicted by the hypothesis, the cholesterolrich VLDL remnnts did not stimulte VLDL secretion bove the control level (Fig. 3). The compositions (trig1yceride:cholesterol rtios) of the newly secreted VLDL prticles isolted fter perfusions with norml chylomicron remnnts or cholesterol-rich VLDL were comprble to those secreted by livers perfused with lipid-free medium (Tble 3). However, perfusion of triglyceride-rich chylomicron remnnts or high dose oleic cid infusions resulted in secretion of VLDL prticles with significntly incresed trig1yceride:cholesterol rtio (Tble 3). Effects of ntive (precursor) lipoproteins on heptic HMG-CoA reductse ctivity in vivo In the intct niml number of fctors re involved in determining the mount nd type of lipid reching the liver. Therefore, it ws importnt to scertin whether the effects of vrious lipoproteins studied in the perfused liver system would be expressed in the regultion of heptic HMG-CoA reductse ctivity in vivo. First, it ws necessry to show tht the three different types of lipoproteins hd similr disppernce rtes in vivo. Trce mounts of 1251-lbeled norml chylomicrons, triglyceride-rich chylomicrons, or cholesterolrich VLDL were injected into the femorl veins of rts nd disppernce of the lbel from TCA-percipitble serum proteins followed. The t1/2 for the first phse of disppernce ws clculted by computer fit to the eqution D(t) = AeCt + BeD'. The initil tl/2 clculted TABLE 3. Effect of lipoprotein remnnts nd oleic cid infusions on the composition of heptic VLDL secretion by perfused livers Trig1yceride:Cholesterol Weight Rtio Control (lipoprotein-free) (1) 9.8 f.6 Chylomicron remnnts (13) 1. f.6 Triglyceride-rich chylomicron remnnts (7) 14.9 f 1.2" Cholesterol-rich VLDL remnnts (6) 8.9 f.7 Oleic cid.5 mm (4) 8.8 f 1. Oleic cid 1. mm (4) 14.8 f 1.'' ' Different from control t P <.1. Perfusions were performed s described in Tble 2. VLDL (d < 1.6 g/ml) ws seprted from the collected finl perfuste by ultrcentrifugtion nd ws resuspended in sline. Triglyceride nd cholesterol contents were determined s described in Methods nd the rtios were computed from the reltive msses. Number of determintions in prentheses. 424 Journl of Lipid Reserch Volume 24, 1983

8 TABLE 4. Effects of lipoproteins in vivo on heptic HMG-CoA reductse Number HMG-CoA Reductse - NF % Control HMG-CoA Reductse + NF 56 Control nmol MVA x mg pro/-' x min-' nmol MVA X mg prot-' X min-' Control f f.1 1 Chylomicrons 9.28 f.4 157O.5 f.1 83 Trig1 yceride-rich chylomicrons 8.33 *.6 187b.5 f.1 83 Cholesterol-rich VLDL 9.5 *.1 3".2 f.1 33 Different from control t P <. 1. Different from control t P <.5. Rts were injected with different lipoproteins through femorl vein ctheter. Ech experimentl niml received bolus of lipoproteins contining 7 mg of cholesterol nd vrying mounts of triglycerides. Control rts received comprble volumes of buffered sline (ph 7.4). After 2 hr, the nimls were killed, the livers were removed, microsomes were prepred in buffer with or without 5 mm NF, nd HMG-CoA reductse ctivities were determined. from C for norml chylomicrons ws 2.4 min; for triglyceride-rich chylomicrons, 3.3 min; nd for cholesterol-rich VLDL,.5 min. This initil disppernce hs been shown previously to be due lrgely to heptic removl for both chylomicrons nd hypercholesterolemic VLDL (19) nd the sme is presumbly true for the triglyceride-rich prticles. Thus, virtully ll of the lipoproteins should hve been removed during the initil period of 2-hr experiment. To study the effects of the composition of these lipoproteins on heptic HMG-CoA reductse, 7 mg of cholesterol s either norml chylomicrons or triglyceride-rich chylomicrons or cholesterol-rich VLDL ws injected into the femorl vein. After 2 hr, the nimls were killed nd heptic HMG-CoA reductse ctivity ws determined. Both the stndrd nd triglyceride-rich chylomicrons stimulted HMG-CoA reductse ctivity (Tble 4). The triglyceride-rich chylomicrons hd more pronounced effect on HMG-CoA reductse ctivity which incresed to 187% of control. The stndrd chylomicrons incresed reductse ctivity to 157% of control. In contrst, the cholesterol-rich prticles inhibited HMG-CoA reductse ctivity to 3% of control. These dt re entirely comptible with the results obtined in the perfused liver studies nd further support the hypothesis tht the cute effect of lipoproteins on heptic HMG-CoA reductse ctivity is function of both the triglyceride nd cholesterol content. When microsomes were prepred in the presence of 5 mm NF, HMG-CoA reductse ctivity ws the sme in control nimls nd in those receiving either norml or triglyceride-rich chylomicrons (Tble 4). From these dt it ppered tht the totl mount of enzyme ctivity hd incresed s well s the mount tht could be ctivted. In contrst, heptic microsomes tht were prepred in 5 mm NF from nimls receiving cholesterol-rich VLDL hd 33% (Tble 4) of the control vlue suggesting tht the totl mount of functionlly ctive enzyme hd been decresed in these nimls. DISCUSSION The decrese in cholesterol synthesis in the liver which occurs in vivo in response to cholesterol feeding (27) or intrvenous injection of chylomicrons (2) hs been well estblished. These phenomen generlly hve been exmined only t reltively long intervls (6-48 hr) fter dministrtion of the stimulus. Becuse the hlf-life of HMG-CoA reductse ctivity, the rte-limiting enzyme for cholesterol biosynthesis, is 1-4 hr (3), nd the ctivity cn respond profoundly to chnges in sterol content of the liver within 1 hr, (28) it should be possible to regulte heptic reductse ctivity cutely with lipoprotein cholesterol. The observtion (9) tht liver perfusion with chylomicron remnnts would suppress the rise in reductse ctivity ordinrily induced by perfusion with sterol-free medium but did not inhibit reductse ctivity below the bseline vlue s hd been observed for 25-hydroxycholestero1, cholesterol nlogue (29), suggested tht the regultion of HMG-CoA reductse by lipoproteins might be complex. Similr findings hve been reported for cultured heptocytes (3). The observtions reported here, tht incresing the mounts of cholesterol crried s chylomicron remnnts did not result in ny further inhibition of reductse ctivity, lso supported this suggestion. The results of Goh nd Heimberg (6) showing tht HMG-CoA reductse ctivity nd VLDL secretion were incresed by ftty cid infusions suggested to us tht the response of HMG-CoA reductse to lipoproteins might be the net result of two opposing phenomen; one, the uptke of cholesterol nd two, the uptke of triglycerides, which re rich source of ftty cids. If this is Vn Zuidrn, Erickson, nd Cooper Lipoprotein composition, HMG-CoA reductse, nd VLDL secretion 425

9 correct, we resoned tht lipoproteins of different cholesterol nd triglyceride compositions should hve different effects on heptic HMG-CoA reductse ctivity cutely, nd tht these effects should reflect the needs of the cell for cholesterol. To study this hypothesis, we chose to prepre lipoproteins of three very different trig1yceride:cholesterol rtios. These were the stndrd chylomicrons tht hve been previously used in this lbortory nd by others (2, 9, 12, 18, 19, 31) with trig1yceride:cholesterol weight rtio of 35, triglyceride-rich chylomicrons with rtio of 225, nd cholesterol-rich VLDL with rtio of.64. Despite the differences in cholesterol nd triglyceride contents, the precursor lipoproteins were shown to be resonbly comprble in their disppernce rtes in vivo, suggesting tht it would be possible to interpret ny differences in their effects s being due minly to the different trig1yceride:cholesterol rtios. Using remnnts prepred from these lipoproteins nd the perfused liver system, strong support for this hypothesis ws obtined. Despite the delivery to the liver of comprble mounts of cholesterol, prticles rich in triglycerides nd poor in cholesterol stimulted HMG- CoA reductse while those poor in triglycerides nd rich in cholesterol inhibited reductse below the pre-perfusion level. Finlly, prticles of intermedite composition were ble to mintin HMG-CoA reductse t its bse line level. The mgnitude of the chnges induced by the lipoproteins ws modest but consistent with wht would be expected over 2.5-hr period for n enzyme with hlf-life in the 1-4 hr rnge, especilly considering the requirement for intrcellulr processing of cholesteryl ester which is slow reltive to uptke (1 8). Becuse in vivo there re lrge number of other metbolic nd hormonl fctors tht could ffect HMG- CoA reductse ctivity, it ws of interest to compre the cute effects of the vrious lipoproteins dministered in vivo with those of their remnnts in vitro. In fct there ws good greement. Triglyceride-rich lipoproteins stimulted nd cholesterol-rich lipoproteins suppressed HMG-CoA reductse ctivity within 2 hr of dministrtion. Interestingly, the stndrd chylomicrons stimulted reductse ctivity in vivo rther thn leving it unchnged s ws observed in the perfused liver system. A possible explntion for this observtion is tht the lrge bolus of lipoprotein triglyceride dministered might not be hydrolyzed in vivo s completely before uptke of the prticle by the liver s it is with the 3 hr recircultion in n eviscerted rt used to prepre remnnts in vitro. Thus in vivo these remnnts could deliver more triglyceride per prticle to the liver thn ws delivered by the stndrd chylomicron remnnts prepred for use in the liver perfusion studies. This is consistent with the observtion (3 1) tht lrge chylomicrons re not s effective in suppressing cholesterol biosynthesis s re smller chylomicrons. Comprison of the reductse ctivities obtined when microsomes were prepred in the presence nd bsence of 5 mm NF suggested tht the rise induced in vivo of reductse ctivity in response to norml or triglyceride-rich chylomicrons ws due in prt to ctivtion of the enzyme while inhibition of reductse by cholesterolrich VLDL involved decrese in the mount of net enzyme ctivity. Cholesterol-induced chnges in the ctivtion stte of reductse hve been observed by some but not ll investigtors (32, 33). The mechnism of these modultions remins to be elucidted. Tken together these results suggest tht HMG-CoA reductse regultion involves effects on regultors tht re sensitive not only to cholesterol influx into the cell but lso to ftty cid (triglyceride) influx. One strightforwrd explntion for the effects of the different lipoproteins observed here ws suggested by the work of Goh nd Heimberg (6) in which they showed tht VLDL secretion incresed with incresing mounts of ftty cid infused. In the present study it ws shown tht remnnts of triglyceride-rich nd stndrd chylomicrons stimulted VLDL secretion s compred to the rte in lipidfree (control) perfusions or perfusions contining cholesterol-rich, triglyceride-poor VLDL remnnts. When chylomicron remnnts were dded to the perfuste, the rte of VLDL secretion observed ws comprble with tht induced by similr mounts of infused oleic cid. This suggested tht over the 2-hr period, much of the triglyceride from remnnts ws hydrolyzed nd tht the free ftty cids were vilble for synthesis nd secretion of lipoprotein triglycerides. The triglyceride-rich chylomicron remnnts did not hve s potent n effect on VLDL secretion s would hve been predicted from the mount of ftty cids theoreticlly vilble. It is possible tht either the triglycerides were not completely hydrolyzed during the period of perfusion or tht some other fctor such s cholesterol or poprotein vilbility my hve become rte-limiting. These effects of remnnt triglyceride content on VLDL production provide very plusible explntion for the response of HMG-CoA reductse becuse it hs been estblished (6, 34) tht concommitnt with incresed VLDL secretion, there is stimultion of cholesterol biosynthesis. Presumbly this occurs becuse of the need for cholesterol in the formtion of lipoprotein prticles. The sme phenomenon my occur in vivo where postprndil rise in VLDL hs been reported (28). The other components of the remnnts my well lso hve effects on heptic lipid metbolism. Thus, the prticulr phospholipid nd glyceride composition of prticle my lso ffect metbolic responses of the liver. It is less likely tht the poprotein components will ffect 426 Journl of Lipid Reserch Volume 24, 1983

10 metbolism other thn by determining the rte of prticle uptke becuse most evidence suggests tht this component is metbolized to free mino cids nd smll peptides. However, direct effect of the poproteins cnnot be excluded. It is possible tht the results reported here on the regultion of reductse my not be entirely pplicble to the overll rte of cholesterol synthesis, which under some circumstnces my be dissocited from the level of reductse ctivity (35). The present results provide simple, unifying mechnism to explin number of discrepnt observtions in the literture. They explin how severl dietry constituents cn hve prompt effects on heptic cholesterol homeostsis vi the remnnt lipoprotein pthwy nd stress the importnce of considering ll dietry lipid components when trying to study or predict effects on heptic, nd thus whole body, cholesterol homeostsis. They lso emphsize the fct tht the heptocyte hs substntil number of unique pthwys of lipid metbolism tht must be tken into ccount when studying regultion of cholesterol homeostsis in this orgn. In summry, we hve demonstrted tht remnnt lipoprotein cn hve two opposing effects on heptic HMG-CoA reductse. The first, due to the cholesterol content, is inhibitory nd the second, due to the triglyceride content, is stimultory. Thus, the net effect on the short-term regultion of HMG-CoA reductse ctivity by lipoprotein will depend upon the reltive mounts of cholesterol nd triglyceride present in the prticles.ii This work ws supported by grnts #AM nd #HL 536 from the USPHS Ntionl Institutes of Helth. Dr. Vn Zuiden ws trinee under Grnt AM 756 from the USPHS Ntionl Institutes of Helth, nd Dr. Cooper ws the recipient of Reserch Creer Development Awrd #AM 53 from the USPHS Ntionl Institutes of Helth. We wish to cknowledge helpful disscussions with Dr. Shigeru Ozs. We would lso like to thnk Adeline Shrewsbury, Puline Yu, nd Gretchen Frntz for excellent ssistnce with some of the experiments nd Ms. Jn Wick, Ms. Beverly Willims, nd Ms. Jennifer Frvelli for typing the mnuscipt.,mniiusrript received 8 Februry 1982, in revised form 16 August 1982, nd rtr re-ruurcud form 6 December 1982 REFERENCES 1. Vn Zuiden, P., A. Cooper, nd S. Erickson Effects of lipoprotein composition on regultion of 3-hydroxy-3- methylglutryl coenzyme A reductse nd cyl coenzyme A: cholesterol cyltrnsferse. Circultion 64: 127 (Abstrct). 2. Nervi, F. O., H. J. Weiss, nd J. M. Dietschy The kinetic chrcteristics of inhibition of heptic cholesterogenesis by lipoproteins of intestinl origin. J. Biol. Chem Dugn, R. E., L. S. Slkey, A. V. Breidis, nd J. W. Porter Fctors ffecting the diurnl vrition in the level of 3-hydroxy-3-methylglutryl coenzyme A reductse nd cholesterol-synthesizing ctivity in rt liver. Arch. Biochem. Biophys. 152: Edwrds, P. A., nd R. G. Could Turnover rte of heptic 3-hydroxy-3-methylglutryl coenzyme A reductse s determined by use of cycloheximide. J. Biol. Chem. 247: Cooper, A. D The regultion of 3-hydroxy-3- methylglutryl coenzyme A reductse in the isolted perfused rt liver.]. Clin. Invest. 57: Goh, E. H., nd M. Heimberg Reltionship between ctivity of heptic 3-hydroxy-3-methylglutryl coenzyme A reductse nd secretion of very-low-density lipoprotein cholesterol by the isolted perfused liver nd in the intct rt. Biochem. J. 184: Mhley, R. W., nd K. S. Holcombe Altertions of the plsm lipoproteins nd poproteins following cholesterol feeding in the rt. J. Lipid. Res. 18: Redgrve, T. G Formtion of cholesteryl ester-rich prticulte lipid during metbolism of chylomicrons. J. Clin. Invest Cooper, A. D The metbolism of chylomicron remnnts by isolted perfused rt liver. Biochim. Biophys..4rt. 488: Clrk, S. B Chylomicron composition during duodenl triglyceride nd lecithin infusion. Am.J. Physiol. 235: E St, T., R. J. Hvel, nd A. L. 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11 21. Erickson, S. K., A. D. Cooper, S. M. Mtsui, nd R. G. Could Keto cholesterol. Its effect on heptic cholesterogenesis nd its heptic metbolism in vivo nd in vitro. J. Bid. Chem. 252: Lowry,. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: Gornll, A. G., C. J. Brdwill, nd M. Dvid Determintion of serum proteins by mens of the biuret rection. J. Biol. Chem. 177: Brtlett, G. R Phosphorus ssy in column chromtogrphy. J. Biol. Chem. 234: Eggstein, M., nd F. H. Kreutz Eine neue Bestimmung der Neutrlfette im Blutserum und Gewebe. I. Mitteilung. Prinzip, Durchfuhrung und Besprechung der Methode. Klin. Wschr. 44: Ishikw, T. T., J. McGee, J. A. Morrison, nd C. J. Glueck, Quntittive nlysis of cholesterol in 5 to 2 fi1 of plsm. J. Lipid. Res. 15: Could, R. G Some spects of the control of heptic cholesterol biosynthesis. In Cholesterol Metbolism nd Lipolytic Enzymes. J. Polonovski, editor. Msson Publishing, Inc., New York Olefsky, J. M., P. Crpo, nd G. M. Reven Postprndil plsm triglyceride nd cholesterol responses to low-ft mel. Am. J. Clin. Nutr. 29: Erickson, S. K., S. M. Mtsui, M. A. Shrewsbury, A. D. Cooper, nd R. G. Could Effects of 25-hydroxycholesterol on rt heptic 3-hydroxy-3-methylglutryl coenzyme A reductse ctivity in vivo, in perfused liver nd in heptocytes. J. Biol. Chem. 253: Breslow, J. L., D. A. Lothrop, A. W. Clowes, nd S. E. Lux Lipoprotein regultion of 3-hydroxy-3-methylglutryl coenzyme A reductse ctivity in rt liver cell culture. J. Biol. Chem. 252: Nervi, F. O., nd J. M. Dietschy Ability of six different lipoprotein frctions to regulte the rte of heptic cholesterogenesis in vivo. J. Biol. Chem Areblo, R. E., J. E. Hrdgrve, nd T. J. Scllen The in vivo regultion of rt liver 3-hydroxy-3-methylglutryl coenzyme A reductse: phosphoryltion of the enzyme s n erly regultory response following cholesterol feeding. J. Biol. Chem Jenke, H-S., M. Lowel, nd J. Berndt In vivo effect of cholesterol feeding on the short term regultion of heptic 3-hydroxy-3-methylglutryl coenzyme A reductse during the diurnl cycle. J. Biol. Chem Ide, T., nd J. A. Ontko Incresed secretion of very low density lipoprotein triglyceride following inhibition of long chin ftty cid oxidtion in isolted rt liver. J. Biol. Chem Nervi, F. O., M. Crrell, nd J. M. Dietschy Dissocition of 3-hydroxy-3-methylglutryl CoA reductse ctivity from the overll rte of cholesterol biosynthesis in the liver following the intrvenous dministrtion of lipid. J. Bid. Chem. 251: Journl of Lipid Reserch Volume 24, 1983