464
ljiochimica et Biophysics 0 Elsevier/North-Holland
Acta,
488
Biomedical
(1977)
464-474
Press
BBA 57052
THE METABOLISM OF CHYLOMICRON PERFUSED RAT LIVER
ALLEN
REMNANTS
BY ISOLATED
D. COOPER
Department of Medicine, Division of Gastroenterology, Medicine, Stanford, Calif. 94305 (U.S.A.)
(Received February (Revised manuscript
Stanford
University
School
of
2nd, 1977) received May 31st, 1977)
Summary When whole chylomicrons containing radiolabeled lipids were added to the perfusate of an isolated liver, virtually no cholesterol ester or triacylglycerol was removed during the first hour, while a small amount of free cholesterol disappeared. After 3 h, 40% of the cholesterol ester was removed by the liver. In contrast, when chylomicron remnants, prepared by incubation of chylomicrons with post heparin plasma and containing the same amount of cholesterol, were added to the perfusate, 76 f 7% of the cholesterol ester was removed in the first hour. Moreover, free cholesterol and triacylcerol disappeared from the perfusate at the same rate as the cholesterol ester and during the early phase of the perfusion, the total perfusate cholesterol content declined by the same amount as the radioactive cholesterol content. These results suggest a specific removal of the intact lipoprotein particle. Of the cholesterol ester removed, 42% was hydrolyzed to free cholesterol after three hours. When whole chylomicrons, containing the same amount of cholesterol, were injected into rats in vivo, 71 f 4% of the cholesterol ester was removed by the liver in three hours and 53% of this was hydrolyzed. Finally, the activity of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis, increased during liver perfusion with plasma-free, or chylomicron-containing perfusate while addition of chylomicron remnants to the perfusate significantly diminished the effect. It is concluded that the chylomicron remnant is the lipoprotein of alimentary origin which regulates hepatic cholesterol synthesis, and that its metabolism by isolated liver appears to reflect the hepatic component of chylomicron metabolism in vivo.
Abbreviations Sf:
Svedberg
used: flotation
HMG-CoA rate
reductase:
3-hydroxy-3-methylglutaryl-coenzyme
A
reductase:
465
It has been known for almost three decades that the ingestion of cholesterol results in a decrease in hepatic cholesterol synthesis [ 11. Moreover, it is well documented that dietary cholesterol is incorporated into chylomicrons, the large lipoproteins of intestinal origin, and that the cholesterol from these particles ultimately appears in the liver [ 21. Finally, it has been demonstrated that the intravenous infusion of lymph [3] or chylomicrons [4] in vivo effectively reduces the rate of bepatic cholesterol synthesis and that the degree of suppression of cholesterol synthesis is proportional to the amount of cholesterol removed by the liver [5,6]. However, it has been shown that unmodified chylomicrons are not removed rapidly by isolated livers [7]. Moreover, the work of Bergman et al. [S] demonstrated that although the cholesterol from chylomicrons appeared in liver, the triglycerides did not. These results suggested that the chylomicron itself was not the lipoprotein particle responsible for transporting dietary cholesterol to the liver and suppressing hepatic cholesterol synthesis. Recently, several groups have produced partially metabolized chylomicrons in vitro, or in surgically modified animals [g-12]. These lipoprotein particles have been termed chylomicron “remnants” or “skeletons”. They differ from chylomicrons in several important respects : they have less triacylglycerol [9,10,12], are smaller [E&13], have lower Sf [9] coefficients and have an altered surface structure [13]. In addition, there is a change in their apoprotein composition [10,12] and they seem to contain lipoprotein lipase and perhaps heparin [ 111. Moreover, it has been shown that isolated livers can remove the triacylglycerol of the remnant particles more rapidly than that of chylomicrons when the lipoprotein is added to the perfusate [lO,ll]. However, some questions have been raised about the role of exchange of lipid as opposed to net removal in this process [ 10,141. The hypothesis that the chylomicron remnant is actually the lipoprotein particle metabolized by the liver would be strengthened if some of the metabolic consequences of lipid ingestion could be reproduced by remnant particles in the absence of potential extrahepatic effecters. We have previously demonstrated the usefulness of the isolated perfused rat liver for studying the regulation of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis and found that pure free cholesterol could rapidly affect the rate of hepatic cholesterol synthesis in this preparation [ 151. In the present report, the isolated perfused rat liver was utilized to study the hepatic metabolism of chylomicron remnants and to assess the hypothesis that they are the lipoprotein particles of dietary origin which can exert physiologic regulation of hepatic cholesterol synthesis [ 161. Methods Animals. Male Sprague-Dawley rats were housed in windowless rooms, illuminated from 7:00 am until 7:00 pm and were fed Berkeley Diet Rat and Mouse Food (Feedstuff Processing Company, San Francisco, Ca.) ad lib until killing or operation. Liver donors weighed 160-200 g and were obtained at least 10 days before use. Lymph donors weighed 300-400 g and blood donors were retired breeders. Liver perfusion. Liver perfusion was performed as previously described [ 151
466
except that the perfusate consisted of 22% washed rat red blood cells in Eagle’s Basal Medium with glutamine and Earle’s salts (Grand Island Biological Company, Grand Island, N.Y .) plus 3 g% bovine serum albumin (Cohn fraction V, Sigma, St. Louis, MO.). In lipoprotein removal experiments, the perfusions were established with a lipoprotein free perfusate and then switched to the lipoprotein containing media. In experiments designed to study the effect of lipoprotein on HMG-CoA reductase the lipoproteins were added to the perfusate after the perfusion was established. Start-up perfusate volume was 60 ml and the flow rate was 1.1 ml/min per g liver. Following each perfusion, the liver was flushed with 50 ml of homogenizing buffer (see below). Lipoprotein preparation. Mesenteric lymph fistulas were prepared and the duodenum was continuously perfused at 2 ml per hour with whole egg homogenized in 0.9 NaCl. One egg was diluted to 125 ml and 0.75 mCi [3H]cholesterol (60 Ci/mmol) and in some experiments 0.25 mCi [ “C]palmitate (10 mCi/ mmol) was added to this homogenate in 1 ml of ethanol. The lymph was collected on ice, defibrinated, layered under 0.9% NaCl and spun in a Beckman model L2-65 ultracentrifuge in a SW41 rotor at 1.1 * 10’ X g for 45 min. The supernatant lipid layer was separated by a tube slicer, resuspended in 0.9% NaCl with 0.01% EDTA, and spun again under the same conditions. This supernatant lipoprotein fraction was considered to be whole chylomicrons. Chylomicron “remnants” were prepared by a modification of the methods of Felts et al. [ 111, and Eisenberg et al. ]17]. Retired male breeder rats were injected with 100 units of heparin IV and 10 min later exsanguinated. To 10 ml of this postheparin plasma, 2 ml of a solution of fatty acid poor albumin (0.5%, pH 8.6) and chylomicrons containing 1 mg of cholesterol were added. The mixture was incubated at 37°C for 1 h and the lipoproteins isolated by centrifugation at 1.6 * lo6 X g for 120 min. The supe~atant lipoprotein was resuspended in 0.9% NaCl by drawing it back and forth through a 25 gauge needle. All of the glassware used in preparing and handling the remnants was siliconized. In the chylomicrons an average of 16% of the cholesterol was unesterified and in the remnants about 12%. HMG-CoA reductase determination. Microsomes were prepared as previously described [ 151 and resuspended in the homogenizing buffer (0.04 M potassium phosphate, 0.1 M sucrose, 0.05 M KCl, 0.025 M EDTA, pH 7.2) to result in a protein concentration of about 3 mglml. l/10 ml of this and 0.05 ml of a co-factor mixture (96 mM glucose g-phosphate, 9.6 mM NADP, 7 units/ml glucose-g-phosphate dehydrogenase, 10 mM dithiothreitol, 70 mM NaCl) were preincubated at 37°C for 5 min. The reaction was started by adding 60 pmol of [ 14C]HMG-CoA (specific activity 2.3 pCi/@mol) and after 20 min terminated by adding 0.025 ml cont. HCl. [ 3H]mevalonate, 10 pmol (specific activity 1 nCi/pmol) was added as a recovery standard and the protein was removed by centrifugation. The aqueous supernate (0.1 ml) was applied directly to a mylarbacked silica gel chromatography sheet (Eastman Kodak, Rochester, N.Y .) and run in acetone/benzene (1 : 1, v : v). On each plate a background standard consisting of the incubation mixture without added protein was also applied, The area corresponding to mevalonate was scraped and counted in Aquasol (New England Nuclear) and the results calculated as previously described [15]. The formation of mevalonate was linear with time between 0 and 40 min and with
167
protein between 0.1 and 1 mg per incubation. Chemical methods. Lipids were extracted in chloroform/methanol (2 : 1) [18]. Free and ester cholesterol and triacylglycerols were separated by thin layer chromatography on silica gel H using the solvent system hexane/ether/ acetic acid (85 : 15 : 2, v : v : v). Triacylglycerol was measured by the method of Eggstein and Kreutz [19] using reagents purchased from Sigma Chemical, phospholipids by the method of Bartlett [20] and protein by the method of Lowry et al. [21]. Cholesterol was determined by the method of Zlatkis and Zak [ 221, or, in the case of perfusate, by gas liquid chromatography on a 6-ft. column of 3% 0V17 on SO/l00 Gas Chrom Q (Applied Science, College Park, Pa., U.S.A.) at 230°C using 5-a-cholestane as an internal standard. Statistics. Statistical significance of results was determined by either paired or grouped T test using the program supplied with an HP-65 computer (Hewlett Packard, Cupertino, Calif., U.S.A.). Electron microscopy. Electron microscopy of lipoproteins was performed using 2% phosphotungstic acid for negative staining [ 231. Photographs from two separate batches of lymph were prepared and 50 particles of each lipoprotein class from each batch were measured. Results Composition of chylomicrons and their remnants. The chemical composition of chylomicrons and their remnants is given in Table I. The major differences between the two particles are size and triacylglycerol content. The degree of triacylglycerol depletion achieved by the in vitro method of production was somewhat less than that reported when “supra-diaphragmatic” rats or isolated perfused rat hearts [ 12,171 are used to produce remnants. There was, however, a close correlation between the change in triacylglycerol content and the change in mean particle size. While this was not unexpected, apparently this simple relationship does not obtain when remnants are produced from smaller lipoproteins [12]. It is important to note that although major changes were largely confined to the triacylglycerol content, minor changes in the other com-
TABLE I RELATIVE
WEIGHT COMPOSITION
OF CHYLOMICRONS
AND THEIR REMNANTS
Lipoproteins were prepared as described suspended in saline and the lipids extracted. puted from the relative masses.
Chylomicrons Remnant Remnant : Chylomicron
Ratios were com~-
Cholesterol ester j total cholesterol *
Protein + total cholesterol *
Triacylglycero1+ total cholesterol * *
(‘Q
Volume * * * (it3 t 10-8)
0.62 2 0.02 0.61 + 0.01 0.98
1.13 ? 0.27 1.38 t 0.10 1.22
42 ? 13 t 3 17 0.41
1330 !: 75 990 ?- 40 _
12.3 5.08 0.44
* Mean ? SEM 3 determinations. ** Mean ? SEM 5 determinations. * * * Measured and calculated as described under Methods.
Diameter * * *
468
WHOLE
CHY 40
CHYLOMICRON
LO
REMNANT
-
20 -
i
OI 0
15
30
60
45
MINUTES
Fig. 1. Removal of lipoprotein cholesterol ester by t,he isolated perfused rat liver. Chy!omicrons or remnants (prepared from lymph of animals fed [3H]cholesterol) containing a total of 1 mg cholesterol were added to the perfusate of an isolated liver and the [3H]cholesterol ester in samples of perfusate were determined at the intervals shown. Mean +S.E., n = 4.
ponents of the lipoprotein, ably did occur [ 10,12,17].
especially
the apoproteins,
could have and presum-
Removal of chylomicrons by isolated perfused liver. Chylomicrons containing 1 mg of total cholesterol were added to the perfusate of an isolated liver and the [3H]cholestero1 ester content of the perfusate measured in samples obtained after 0, 15, 30, 60, 120 and 180 min of perfusion. During the first hour virtually no cholesterol ester was removed from the perfusate (Fig. 1); some cholesterol ester was removed later in the perfusion with a total of 40% removed by the conclusion of the experiment. In sharp contrast are the results obtained when chylomicron remnants containing the same amount of cholesterol were added. After the first hour 76 f 7% (Fig. 1) of the labeled cholesterol ester had been removed from the perfusate with a total of 89 + 9% removed by the end of the perfusion. In a series of four experiments with both whole chylomicrons and their remnants, the radioactivity appearing in the liver accounted for 97 f 3% of the amount removed from the perfusate. In order to insure that the removal of isotope reflected a net clearance of lipoprotein and not isotope exchange as has been suggested [14], the total
TABLE
II
COMPARISON
OF REMOVAL
OF LABELED
AND TOTAL
CHOLESTEROL
The decrease in perfusate cholesterol concentration was determined by the change in perfusate radioactivity or by the change in mass of cholesterol measured by gas liquid chromatography. The initial cholesterol concentration was 94 fig/ml in 1, 58 Mg/ml in 2 and 19 pg/ml in 3. The duration of perfusion was 10 min. Experiment (lo-min perfusion)
(fig cholesterol ~Isotope
1 2 3
36.5 38.0 9.0
removed
per ml) Mass removed 28.9 34.4 16.6
perfusate cholesterol concentration was determined during the first 10 min of three perfusions. In the first two experiments the initial amount of remnant added was greater than in the preceding experiment in order to allow accurate determination of the mass of cholesterol. The close correlation between isotope and mass removal is shown by the results in Table II. In contrast during the second hour of perfusion, the bulk of the remnants had been removed and the net perfusate cholesterol concentration increased despite a constant or decreasing isotope content. This is probably due to the fact that the liver secretes lipoprotein, which has a low specific activity. Failure to distinguish the processes of removal and secretion could result in the conclusion that isotope removal had occurred by exchange [ 10,141. To ascertain whether the chylomicron remnants were removed as a unit, particles containing [ 3H] cholesterol and 14C-labelled fatty acids were added to the perfusate and the rate of disappearance of free cholesterol, cholesterol ester, and triacylglycerol were measured. It was found that the rate of removal of the three lipids was virtually identical. This is in contrast to the fate of lipid in the whole chylomicron where free [ 3H]cholesterol disappeared from the particle more rapidly than the [3H]~holesterol ester, probably by exchange. For example, the ratio of % free [3H]~holesterol remaining: o/o ~3H]cholesterol ester remaining after 30 min was 0.97 i 0.04 for remnants and 0.83 t 0.06 for chylomicrons. During the second and third hours of perfusion with chylomicron remnants some divergence from the initial isotope ratios of free cholesterol, cholesterol ester and triacylglycerol were observed. This may have been due to resecretion of labeled lipid as well as the action of lecithin : cholesterol acyltransferase and the hepatic lipases. The amounts of radioactivity present at this point, however, were too small to allow any conclusions. On the basis of the preceeding data, it is clear that si~ific~t differences exist between the way an intact isolated liver treats chylomicrons before and after they are exposed to serum containing lipoprotein lipase. It appears that the liver rapidly removes the chylomicron remnant as a discrete particle while intact chylomicrons are removed more slowly. Comparison of in vivo chylomicron removal and subsequent cholesterol ester hydrolysis with in vitro remnant removal and cholesterol ester hydrolysis. To asses how closely the metabolism of remnants in the isolated perfused liver reflects the hepatic phase of chylomicron metabolism in vivo, rats were injected with whole ~hylomicrons prepared from lymph of animals fed [3H]~hoiesterol and [14C]palmitate containing 1 mg of cholesterol (the same amount used in the remnant removal experiments) and were killed 3 h later. Their livers were flushed with saline and the radioactivity measured. The livers contained ‘71 2 4% of the injected radioactive cholesterol. This is somewhat lower than the 81% removal in 3 h observed with addition of remnants to the isolated liver perfusate. It is noteworthy that in these experiments, although the total amount of cholesterol added in vivo and in vitro was the same, the initial serum and perfusate concentrations were different because of the different volumes in which they are dist~buted. However, the rate of removal of remnants is proportions to perfusate concentration at both of these concentration levels (Cooper, AD.
470 TABLI?
111
COMPARISON
OF HEPATIC
CHYLOMICRON
AND REMNANT
METABOLISM
Chylomicrons were prepared from the lymph of rats fed [14C]palmitate and [3H]cholesterol. Remnants were prepared as described in methods. The experiments were performed with lipoproteins from the samr batch of lymph. Chylomicrons with 1 mg of cholesterol were injected intravenously or remnants with 1 mg of cholesterol were added to the perfusate of an isolated liver. 3 h later the livers were flushed with saline, homogenized and the lipids extracted and assayed as described in methods. The amount of cholesterol hydrolyzed was calculated by multiplying the amount of t3H]cholesterol ester removed by the difference in percent of [3H]cholesterol present as ester in particles before perfusion and in liver after perfusion. -.-_ . Ratio of dpm triglyceride dpm cholesterol
Remnant (in isolated liver) Chylomicron (in viva)
to
% [-‘Hlcholesterol
as ester
mg cholesterol ester hydrolyzed (calculated)
Lipoprotein
Liver
Lipoprotrin
Liver
3.5 *
3.3 *
81 *
39 +_11 **
0.34
9.0
2.9 + 0.2 **
78 *
252
0.38
*
--.._
3**
_.
* Mean of 2 experiments. * * Mean +S.E., 3 experiments.
and Yu, P., unpublished observations) and is sufficiently rapid at both levels so that the period during which the bulk of removal occurs is short relative to the overall duration of the experiment. Comparison of [ 14C]triacylglycerol to [ 3H] cholesterol ratio in the particles and the livers (Table III) revealed that when the remnants were removed little change in this ratio occurred. This adds further support for the concept that the particle was removed intact. As expected when whole chylomicrons were injected in vivo, considerable triacylglycerol removal occurred before the particles were removed by the liver. Interestingly, the triacylglycerol to cholesterol ratio in the liver, which presumably reflects the composition of the ‘real’ chylomicron remnant, was only slightly lower than that of the remnants produced by the in vitro method from the same batch of chylomicrons. Finally, the amount of removed [ 3H]cholesterol ester which was hydrolyzed during 3 h by liver in vitro and in vivo was compared. In vivo the liver converted relatively more of the removed cholesterol ester to free cholesterol than the isolated liver (Table III). However, since more cholesterol was removed by the isolated liver, the actual mass of cholesterol ester hydrolyzed was similar in vivo and in vitro. In neither case was a si~ificant amount of the hydrolyzed cholesterol ester re-esterified as judged by the fact that the 14C-labelled fatty acid to [3H]cholestero1 ratio of the cholesterol ester was the same in the perfusate and in the liver (data not shown). Overall these data suggest that the metabolism by the isolated perfused liver of chylomicron remnants which are produced in vitro is very similar to the hepatic component of chylomicron metabolism in vivo. Effect of chyiomicrons and their remnants on hepati&HMG-CoA reductase. If the chylomicron remnant is indeed the vehicle by which dietary cholesterol reaches the liver, it should be able to regulate hepatic cholesterol synthesis. We have previously demonstrated that if the liver of a rat is removed and per-
471 TABLE EFFECT
IV OF CHYLOMICRONS
AND
Effect of lipoproteins on hepatic the same liver before and after a and minimum essential medium 3.5 mg cholesterol or chylomicron _.
THEIR
HMG-CoA
_..-.
Control (?I= 5) Chylomicron fn = 3)’ Remnant (n = 5)
REMNANTS
ON HEPATIC
HMG-CoA
REDUCTASE
HMG-COA reductase. HMG-CoA reductase activity was determined in 3-h perfusion. The perfusate contained washed red blood cells, albumin to which was added nothing (control), whole chylomicrons containing remnants containing 3.5 mg cholesterol. reductase (Ctmol/mg per min)
Pre-perfusion
Post-perfusion
% increase
0.16 z 0.04 0.19 t 0.04 0.17 t 0.02 .
0.40 ? 0.03 0.49 +_0.06 0.26 + 0.04 *
182 ? 48 162? 28 50? 14 **
._.
I--
* The post perfusion value of the remnant group was significantly different from the other post perfusion groups, P < 0.05. ** The remnant group was significantly different from the other groups. P < 0.025.
fused during the nadir of its diurnal cycle, there is a rise in the activity of hepatic HMG-CoA reductase. Moreover, the rise can be prevented by the addition of cycloheximide, pure cholesterol, or other sterols to the perfusate (15, 241. If the ‘derepression’ of HMG-CoA reductase induced by liver perfusion with a plasma-free medium is similar to that which occurs when fibroblasts [25] or leukocytes [26] are grown in serum-free medium, it would be of importance to ascertain whether addition of cholesterol in the form of lipoprotein could prevent it. When whole chylomicrons containing 3.5 mg of cholesterol were added to the perfusate, the increase in HMG-CoA reductase induced by perfusion occurred and was of the same magnitude as in control (no lipoprotein added) perfusion (Table IV). In contrast, when chylomicron remnants with the same amount of cholesterol were added to the perfusate, the magnitude of the increase in reductase with perfusion was significantly less (p < 0.025) than occurred with control or chylomicron added perfusions and the final reductase activity was less than that of the other groups (p < 0.05) (Table IV). Discussion Our current understanding of chylomicron metabolism suggests that, following their formation by the intestine and excretion in the lymph, they reach the general circulation where they attach to capillary endothelial cells [ 271. Were, following activation by the apoprotein CII [ 281, the enzyme lipoprotein lipase causes hydrolysis of the chylomicron triacylglycerol. This, probably, along with the loss of apoproteins [10,12], and the addition of lipoprotein lipase and heparin [ 111, results in the formation of a lipoprotein particle called the chylomicron ‘remnant’ or ‘skeleton’. This particle is then rapidly removed from the circulation by the liver. Because the remnants have a very short half-life in the circulation and the fact that their size and density does not allow their separation from the other lipoprotein classes by conventional techniques, they have not been isolated and identified in serum from normal animals. Rather, surgically modified animals or
472
in vitro systems have been used to produce these particles. It is not yet clear which method results in a particle that is most similar to the ‘real’ chylomicron remnant. We have chosen to incubate chylomicrons with post-heparin plasma and albumin to produce remnants. The particles produced this way are larger, contain more triacylglycerol and have a different free to esterified cholesterol ratio than reported by Mjos et al. [12] for remnants produced in “suprato the diaphragmatic rats”. However, they have similar chemical composition remnants produced by Noel et al. [lo] using isolated heart perfusion. Similar changes in apoprotein composition have been reported with all three methods of remnant production [ 10,12,17]. As pointed out by Noel et al. [lo], the isolated perfused liver is potentially useful for studying the metabolism of these particles. These authors, as well as Felts et al. [ll] reported that the triacylglycerol of chylomicron remnants disappeared from the perfusate of an isolated liver more rapidly than the triacylglycerol of intact chylomicrons. We have confirmed this observation and extended it in several important respects. First we have addressed the question of whether the remnant particles are removed as a unit or whether the individual components are removed separately, at different rates. Strong support for the former concept is provided by the data demonstrating that labeled cholesterol ester, free cholesterol and triacylglycerol disappear at virtually identical rates. That the fall in radioactivity is not simply due to exchange of lipid was demonstrated by a decrease in perfusate cholesterol content proportional to the fall in isotope content. This is in contrast to the finding of Noel et al. [lo] and Lequire et al. [ 141 where lipid composition was examined after longer perfusions. The discrepancy is probably due to the fact that in the longer experiments the amount of lipid present represents not only what was removed but what had been secreted by the liver. This concept is further strengthened by the finding that the cholesterol to triacylglycerol ratio of the removed particle and that appearing in the liver are the same. The relative rapidity and specificity of the removal process suggests that a specific receptor for the remnant may exist on the surface of the hepatocyte. This conclusion is further supported by the abstract of Sherrill and Dietschy [29] and our unpublished data demonstrating that the removal process is concentration-dependent and saturable. The exact requirements for a particle’s recognition by the liver are not yet clear. Differences in size, lipid content, and apoprotein composition exist and some combination of these may be critical. The suggestion [ll] that attached lipoprotein lipase and heparin are the main factors in recognition awaits experimental confirmation. The results with perfusion of whole chylomicrons are in sharp contrast. Early in the perfusion whole chylomicrons do not seem to be actively removed at all since there is a progressive loss of radioactivity only from free cholesterol. This probably occurs by the well recognized phenomenon of exchange of free cholesterol between the lipoprotein and red cell and liver membranes [ 301. The removal of whole chylomicrons which was noted late in the perfusion may occur by trapping of these particles in sinusoids, by phagocytosis by Kupffer cells, or as the result of activation of the particles by hepatic lipase with resultant remnant formation and then clearance. The ability of isolated livers to remove chylomicrons after prolonged exposure may explain the discrepancies
473
in the literature concerning the ability of isolated perfused livers to remove chylomicrons since varying perfusion lengths and lipid concentration were not taken into account [31,32]. Moreover, this may explain the fact that patients with type I hyperlipidemia are able to clear chylomicrons from their circulation albeit slowly [33]. Following their removal from the circulation, it appears that the lipoprotein is at least partially disassembled and its various constituents metabolized individually. Previous work by Stein et al. has established that the cholesterol ester from injected chylomicrons is initially localized at the cell surface and then diffuses into the cell [ 341, presumably following hydrolysis [ 351. The concept that the remnant represents an intermediate particle in chylomicron metabolism would be further strengthened by comparing quantitatively its metabolism by isolated liver with the hepatic phase of chylomicron metabolism. These studies provide such evidence. Comparison of the process of hydrolysis of cholesterol ester in vitro and in vivo revealed that when the same amount of chylomicron cholesterol ester was injected into the intact rat, as was added to the perfusate as remnants, similar amounts were removed, and virtually the same amount was hydrolyzed by the liver. To ascertain whether the cholesterol of the chylomicron remnant exerted the same metabolic effect as ingested cholesterol we studied its effect on hepatic cholesterol synthesis. We have previously reported [ 151 that when liver is removed at the basal period of its diurnal cycle of cholesterol synthesis and perfused with a lipid-free medium an increase in the activity of HMG-CoA reductase occurs. The derepression may be similar to that seen when a variety of tissues, including fibroblasts [25], leukocytes [ 261 and intestine [ 361 are cultured in lipid-free medium. In fibroblasts, receptor mediated removal of low density lipoproteins results in a reduction of HMG-CoA reductase [25] after lysosomal hydrolysis of the cholesterol ester. The ability of chylomicrons or their remnants to produce such an effect in an isolated liver has not been previously reported. The finding in the isolated liver system that chylomicrons fail to reduce enzyme activity while their remnants are effective adds further strength to the hypothesis that the remnant is the alimentary lipoprotein recognized and metabolized by the liver and that this is the mechanism by which dietary cholesterol exerts its feedback inhibition of hepatic cholesterol synthesis. Acknowledgements Electronmicroscopy was kindly performed by Dr. E. Reaven. I am grateful to Mrs. A. Garst for technical assistance, to Drs. R.G. Gould and G.M. Gray for helpful discussions and Ms. J. Wick for help in preparing the manuscript. This work was supported by grant number AM 18774 from the National Institutes of Health. A.D.C. is the recipient of National Institutes of Health Clinical Investigator Award AM 00137. References 1
Gould, 519-528
R.G..
Taylor,
C.G..
Hqgerman.
J.S.,
Warner,
I. and
Campbell.
D.J.
(1953)
J. Biol.
Chem.
201,
474
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
BorgstrBm, B.. Lindhe. B.A. and Wiodawer, P. (1958) Proc. Sot. Exp. Biol. Med. 99. 365-368 Cooper, A.D. and Ockner. R.K. (1974) Gastroenterology 66, 586-595 Weis. H.J. and Dietschy. J.M. (1969) J. Clin. Invest. 48, 2398-2408 Nervi, F.O. and Dietschy, J.M. (1975) J. Biol. Chem. 250, 8704-8711 Ncrvi, F.O., Weis, H.J. and Dietschy, J.M. (1975) J. Biol. Chem. 250, 4145-4151 Felts, J.M. (1965) Ann. N.Y. Acad. Sci. 131, 24-33 Bergman, E.N., Navel, R.J., Wolfe, B.M. and Bphmer, T. (1971) J. Clin. Invest. 50, 1831-1839 Redgrave. T.G. (1970) J. Clin. Invest. 49, 465-471 Noel. S., Dolphin. P.J. and Rubinstcin, D. (1975) Biochem. Biophys. Res. Commun. 63, 764-772 Felts, J.. Itakura. H. and Crane. R.T. (1975) Biochem. Biophys. Res. Commun. 66.1467-1475 Mj$s. O.D., Faergeman. O., Hamilton, R.L. and Havel. R.J. (1975) J. Clin. Invest. 56. 603-615 Blanchette-Mackie. E.J. and Scow. R.O. (1973) J. Cell Biol. 58, 689-708 Goodman, Z.D. and Lequire, V.S. (1975) Biochim. Biophys. Acta 398.325-336 Cooper, A.D. (1976) J. Clin. Invest. 57, 1461-1470 Cooper, A.D. and Garst. A. (1976) Gastrocnterology 70, 873, abstract Eisenberg, S. and Rachmilowitz, D. (1975) J. Lipid Res. 16, 341-351 Folch, J., Lees, M. and SloaneStanley. G.H. (1957) J. Biol. Chem. 226. 497-509 Eggstein. M. and Krrutz. F.H. (1966) Klin. Wochenschr. 44. 262-267 Bartlett, G.R. (1959) J. Biol. Chem. 234.466-468 Lowry, D.M., Rosebrough. N.J., Fan. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Zlatkis. A.. Zak, B. and Boyle, A.J. (1953) J. Lab. Clin. Med. 41.486-492 Forte, G.M., Nichols. A.V. and Glaeser, R.M. (1969) Chem. Phys. Lipids. 2, 396-408 Erickson. S.. Cooper. A.D.. Matsui, S. and Gould. R. (1977) J. Biol. Chem., in the press Brown, M.S.. Dana. S.E. and Goldstein, J.L. (1974) J. Biol. Chem. 249. 789-796 Fogleman. A.M., Edmond, J.. Seagcr. J. and Popjak. G. (1975) J. Biol. Chem. 250, 2045-2055 Blanchette-Mackie, E.J. and Scow. R.D. (1971) J. Cell Biol. 51. l--25 Bier. D.M. and Havel, R.J. (1970) J. Lipid Res. 11. 565-570 Sherrill. B.C. and Dietschy. J.M. (1976) Circulation 54. II-91 Hagerman, J.S. and Gould, R.G. (1951) Proc. Sot. Exp. Biol. N.Y. 78, 329-332 Ontko, J.A. and Zilversmit, D.B. (1967) J. Lipid Res. 8, SO-96 Quarfordt. S.H. and Goodman. D.S. (1969) Biochim. Biophys. Acta 176, 863-872 Krauss, R&l.?.. Levy, R.I. and Fredrickson, D.S. (1974) J. Clin. Invest. 54, 1107-1124 Stein. 0.. Stein. Y.. Goodman, D.S. and Fidge, N.H. (1969) J. Cell Biol. 43. 410-431 Quarfordt. S.H. and Goodman, D.S. (1967) J. Lipid Res. 8, 263-273 Gebhard, R. and Cooper. A. (1976) Clin. Res. 25, 109
ljiochimica et Biophysics 0 Elsevier/North-Holland
Acta,
488
Biomedical
(1977)
464-474
Press
BBA 57052
THE METABOLISM OF CHYLOMICRON PERFUSED RAT LIVER
ALLEN
REMNANTS
BY ISOLATED
D. COOPER
Department of Medicine, Division of Gastroenterology, Medicine, Stanford, Calif. 94305 (U.S.A.)
(Received February (Revised manuscript
Stanford
University
School
of
2nd, 1977) received May 31st, 1977)
Summary When whole chylomicrons containing radiolabeled lipids were added to the perfusate of an isolated liver, virtually no cholesterol ester or triacylglycerol was removed during the first hour, while a small amount of free cholesterol disappeared. After 3 h, 40% of the cholesterol ester was removed by the liver. In contrast, when chylomicron remnants, prepared by incubation of chylomicrons with post heparin plasma and containing the same amount of cholesterol, were added to the perfusate, 76 f 7% of the cholesterol ester was removed in the first hour. Moreover, free cholesterol and triacylcerol disappeared from the perfusate at the same rate as the cholesterol ester and during the early phase of the perfusion, the total perfusate cholesterol content declined by the same amount as the radioactive cholesterol content. These results suggest a specific removal of the intact lipoprotein particle. Of the cholesterol ester removed, 42% was hydrolyzed to free cholesterol after three hours. When whole chylomicrons, containing the same amount of cholesterol, were injected into rats in vivo, 71 f 4% of the cholesterol ester was removed by the liver in three hours and 53% of this was hydrolyzed. Finally, the activity of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis, increased during liver perfusion with plasma-free, or chylomicron-containing perfusate while addition of chylomicron remnants to the perfusate significantly diminished the effect. It is concluded that the chylomicron remnant is the lipoprotein of alimentary origin which regulates hepatic cholesterol synthesis, and that its metabolism by isolated liver appears to reflect the hepatic component of chylomicron metabolism in vivo.
Abbreviations Sf:
Svedberg
used: flotation
HMG-CoA rate
reductase:
3-hydroxy-3-methylglutaryl-coenzyme
A
reductase:
465
It has been known for almost three decades that the ingestion of cholesterol results in a decrease in hepatic cholesterol synthesis [ 11. Moreover, it is well documented that dietary cholesterol is incorporated into chylomicrons, the large lipoproteins of intestinal origin, and that the cholesterol from these particles ultimately appears in the liver [ 21. Finally, it has been demonstrated that the intravenous infusion of lymph [3] or chylomicrons [4] in vivo effectively reduces the rate of bepatic cholesterol synthesis and that the degree of suppression of cholesterol synthesis is proportional to the amount of cholesterol removed by the liver [5,6]. However, it has been shown that unmodified chylomicrons are not removed rapidly by isolated livers [7]. Moreover, the work of Bergman et al. [S] demonstrated that although the cholesterol from chylomicrons appeared in liver, the triglycerides did not. These results suggested that the chylomicron itself was not the lipoprotein particle responsible for transporting dietary cholesterol to the liver and suppressing hepatic cholesterol synthesis. Recently, several groups have produced partially metabolized chylomicrons in vitro, or in surgically modified animals [g-12]. These lipoprotein particles have been termed chylomicron “remnants” or “skeletons”. They differ from chylomicrons in several important respects : they have less triacylglycerol [9,10,12], are smaller [E&13], have lower Sf [9] coefficients and have an altered surface structure [13]. In addition, there is a change in their apoprotein composition [10,12] and they seem to contain lipoprotein lipase and perhaps heparin [ 111. Moreover, it has been shown that isolated livers can remove the triacylglycerol of the remnant particles more rapidly than that of chylomicrons when the lipoprotein is added to the perfusate [lO,ll]. However, some questions have been raised about the role of exchange of lipid as opposed to net removal in this process [ 10,141. The hypothesis that the chylomicron remnant is actually the lipoprotein particle metabolized by the liver would be strengthened if some of the metabolic consequences of lipid ingestion could be reproduced by remnant particles in the absence of potential extrahepatic effecters. We have previously demonstrated the usefulness of the isolated perfused rat liver for studying the regulation of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis and found that pure free cholesterol could rapidly affect the rate of hepatic cholesterol synthesis in this preparation [ 151. In the present report, the isolated perfused rat liver was utilized to study the hepatic metabolism of chylomicron remnants and to assess the hypothesis that they are the lipoprotein particles of dietary origin which can exert physiologic regulation of hepatic cholesterol synthesis [ 161. Methods Animals. Male Sprague-Dawley rats were housed in windowless rooms, illuminated from 7:00 am until 7:00 pm and were fed Berkeley Diet Rat and Mouse Food (Feedstuff Processing Company, San Francisco, Ca.) ad lib until killing or operation. Liver donors weighed 160-200 g and were obtained at least 10 days before use. Lymph donors weighed 300-400 g and blood donors were retired breeders. Liver perfusion. Liver perfusion was performed as previously described [ 151
466
except that the perfusate consisted of 22% washed rat red blood cells in Eagle’s Basal Medium with glutamine and Earle’s salts (Grand Island Biological Company, Grand Island, N.Y .) plus 3 g% bovine serum albumin (Cohn fraction V, Sigma, St. Louis, MO.). In lipoprotein removal experiments, the perfusions were established with a lipoprotein free perfusate and then switched to the lipoprotein containing media. In experiments designed to study the effect of lipoprotein on HMG-CoA reductase the lipoproteins were added to the perfusate after the perfusion was established. Start-up perfusate volume was 60 ml and the flow rate was 1.1 ml/min per g liver. Following each perfusion, the liver was flushed with 50 ml of homogenizing buffer (see below). Lipoprotein preparation. Mesenteric lymph fistulas were prepared and the duodenum was continuously perfused at 2 ml per hour with whole egg homogenized in 0.9 NaCl. One egg was diluted to 125 ml and 0.75 mCi [3H]cholesterol (60 Ci/mmol) and in some experiments 0.25 mCi [ “C]palmitate (10 mCi/ mmol) was added to this homogenate in 1 ml of ethanol. The lymph was collected on ice, defibrinated, layered under 0.9% NaCl and spun in a Beckman model L2-65 ultracentrifuge in a SW41 rotor at 1.1 * 10’ X g for 45 min. The supernatant lipid layer was separated by a tube slicer, resuspended in 0.9% NaCl with 0.01% EDTA, and spun again under the same conditions. This supernatant lipoprotein fraction was considered to be whole chylomicrons. Chylomicron “remnants” were prepared by a modification of the methods of Felts et al. [ 111, and Eisenberg et al. ]17]. Retired male breeder rats were injected with 100 units of heparin IV and 10 min later exsanguinated. To 10 ml of this postheparin plasma, 2 ml of a solution of fatty acid poor albumin (0.5%, pH 8.6) and chylomicrons containing 1 mg of cholesterol were added. The mixture was incubated at 37°C for 1 h and the lipoproteins isolated by centrifugation at 1.6 * lo6 X g for 120 min. The supe~atant lipoprotein was resuspended in 0.9% NaCl by drawing it back and forth through a 25 gauge needle. All of the glassware used in preparing and handling the remnants was siliconized. In the chylomicrons an average of 16% of the cholesterol was unesterified and in the remnants about 12%. HMG-CoA reductase determination. Microsomes were prepared as previously described [ 151 and resuspended in the homogenizing buffer (0.04 M potassium phosphate, 0.1 M sucrose, 0.05 M KCl, 0.025 M EDTA, pH 7.2) to result in a protein concentration of about 3 mglml. l/10 ml of this and 0.05 ml of a co-factor mixture (96 mM glucose g-phosphate, 9.6 mM NADP, 7 units/ml glucose-g-phosphate dehydrogenase, 10 mM dithiothreitol, 70 mM NaCl) were preincubated at 37°C for 5 min. The reaction was started by adding 60 pmol of [ 14C]HMG-CoA (specific activity 2.3 pCi/@mol) and after 20 min terminated by adding 0.025 ml cont. HCl. [ 3H]mevalonate, 10 pmol (specific activity 1 nCi/pmol) was added as a recovery standard and the protein was removed by centrifugation. The aqueous supernate (0.1 ml) was applied directly to a mylarbacked silica gel chromatography sheet (Eastman Kodak, Rochester, N.Y .) and run in acetone/benzene (1 : 1, v : v). On each plate a background standard consisting of the incubation mixture without added protein was also applied, The area corresponding to mevalonate was scraped and counted in Aquasol (New England Nuclear) and the results calculated as previously described [15]. The formation of mevalonate was linear with time between 0 and 40 min and with
167
protein between 0.1 and 1 mg per incubation. Chemical methods. Lipids were extracted in chloroform/methanol (2 : 1) [18]. Free and ester cholesterol and triacylglycerols were separated by thin layer chromatography on silica gel H using the solvent system hexane/ether/ acetic acid (85 : 15 : 2, v : v : v). Triacylglycerol was measured by the method of Eggstein and Kreutz [19] using reagents purchased from Sigma Chemical, phospholipids by the method of Bartlett [20] and protein by the method of Lowry et al. [21]. Cholesterol was determined by the method of Zlatkis and Zak [ 221, or, in the case of perfusate, by gas liquid chromatography on a 6-ft. column of 3% 0V17 on SO/l00 Gas Chrom Q (Applied Science, College Park, Pa., U.S.A.) at 230°C using 5-a-cholestane as an internal standard. Statistics. Statistical significance of results was determined by either paired or grouped T test using the program supplied with an HP-65 computer (Hewlett Packard, Cupertino, Calif., U.S.A.). Electron microscopy. Electron microscopy of lipoproteins was performed using 2% phosphotungstic acid for negative staining [ 231. Photographs from two separate batches of lymph were prepared and 50 particles of each lipoprotein class from each batch were measured. Results Composition of chylomicrons and their remnants. The chemical composition of chylomicrons and their remnants is given in Table I. The major differences between the two particles are size and triacylglycerol content. The degree of triacylglycerol depletion achieved by the in vitro method of production was somewhat less than that reported when “supra-diaphragmatic” rats or isolated perfused rat hearts [ 12,171 are used to produce remnants. There was, however, a close correlation between the change in triacylglycerol content and the change in mean particle size. While this was not unexpected, apparently this simple relationship does not obtain when remnants are produced from smaller lipoproteins [12]. It is important to note that although major changes were largely confined to the triacylglycerol content, minor changes in the other com-
TABLE I RELATIVE
WEIGHT COMPOSITION
OF CHYLOMICRONS
AND THEIR REMNANTS
Lipoproteins were prepared as described suspended in saline and the lipids extracted. puted from the relative masses.
Chylomicrons Remnant Remnant : Chylomicron
Ratios were com~-
Cholesterol ester j total cholesterol *
Protein + total cholesterol *
Triacylglycero1+ total cholesterol * *
(‘Q
Volume * * * (it3 t 10-8)
0.62 2 0.02 0.61 + 0.01 0.98
1.13 ? 0.27 1.38 t 0.10 1.22
42 ? 13 t 3 17 0.41
1330 !: 75 990 ?- 40 _
12.3 5.08 0.44
* Mean ? SEM 3 determinations. ** Mean ? SEM 5 determinations. * * * Measured and calculated as described under Methods.
Diameter * * *
468
WHOLE
CHY 40
CHYLOMICRON
LO
REMNANT
-
20 -
i
OI 0
15
30
60
45
MINUTES
Fig. 1. Removal of lipoprotein cholesterol ester by t,he isolated perfused rat liver. Chy!omicrons or remnants (prepared from lymph of animals fed [3H]cholesterol) containing a total of 1 mg cholesterol were added to the perfusate of an isolated liver and the [3H]cholesterol ester in samples of perfusate were determined at the intervals shown. Mean +S.E., n = 4.
ponents of the lipoprotein, ably did occur [ 10,12,17].
especially
the apoproteins,
could have and presum-
Removal of chylomicrons by isolated perfused liver. Chylomicrons containing 1 mg of total cholesterol were added to the perfusate of an isolated liver and the [3H]cholestero1 ester content of the perfusate measured in samples obtained after 0, 15, 30, 60, 120 and 180 min of perfusion. During the first hour virtually no cholesterol ester was removed from the perfusate (Fig. 1); some cholesterol ester was removed later in the perfusion with a total of 40% removed by the conclusion of the experiment. In sharp contrast are the results obtained when chylomicron remnants containing the same amount of cholesterol were added. After the first hour 76 f 7% (Fig. 1) of the labeled cholesterol ester had been removed from the perfusate with a total of 89 + 9% removed by the end of the perfusion. In a series of four experiments with both whole chylomicrons and their remnants, the radioactivity appearing in the liver accounted for 97 f 3% of the amount removed from the perfusate. In order to insure that the removal of isotope reflected a net clearance of lipoprotein and not isotope exchange as has been suggested [14], the total
TABLE
II
COMPARISON
OF REMOVAL
OF LABELED
AND TOTAL
CHOLESTEROL
The decrease in perfusate cholesterol concentration was determined by the change in perfusate radioactivity or by the change in mass of cholesterol measured by gas liquid chromatography. The initial cholesterol concentration was 94 fig/ml in 1, 58 Mg/ml in 2 and 19 pg/ml in 3. The duration of perfusion was 10 min. Experiment (lo-min perfusion)
(fig cholesterol ~Isotope
1 2 3
36.5 38.0 9.0
removed
per ml) Mass removed 28.9 34.4 16.6
perfusate cholesterol concentration was determined during the first 10 min of three perfusions. In the first two experiments the initial amount of remnant added was greater than in the preceding experiment in order to allow accurate determination of the mass of cholesterol. The close correlation between isotope and mass removal is shown by the results in Table II. In contrast during the second hour of perfusion, the bulk of the remnants had been removed and the net perfusate cholesterol concentration increased despite a constant or decreasing isotope content. This is probably due to the fact that the liver secretes lipoprotein, which has a low specific activity. Failure to distinguish the processes of removal and secretion could result in the conclusion that isotope removal had occurred by exchange [ 10,141. To ascertain whether the chylomicron remnants were removed as a unit, particles containing [ 3H] cholesterol and 14C-labelled fatty acids were added to the perfusate and the rate of disappearance of free cholesterol, cholesterol ester, and triacylglycerol were measured. It was found that the rate of removal of the three lipids was virtually identical. This is in contrast to the fate of lipid in the whole chylomicron where free [ 3H]cholesterol disappeared from the particle more rapidly than the [3H]~holesterol ester, probably by exchange. For example, the ratio of % free [3H]~holesterol remaining: o/o ~3H]cholesterol ester remaining after 30 min was 0.97 i 0.04 for remnants and 0.83 t 0.06 for chylomicrons. During the second and third hours of perfusion with chylomicron remnants some divergence from the initial isotope ratios of free cholesterol, cholesterol ester and triacylglycerol were observed. This may have been due to resecretion of labeled lipid as well as the action of lecithin : cholesterol acyltransferase and the hepatic lipases. The amounts of radioactivity present at this point, however, were too small to allow any conclusions. On the basis of the preceeding data, it is clear that si~ific~t differences exist between the way an intact isolated liver treats chylomicrons before and after they are exposed to serum containing lipoprotein lipase. It appears that the liver rapidly removes the chylomicron remnant as a discrete particle while intact chylomicrons are removed more slowly. Comparison of in vivo chylomicron removal and subsequent cholesterol ester hydrolysis with in vitro remnant removal and cholesterol ester hydrolysis. To asses how closely the metabolism of remnants in the isolated perfused liver reflects the hepatic phase of chylomicron metabolism in vivo, rats were injected with whole ~hylomicrons prepared from lymph of animals fed [3H]~hoiesterol and [14C]palmitate containing 1 mg of cholesterol (the same amount used in the remnant removal experiments) and were killed 3 h later. Their livers were flushed with saline and the radioactivity measured. The livers contained ‘71 2 4% of the injected radioactive cholesterol. This is somewhat lower than the 81% removal in 3 h observed with addition of remnants to the isolated liver perfusate. It is noteworthy that in these experiments, although the total amount of cholesterol added in vivo and in vitro was the same, the initial serum and perfusate concentrations were different because of the different volumes in which they are dist~buted. However, the rate of removal of remnants is proportions to perfusate concentration at both of these concentration levels (Cooper, AD.
470 TABLI?
111
COMPARISON
OF HEPATIC
CHYLOMICRON
AND REMNANT
METABOLISM
Chylomicrons were prepared from the lymph of rats fed [14C]palmitate and [3H]cholesterol. Remnants were prepared as described in methods. The experiments were performed with lipoproteins from the samr batch of lymph. Chylomicrons with 1 mg of cholesterol were injected intravenously or remnants with 1 mg of cholesterol were added to the perfusate of an isolated liver. 3 h later the livers were flushed with saline, homogenized and the lipids extracted and assayed as described in methods. The amount of cholesterol hydrolyzed was calculated by multiplying the amount of t3H]cholesterol ester removed by the difference in percent of [3H]cholesterol present as ester in particles before perfusion and in liver after perfusion. -.-_ . Ratio of dpm triglyceride dpm cholesterol
Remnant (in isolated liver) Chylomicron (in viva)
to
% [-‘Hlcholesterol
as ester
mg cholesterol ester hydrolyzed (calculated)
Lipoprotein
Liver
Lipoprotrin
Liver
3.5 *
3.3 *
81 *
39 +_11 **
0.34
9.0
2.9 + 0.2 **
78 *
252
0.38
*
--.._
3**
_.
* Mean of 2 experiments. * * Mean +S.E., 3 experiments.
and Yu, P., unpublished observations) and is sufficiently rapid at both levels so that the period during which the bulk of removal occurs is short relative to the overall duration of the experiment. Comparison of [ 14C]triacylglycerol to [ 3H] cholesterol ratio in the particles and the livers (Table III) revealed that when the remnants were removed little change in this ratio occurred. This adds further support for the concept that the particle was removed intact. As expected when whole chylomicrons were injected in vivo, considerable triacylglycerol removal occurred before the particles were removed by the liver. Interestingly, the triacylglycerol to cholesterol ratio in the liver, which presumably reflects the composition of the ‘real’ chylomicron remnant, was only slightly lower than that of the remnants produced by the in vitro method from the same batch of chylomicrons. Finally, the amount of removed [ 3H]cholesterol ester which was hydrolyzed during 3 h by liver in vitro and in vivo was compared. In vivo the liver converted relatively more of the removed cholesterol ester to free cholesterol than the isolated liver (Table III). However, since more cholesterol was removed by the isolated liver, the actual mass of cholesterol ester hydrolyzed was similar in vivo and in vitro. In neither case was a si~ificant amount of the hydrolyzed cholesterol ester re-esterified as judged by the fact that the 14C-labelled fatty acid to [3H]cholestero1 ratio of the cholesterol ester was the same in the perfusate and in the liver (data not shown). Overall these data suggest that the metabolism by the isolated perfused liver of chylomicron remnants which are produced in vitro is very similar to the hepatic component of chylomicron metabolism in vivo. Effect of chyiomicrons and their remnants on hepati&HMG-CoA reductase. If the chylomicron remnant is indeed the vehicle by which dietary cholesterol reaches the liver, it should be able to regulate hepatic cholesterol synthesis. We have previously demonstrated that if the liver of a rat is removed and per-
471 TABLE EFFECT
IV OF CHYLOMICRONS
AND
Effect of lipoproteins on hepatic the same liver before and after a and minimum essential medium 3.5 mg cholesterol or chylomicron _.
THEIR
HMG-CoA
_..-.
Control (?I= 5) Chylomicron fn = 3)’ Remnant (n = 5)
REMNANTS
ON HEPATIC
HMG-CoA
REDUCTASE
HMG-COA reductase. HMG-CoA reductase activity was determined in 3-h perfusion. The perfusate contained washed red blood cells, albumin to which was added nothing (control), whole chylomicrons containing remnants containing 3.5 mg cholesterol. reductase (Ctmol/mg per min)
Pre-perfusion
Post-perfusion
% increase
0.16 z 0.04 0.19 t 0.04 0.17 t 0.02 .
0.40 ? 0.03 0.49 +_0.06 0.26 + 0.04 *
182 ? 48 162? 28 50? 14 **
._.
I--
* The post perfusion value of the remnant group was significantly different from the other post perfusion groups, P < 0.05. ** The remnant group was significantly different from the other groups. P < 0.025.
fused during the nadir of its diurnal cycle, there is a rise in the activity of hepatic HMG-CoA reductase. Moreover, the rise can be prevented by the addition of cycloheximide, pure cholesterol, or other sterols to the perfusate (15, 241. If the ‘derepression’ of HMG-CoA reductase induced by liver perfusion with a plasma-free medium is similar to that which occurs when fibroblasts [25] or leukocytes [26] are grown in serum-free medium, it would be of importance to ascertain whether addition of cholesterol in the form of lipoprotein could prevent it. When whole chylomicrons containing 3.5 mg of cholesterol were added to the perfusate, the increase in HMG-CoA reductase induced by perfusion occurred and was of the same magnitude as in control (no lipoprotein added) perfusion (Table IV). In contrast, when chylomicron remnants with the same amount of cholesterol were added to the perfusate, the magnitude of the increase in reductase with perfusion was significantly less (p < 0.025) than occurred with control or chylomicron added perfusions and the final reductase activity was less than that of the other groups (p < 0.05) (Table IV). Discussion Our current understanding of chylomicron metabolism suggests that, following their formation by the intestine and excretion in the lymph, they reach the general circulation where they attach to capillary endothelial cells [ 271. Were, following activation by the apoprotein CII [ 281, the enzyme lipoprotein lipase causes hydrolysis of the chylomicron triacylglycerol. This, probably, along with the loss of apoproteins [10,12], and the addition of lipoprotein lipase and heparin [ 111, results in the formation of a lipoprotein particle called the chylomicron ‘remnant’ or ‘skeleton’. This particle is then rapidly removed from the circulation by the liver. Because the remnants have a very short half-life in the circulation and the fact that their size and density does not allow their separation from the other lipoprotein classes by conventional techniques, they have not been isolated and identified in serum from normal animals. Rather, surgically modified animals or
472
in vitro systems have been used to produce these particles. It is not yet clear which method results in a particle that is most similar to the ‘real’ chylomicron remnant. We have chosen to incubate chylomicrons with post-heparin plasma and albumin to produce remnants. The particles produced this way are larger, contain more triacylglycerol and have a different free to esterified cholesterol ratio than reported by Mjos et al. [12] for remnants produced in “suprato the diaphragmatic rats”. However, they have similar chemical composition remnants produced by Noel et al. [lo] using isolated heart perfusion. Similar changes in apoprotein composition have been reported with all three methods of remnant production [ 10,12,17]. As pointed out by Noel et al. [lo], the isolated perfused liver is potentially useful for studying the metabolism of these particles. These authors, as well as Felts et al. [ll] reported that the triacylglycerol of chylomicron remnants disappeared from the perfusate of an isolated liver more rapidly than the triacylglycerol of intact chylomicrons. We have confirmed this observation and extended it in several important respects. First we have addressed the question of whether the remnant particles are removed as a unit or whether the individual components are removed separately, at different rates. Strong support for the former concept is provided by the data demonstrating that labeled cholesterol ester, free cholesterol and triacylglycerol disappear at virtually identical rates. That the fall in radioactivity is not simply due to exchange of lipid was demonstrated by a decrease in perfusate cholesterol content proportional to the fall in isotope content. This is in contrast to the finding of Noel et al. [lo] and Lequire et al. [ 141 where lipid composition was examined after longer perfusions. The discrepancy is probably due to the fact that in the longer experiments the amount of lipid present represents not only what was removed but what had been secreted by the liver. This concept is further strengthened by the finding that the cholesterol to triacylglycerol ratio of the removed particle and that appearing in the liver are the same. The relative rapidity and specificity of the removal process suggests that a specific receptor for the remnant may exist on the surface of the hepatocyte. This conclusion is further supported by the abstract of Sherrill and Dietschy [29] and our unpublished data demonstrating that the removal process is concentration-dependent and saturable. The exact requirements for a particle’s recognition by the liver are not yet clear. Differences in size, lipid content, and apoprotein composition exist and some combination of these may be critical. The suggestion [ll] that attached lipoprotein lipase and heparin are the main factors in recognition awaits experimental confirmation. The results with perfusion of whole chylomicrons are in sharp contrast. Early in the perfusion whole chylomicrons do not seem to be actively removed at all since there is a progressive loss of radioactivity only from free cholesterol. This probably occurs by the well recognized phenomenon of exchange of free cholesterol between the lipoprotein and red cell and liver membranes [ 301. The removal of whole chylomicrons which was noted late in the perfusion may occur by trapping of these particles in sinusoids, by phagocytosis by Kupffer cells, or as the result of activation of the particles by hepatic lipase with resultant remnant formation and then clearance. The ability of isolated livers to remove chylomicrons after prolonged exposure may explain the discrepancies
473
in the literature concerning the ability of isolated perfused livers to remove chylomicrons since varying perfusion lengths and lipid concentration were not taken into account [31,32]. Moreover, this may explain the fact that patients with type I hyperlipidemia are able to clear chylomicrons from their circulation albeit slowly [33]. Following their removal from the circulation, it appears that the lipoprotein is at least partially disassembled and its various constituents metabolized individually. Previous work by Stein et al. has established that the cholesterol ester from injected chylomicrons is initially localized at the cell surface and then diffuses into the cell [ 341, presumably following hydrolysis [ 351. The concept that the remnant represents an intermediate particle in chylomicron metabolism would be further strengthened by comparing quantitatively its metabolism by isolated liver with the hepatic phase of chylomicron metabolism. These studies provide such evidence. Comparison of the process of hydrolysis of cholesterol ester in vitro and in vivo revealed that when the same amount of chylomicron cholesterol ester was injected into the intact rat, as was added to the perfusate as remnants, similar amounts were removed, and virtually the same amount was hydrolyzed by the liver. To ascertain whether the cholesterol of the chylomicron remnant exerted the same metabolic effect as ingested cholesterol we studied its effect on hepatic cholesterol synthesis. We have previously reported [ 151 that when liver is removed at the basal period of its diurnal cycle of cholesterol synthesis and perfused with a lipid-free medium an increase in the activity of HMG-CoA reductase occurs. The derepression may be similar to that seen when a variety of tissues, including fibroblasts [25], leukocytes [ 261 and intestine [ 361 are cultured in lipid-free medium. In fibroblasts, receptor mediated removal of low density lipoproteins results in a reduction of HMG-CoA reductase [25] after lysosomal hydrolysis of the cholesterol ester. The ability of chylomicrons or their remnants to produce such an effect in an isolated liver has not been previously reported. The finding in the isolated liver system that chylomicrons fail to reduce enzyme activity while their remnants are effective adds further strength to the hypothesis that the remnant is the alimentary lipoprotein recognized and metabolized by the liver and that this is the mechanism by which dietary cholesterol exerts its feedback inhibition of hepatic cholesterol synthesis. Acknowledgements Electronmicroscopy was kindly performed by Dr. E. Reaven. I am grateful to Mrs. A. Garst for technical assistance, to Drs. R.G. Gould and G.M. Gray for helpful discussions and Ms. J. Wick for help in preparing the manuscript. This work was supported by grant number AM 18774 from the National Institutes of Health. A.D.C. is the recipient of National Institutes of Health Clinical Investigator Award AM 00137. References 1
Gould, 519-528
R.G..
Taylor,
C.G..
Hqgerman.
J.S.,
Warner,
I. and
Campbell.
D.J.
(1953)
J. Biol.
Chem.
201,
474
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
BorgstrBm, B.. Lindhe. B.A. and Wiodawer, P. (1958) Proc. Sot. Exp. Biol. Med. 99. 365-368 Cooper, A.D. and Ockner. R.K. (1974) Gastroenterology 66, 586-595 Weis. H.J. and Dietschy. J.M. (1969) J. Clin. Invest. 48, 2398-2408 Nervi, F.O. and Dietschy, J.M. (1975) J. Biol. Chem. 250, 8704-8711 Ncrvi, F.O., Weis, H.J. and Dietschy, J.M. (1975) J. Biol. Chem. 250, 4145-4151 Felts, J.M. (1965) Ann. N.Y. Acad. Sci. 131, 24-33 Bergman, E.N., Navel, R.J., Wolfe, B.M. and Bphmer, T. (1971) J. Clin. Invest. 50, 1831-1839 Redgrave. T.G. (1970) J. Clin. Invest. 49, 465-471 Noel. S., Dolphin. P.J. and Rubinstcin, D. (1975) Biochem. Biophys. Res. Commun. 63, 764-772 Felts, J.. Itakura. H. and Crane. R.T. (1975) Biochem. Biophys. Res. Commun. 66.1467-1475 Mj$s. O.D., Faergeman. O., Hamilton, R.L. and Havel. R.J. (1975) J. Clin. Invest. 56. 603-615 Blanchette-Mackie. E.J. and Scow. R.O. (1973) J. Cell Biol. 58, 689-708 Goodman, Z.D. and Lequire, V.S. (1975) Biochim. Biophys. Acta 398.325-336 Cooper, A.D. (1976) J. Clin. Invest. 57, 1461-1470 Cooper, A.D. and Garst. A. (1976) Gastrocnterology 70, 873, abstract Eisenberg, S. and Rachmilowitz, D. (1975) J. Lipid Res. 16, 341-351 Folch, J., Lees, M. and SloaneStanley. G.H. (1957) J. Biol. Chem. 226. 497-509 Eggstein. M. and Krrutz. F.H. (1966) Klin. Wochenschr. 44. 262-267 Bartlett, G.R. (1959) J. Biol. Chem. 234.466-468 Lowry, D.M., Rosebrough. N.J., Fan. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Zlatkis. A.. Zak, B. and Boyle, A.J. (1953) J. Lab. Clin. Med. 41.486-492 Forte, G.M., Nichols. A.V. and Glaeser, R.M. (1969) Chem. Phys. Lipids. 2, 396-408 Erickson. S.. Cooper. A.D.. Matsui, S. and Gould. R. (1977) J. Biol. Chem., in the press Brown, M.S.. Dana. S.E. and Goldstein, J.L. (1974) J. Biol. Chem. 249. 789-796 Fogleman. A.M., Edmond, J.. Seagcr. J. and Popjak. G. (1975) J. Biol. Chem. 250, 2045-2055 Blanchette-Mackie, E.J. and Scow. R.D. (1971) J. Cell Biol. 51. l--25 Bier. D.M. and Havel, R.J. (1970) J. Lipid Res. 11. 565-570 Sherrill. B.C. and Dietschy. J.M. (1976) Circulation 54. II-91 Hagerman, J.S. and Gould, R.G. (1951) Proc. Sot. Exp. Biol. N.Y. 78, 329-332 Ontko, J.A. and Zilversmit, D.B. (1967) J. Lipid Res. 8, SO-96 Quarfordt. S.H. and Goodman. D.S. (1969) Biochim. Biophys. Acta 176, 863-872 Krauss, R&l.?.. Levy, R.I. and Fredrickson, D.S. (1974) J. Clin. Invest. 54, 1107-1124 Stein. 0.. Stein. Y.. Goodman, D.S. and Fidge, N.H. (1969) J. Cell Biol. 43. 410-431 Quarfordt. S.H. and Goodman, D.S. (1967) J. Lipid Res. 8, 263-273 Gebhard, R. and Cooper. A. (1976) Clin. Res. 25, 109
