1 Lipoprotein Research Group and Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia B3H 4H7; and 2 Health Sciences Laboratory Animal Services and 3 Lipid and Lipoprotein Research Group and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
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ABSTRACT |
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In -naphthylisothiocyanate-treated mice,
plasma phospholipid (PL) levels were elevated 10- and 13-fold at 48 and
168 h, respectively, whereas free cholesterol (FC) levels increased
between 48 h (17-fold) and 168 h (39-fold). Nearly all of these lipids
were localized to lipoprotein X-like particles in the low-density
lipoprotein density range. The PL fatty acyl composition was indicative
of biliary origin. Liver cholesterol and PL content were near normal at
all time points. Hepatic hydroxymethylglutaryl CoA reductase activity
was increased sixfold at 48 h, and cholesterol 7
-hydroxylase activity was decreased by ~70% between 24 and 72 h. These findings suggest a metabolic basis for the appearance of abnormal plasma lipoproteins during cholestasis. Initially, PL and bile acids appear in
plasma where they serve to promote the efflux of cholesterol from
hepatic cell membranes. Hepatic cholesterol synthesis is then likely
stimulated in the response to the depletion of hepatic cell membranes
of cholesterol. We speculate that the enhanced synthesis of cholesterol
and impaired conversion to bile acids, particularly during the early
phase of drug response, contribute to the accumulation of FC in the plasma.
cholesterol 7-hydroxylase; hydroxymethylglutaryl coenzyme A
reductase; lipoproteins; bile acids;
-naphthylisothiocyanate
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INTRODUCTION |
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CHOLESTASIS PRODUCES aberrations in lipid and lipoprotein metabolism in humans and animal models (18). In plasma, the most prominent features are increased free cholesterol (FC) and phospholipid (PL) levels as well as the appearance of abnormal pathological lipoproteins known as lipoprotein X (Lp-X). These protein-depleted bilaminate vesicles have an aqueous core and float at a density comparable to that of low-density lipoproteins (LDL). They do not contain apolipoprotein (apo) B or appreciable levels of hydrophobic core lipids [cholesteryl esters (CE) and triacylglycerols (TG)]. In cholestasis, the origin of the lipids constituting Lp-X is thought to be biliary and to result from a refluxing of bile into the plasma compartment following some form of biliary obstruction.
The compound -naphthylisothiocyanate (ANIT) has been used to induce
experimental cholestasis in a variety of laboratory animals (4). In
rats, a single dose of ANIT (100 mg/kg) administered by gavage results
in a transient and fully reversible intrahepatic cholestasis that is
accompanied by remarkable alterations to the plasma constituents (7).
Specifically, ANIT treatment results in markedly elevated levels of
plasma FC and PL, the appearance of Lp-X-like vesicles, elevations in
plasma apo A-I and apo E, and a pronounced shift of apo E-containing
particles into the LDL density range. These effects peak 48 h after
ANIT administration and return to normal after 168 h. Thus ANIT-induced
experimental cholestasis appears to be a useful nonsurgical approach to
study the metabolism of abnormal cholestatic lipoproteins. Here we
report the effects of ANIT on the metabolism of cholesterol in treated mice. The data demonstrate that ANIT treatment results in significant modification of the activities of enzymes involved in the synthesis and
degradation of cholesterol in the liver. The alteration of lipid
metabolism in the plasma of ANIT-treated mice also appears to evolve
differently and more severely compared with that in ANIT-treated rats.
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MATERIALS AND METHODS |
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Animals. Female C57BL/6 mice (10 wk old) were purchased from Charles River Laboratories. Mice were fed standard laboratory food, given water ad libitum, and housed under a 12:12-h light-dark photoperiod. Food was withdrawn 8-10 h before ANIT treatment. Mice were lightly anesthetized by intraperitoneal administration of 2% ketamine/0.4% xylazine at a dosage of 1-1.5 ml/kg and then given 100 mg/kg ANIT (Sigma Chemical) in a 10 mg/ml corn oil (Mazola) bolus by gavage. The control group received a volume of corn oil that was appropriate for the weight of each mouse. At the indicated experimental time points, fasted mice were deeply anesthetized by intraperitoneal injection of 2% ketamine/0.4% xylazine at a dosage of 2-3 ml/kg, and blood was collected from the descending aorta into tubes containing Na2EDTA, thimerosal, aprotinin, and NaN3 to final respective concentrations of 0.1, 0.005, 0.001, and 0.02%.
Mouse livers and gallbladders were excised at necropsy. Liver pieces were washed in saline, weighed, and then quick frozen in liquid nitrogen or preserved in 10% neutral buffered Formalin until analysis. Gallbladders were quickly spun to the bottom of a 1.5-ml microcentrifuge tube, and the bile was collected with a glass capillary and then quick frozen in liquid nitrogen until analysis.Lipid and lipoprotein analyses. Liver lipids were extracted from homogenized liver sections by the method of Folch et al. (16). Plasma, density gradient fractions, and liver lipid mass and compositional analysis were performed by gas chromatography (28). Bile acid content of plasma and bile as well as bile cholesterol and PL were measured using commercial diagnostic kits (Boehringer Mannheim, Sigma Chemical, and Wako Pure Chemical Industries). Pooled plasma from control and ANIT-treated mice was subjected to density gradient ultracentrifugation and fractionated manually into 400-µl fractions as previously described (7). The protein concentration of density gradient fractions was measured by the bicinchoninic assay (Pierce) modified by the addition of 0.25% deoxycholate (Sigma Chemical). The apolipoprotein composition of isolated density gradient fractions was analyzed by SDS-PAGE on 5-19% gradient gels as previously described (7).
Enzyme activity assays.
Microsomes were prepared from livers of control and ANIT-treated mice
as described previously (5). Cyp7 activity in isolated microsomes was
measured by isotope incorporation using -cyclodextrin encapsulated
[14C]cholesterol as a
substrate (1, 29) and by an HPLC-based method that uses endogenous
microsomal cholesterol as a substrate (5, 6). Hydroxymethylglutaryl CoA
reductase (HMGR) activity in the isolated microsomes was measured by
following the formation of
[14C]mevalonate from
[14C]hydroxymethylglutaryl
CoA (23). The radiolabeled products generated by the enzyme assays were
separated by thin-layer chromatography and then quantitated using a
Fuji BAS1000 PhosphorImager. Exogenous lecithin:cholesterol
acyltransferase (LCAT) activity was measured by the method of Sparks et
al. (43) using recombinant high-density lipoprotein (HDL) particles
(80:10:0:1;
1,2-di[cis-9-octodecanoyl]-sn-glycero-3-phosphocholine:FC:CE:human apo A-I). Aspartate aminotransferase and alanine aminotransferase activities in mouse plasma were measured using commercial diagnostic kits (Boehringer Mannheim).
RNA analyses. Total RNA from mouse livers was purified according to standard procedures (8). Complementary DNA was synthesized from total liver RNA (1 µg/reaction) using Superscript reverse transcriptase (Life Technologies) and random hexamers as primers. The cDNA for mouse cyp7 and mouse glyceraldehyde-3-phosphate dehydrogenase were amplified in vitro using Taq polymerase and primer pairs specific for each of the mRNA species. Amplification products were visualized by ethidium bromide staining of agarose gels after electrophoresis.
Histology. Liver samples were collected at necropsy and preserved in 10% neutral buffered Formalin. Preserved tissue samples were processed routinely, embedded in paraffin, sectioned at 5 µm, and then stained with hematoxylin and eosin using standard techniques.
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RESULTS |
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Bile acids, bilirubin, and liver-specific enzymes in the plasma of
ANIT-treated mice.
To verify the induction of cholestasis in ANIT-treated mice, plasma
collected 48 h after treatment was analyzed for markers of hepatic
damage. Compared with controls, bilirubin levels in the plasma of
ANIT-treated mice were increased by 11.2-fold (Table 1). The activities of alanine
aminotransferase and aspartate aminotransferase in plasma of
ANIT-treated mice were also increased by 7.4- and 4.3-fold,
respectively. Finally, the concentration of bile acids in the plasma of
ANIT-treated mice was increased by 263-fold. These features are
consistent with the changes that have been previously documented to
occur in the plasma of ANIT-treated rats (7). Unlike the ANIT-treated
rat, ANIT-treated mice were LCAT deficient 48 h after ANIT treatment
(Table 1) and had a 5.2-fold reduction in exogenous LCAT activity when
compared with controls.
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Plasma lipids and lipoproteins.
The FC and PL mass in the plasma of ANIT-treated mice were increased
17.3- and 10.4-fold, respectively, at 48 h after treatment (Fig.
1). This result is similar to what has been
previously documented in ANIT-treated rats (7). By 168 h after
treatment, FC and PL levels were 38.9- and 13.4-fold greater,
respectively, than control values. Thus the PL level began to plateau
by 48 h, whereas FC levels continued to rise significantly. This
response differs from that observed in rats where both FC and PL levels
began to decrease after 48 h, reaching near-normal levels by 120 h
after treatment. Smaller but significant changes in TG and CE content were also evident in the plasma of ANIT-treated mice (Fig. 1). The rise
in plasma TG peaked at 24 h after treatment and thereafter returned to
near-normal levels.
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Gallbladder bile lipids.
Unlike rats, mice accumulate bile in the gallbladder. ANIT treatment
did not abolish gallbladder content. In fact, the volume of bile in the
gallbladders of ANIT-treated mice was two- to fourfold greater than in
the controls (data not shown). Bile collected from the gallbladders of
ANIT-treated mice had reduced bile lipid concentrations (Table
4). At 48 h, there was a greater reduction in PL and total bile acid concentrations (62 and 86% decrease, respectively) compared with cholesterol concentration (30% decrease). The volume of bile in the gallbladder of ANIT-treated mice at 168 h was
substantially reduced and did not allow for reliable compositional
analysis.
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Hepatic lipids.
Lipid composition analysis did not show significant changes in total
cholesterol content in the liver of ANIT-treated mice (Table
5). The hepatic PL level was comparable to
controls at 48 h after treatment but was slightly reduced after 168 h.
However, the TG level was doubled at 48 h (Table 5). At 168 h, the
hepatic TG level of ANIT-treated mice was less than that in the
controls. Molecular species analysis of CE and PL did not reveal any
significant changes resulting from ANIT treatment (data not shown).
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Hepatic cholesterol and bile acid metabolism.
The activities of HMGR and cyp7 were determined to study the basis for
the altered levels of cholesterol and bile acids. Measurement of cyp7
activity beginning from the time of ANIT treatment revealed a ~75%
decrease in cyp7 enzyme activity at 24 h that persisted until at least
72 h (Fig.
4A). The
reduction in cyp7 activity was accompanied by a decrease in cyp7 mRNA
to undetectable levels at 48-96 h after ANIT treatment (Fig.
4B). The reduction in cyp7 activity
was confirmed in another experiment by following the conversion of
endogenous cholesterol to 7-hydroxycholesterol (Fig.
5). Interestingly, the cyp7 activity
increased 6.4-fold over the control values at 168 h after treatment.
This increase in cyp7 activity corresponded with the increase in the
cyp7 mRNA abundance (data not shown). HMGR activity in liver microsomes of ANIT-treated mice was enhanced sixfold relative to controls at 48 h
after treatment (Fig. 5). By 168 h after the treatment, the HMGR
activity stimulated by the ANIT treatment decreased but to a level that
was still 2.3-fold higher than the controls. The rise in plasma FC
level may therefore be due to the combined effects of increased hepatic
cholesterol biosynthesis, impaired bile acid synthesis and biliary
cholesterol secretion, and decreased CE formation in the plasma.
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Liver histopathology.
Liver samples from control and ANIT-treated mice were subjected to
histopathological analysis. Liver samples from the control group did
not reveal any significant abnormal features. However, sections made of
the livers collected 48 h after ANIT treatment revealed multiple focal
areas of cell death that appeared to be located randomly within the
liver parenchyma (Fig.
6A). There were no
obvious signs of cell death in the area adjacent to either the portal
triads or the central veins. No inflammation was apparent around the
areas of damage in the parenchyma, but inflammatory cell infiltration
in the periportal areas was evident. In addition, there was
proliferation of fibrocytic cells particularly around the bile ducts
(Fig. 6B). An increased number of
mitotic figures were also evident throughout the liver. The cytoplasm
of hepatocytes in the liver of ANIT-treated mice taken at 48 h after
treatment often contained multiple small vacuoles. Surprisingly, the
changes were largely resolved at 168 h after ANIT treatment. The
vacuoles, which may have contained TG, that were evident at 48 h after
treatment were no longer apparent in liver samples collected at 168 h
after treatment. The reactive fibroplasia that was prominent in the periportal region had entirely disappeared. Small clusters of neutrophils were occasionally present, and the region surrounding these
areas was essentially microscopically normal.
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DISCUSSION |
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The elevation of plasma FC and PL concentrations and the appearance of abnormal vesicular lipoprotein particles known as Lp-X are often prominent features in human cholestasis (18). Experimental cholestasis in animals can be induced by surgical ligation of the common bile duct or administration of drugs such as ethinyl estradiol, chlorpromazine, and ANIT (33). A single dose of ANIT administered to rats by gavage induces transient and fully reversible changes to plasma lipids and lipoproteins that are strikingly similar to those associated with cholestatic liver disease (7). In this study, experimental cholestasis was induced in mice by the administration of ANIT to investigate the metabolic basis underlying the changes in plasma lipid levels.
Administration of ANIT to mice at a dose equivalent to that given to rats resulted in alterations to the plasma compartment that were consistent with human cholestasis (18), experimental cholestasis in the rat (4, 7, 15, 22), and human LCAT deficiency (20, 40, 46). Analysis of plasma, liver, and bile lipids indicated that the modification of lipid levels in these three compartments was distinct. FC and PL levels became elevated in the plasma of ANIT-treated mice, and the rise was progressive over the entire experimental time period (0-168 h). This response differs from ANIT-treated rats where the elevation of plasma FC and PL levels peaks at ~48 h after ANIT treatment and returns to near-normal levels by 120 h (7). In addition, the plasma CE levels in ANIT-treated mice were greatly diminished, and this was likely due, at least in part, to the large reduction in LCAT activity. In humans, secondary LCAT deficiency is not a universal feature of liver disease or cholestasis, but it appears to be related to the severity of liver dysfunction (34, 47). Therefore, the liver damage caused by ANIT in mice may be more severe than that observed in the rat. Indeed, the cholestatic time course in the ANIT-treated mouse is more prolonged, and the changes in plasma lipid composition are consistent with a more severe cholestasis and an accompanying secondary LCAT deficiency.
Although the plasma of ANIT-treated mice was not examined by electron microscopy, the amount and composition of PL and cholesterol in the LDL density range were characteristic of the presence of Lp-X (39). The changes in the density distribution of apolipoproteins associated with lipoproteins after ANIT treatment were analyzed by denaturing gradient gel electrophoresis of lipoprotein fractions from mouse plasma. The most striking changes were associated with apo A-I, apo A-IV, and apo E. The amount of apo A-I in the HDL density range was decreased moderately, in contrast to that observed in ANIT-treated rats (7). A reduction in plasma apo A-I levels is typically observed in cholestasis with secondary LCAT deficiency (41) as well as in human familial LCAT deficiency (20). Thus the difference between rat and mouse species with respect to apo A-I levels after ANIT treatment may be directly related to changes in LCAT activity, either through increased apo A-I catabolism or decreased apo A-I and/or mature HDL production. As in the ANIT rat, apo E was dramatically increased in the LDL density range. Elevated plasma apo E in human liver disease (9) and LCAT deficiency (20) has been previously observed and associated with large discoidal HDL particles. Whether apo E in the ANIT mouse is associated with HDL or Lp-X was not determined in this study. The level of apo A-IV associated with the LDL density range of plasma from ANIT-treated mice also was increased, and this is consistent with that previously observed in ANIT-treated rats (7). The reason for this remains unknown, but based on its location, we speculate that apo A-IV may have a strong affinity for the PL-rich lipoproteins in cholestatic plasma.
The level of CE in the plasma decreased after ANIT treatment and remained depressed throughout the duration of the experiment. Interestingly, the proportion of C-16 and C-18 CE species was increased at the expense of C-20 CE. These changes are probably due to the residual LCAT activity in the plasma and the elevated levels of PL species containing C-16 and C-18 fatty acids (i.e., C-16/18 and C-18/18). Mouse LCAT has been shown to have a preference for long-chain fatty acids in PL (44); however, this specificity appears to be overcome in vivo when large amounts of PL containing C16 and C18 fatty acids are available as substrates.
Although the concentrations of FC, PL, and bile acids in gallbladder bile were decreased at 48 h after ANIT treatment, the volume of bile in the gallbladders of ANIT-treated mice was increased two to four times compared with controls. Based on this, the estimated total amount of bile acids in the gallbladders of ANIT-treated mice was reduced by 0.4- to 0.7-fold compared with the controls, whereas the total amounts of FC and PL were similar or slightly elevated (1.3- to 2.7-fold and 0.7- to 1.4-fold, respectively). It is not known whether ANIT impairs the release of gallbladder bile in mice.
The activity of HMGR in hepatic microsomes of ANIT-treated mice was
increased substantially during the early phase of the response.
Considering that HMGR catalyzes the rate-limiting step in the
biosynthesis of cholesterol (3), it appears that ANIT-induced intrahepatic cholestasis occurs under conditions where liver
cholesterol synthesis is stimulated, and this is consistent with that
observed in rats with extrahepatic cholestasis (11). Osono et al. (31) estimated that hepatic synthesis accounts for ~22% of the total cholesterol synthesis (153 mg · kg1 · day
1)
in normal mice. Because we did not measure HMGR activity in peripheral
tissues, it is not possible to estimate the amount of cholesterol
contributed by extrahepatic sources to the plasma of ANIT-treated mice.
However, on the basis of the stimulated activity of hepatic HMGR
observed at 48 and 168 h, the estimated rate of sterol synthesis in the
liver is sufficient to account for nearly all of the cholesterol
accumulated in the plasma of ANIT-treated mice.
Cyp7 is a cytochrome P-450 enzyme responsible for catalyzing the rate-limiting step in the hepatic conversion of cholesterol into bile acids (35). ANIT has been shown to inhibit hepatic cytochrome P-450 activity (13, 36). In mice, cyp7 mRNA abundance was reduced to undetectable levels after ANIT treatment, but, surprisingly, cyp7 activity was not completely abolished. The large reduction in cyp7 activity nevertheless suggests the impairment of cholesterol catabolism via conversion to bile acids.
The creation of cyp7-deficient mice (24) unequivocally established the
existence of the postulated alternative bile acid biosynthetic pathway
(see Ref. 25 for review). In this pathway, a distinct enzyme known as
oxysterol 7-hydroxylase is responsible for the 7
-hydroxylation of
the steroid nucleus (38). This enzyme exhibits a high degree of
substrate specificity for oxysterols rather than cholesterol (24, 37,
45). The alternate pathway appears to be constitutive, although a
slight reduction in hepatic oxysterol 7
-hydroxylase activity has
been observed in mice fed cholic acid (37). However, the
cyp7-controlled pathway is responsible for the bulk of hepatic bile
acid synthesis (38). In rats, ANIT treatment results in the enrichment
of trihydroxy bile acids (cholic and
-muricholic acids) and the
depletion of mono- and dihydroxy bile acid species in plasma (36). The
second pathway likely does contribute significantly to the conversion
of hepatic cholesterol into bile acids in ANIT-treated mice.
Despite the large increase of cholesterol and phospholipid concentrations in the plasma, the levels of both these lipids in the liver were not significantly altered. A previous study showed an increase in hepatic cholesterol content in bile duct-ligated rats (12). In this regard, it appears that the response of mice to induction of cholestasis by drug treatment differs from that of rats after bile duct ligation. Hepatic PL levels of ANIT-treated mice were also comparable to control levels even though massive amounts of PL accumulated in the plasma. The molecular species of phospholipids that was enriched to the greatest degree contained fatty acyl moieties with 34 carbons (i.e., C-16 and C-18 fatty acids). These PL species are normally found in bile (21), indicating therefore that the majority of the PL in the plasma of ANIT-treated mice originates from the liver. ANIT treatment likely enhances the synthesis of PL in the liver based on the amount of PL accumulated in the plasma. Stimulation of PL synthesis may promote the efflux of bile acids through the formation of PL-bile acid micelles, thereby minimizing bile acid cytotoxicity.
The importance of the mdr2 P-glycoprotein, the protein responsible for the translocation of phosphatidylcholine across the canalicular membrane (42), in the formation of Lp-X was recently investigated (32). Lp-X was not found in the plasma of mdr2-deficient mice after bile duct ligation, suggesting that mdr2 function is necessary for Lp-X formation. In addition, experiments using mice with only one functional allele of the Mdr2 gene and transgenic mice expressing the human MDR3 gene (the homolog of Mdr2) at different levels demonstrated that the amount of Lp-X that appears in the plasma after bile duct ligation is proportional to mdr2 activity in canalicular membranes (32). The abundance of the mdr2 mRNA is increased by cholic acid feeding (17). Thus, during cholestasis, the expression of the Mdr2 gene may actually be stimulated in response to increased intracellular bile acid concentration.
The PL-to-FC ratio of gallbladder bile taken from mice at 48 h after treatment was considerably greater than the PL-to-FC ratio of lipoproteins in the LDL density range of plasma from the same mice. Increased tight junction permeability is postulated to be responsible for the leakage of biliary constituents into the plasma compartment during ANIT-induced cholestasis (27). It was previously suggested that nascent Lp-X particles initially form within the bile canaliculi, then are transferred through liver parenchymal cells to the space of Disse, thereby gaining access to the plasma compartment (14). It is interesting to note that the bile secreted by rat livers perfused with various species of bile acids has nearly a fixed PL-to-FC molar ratio of 12:1 (2), whereas the PL-to-FC molar ratio of Lp-X in plasma is nearly equal (7, 39). Because PL vesicles are known to be efficient acceptors of cellular cholesterol (see Ref. 48 for review), the nascent cholesterol-poor vesicles secreted by the ANIT mouse liver may promote the efficient efflux of cholesterol from hepatic cellular membranes and subsequently accumulate in the plasma. The continuous extraction of cholesterol from hepatic cellular membranes would explain the stimulation of cholesterol synthesis in the liver.
A transient rise of TG that peaked at 24 h after treatment was evident in the plasma of ANIT-treated mice. Previously, Felker et al. (14) reported that the perfusate of bile duct-ligated rat liver contained LDL particles that were abnormally enriched in TG. The production of these particles may represent the very early stages of aberrant lipoprotein metabolism after the induction of cholestasis. The TG level in the plasma of ANIT-treated mice decreased to near-normal levels by 48 h after treatment. This coincided with the large increase in hepatic TG level, which may reflect abnormal assembly and secretion of apo B-containing particles. By the end of the experimental period, the amount of TG in the liver of ANIT-treated mice was less than in controls.
Analysis of liver pathology indicates that the response of the mouse liver to ANIT treatment can also be differentiated histologically. In the early phase of the response, significant pathological features such as focal loss of hepatocytes and hyperplasia of cells surrounding the bile ducts were observed. Injury to hepatocytes and cholangiohepatitis has been documented previously in ANIT-treated rats (10, 19, 26). However, the exact basis for ANIT hepatotoxicity in vivo remains to be defined (30). Liver samples taken from ANIT-treated mice during the latter phase of the response were surprisingly histologically normal and in marked contrast to the remarkable pathological changes evident during the early phase.
In summary, we investigated the metabolism of lipids in mice with drug-induced cholestasis. The ANIT-treated mouse clearly elaborates the aberrant metabolism of plasma lipids and lipoproteins associated with cholestasis and human LCAT deficiency. It is also apparent that the response of mice to ANIT treatment is complex and produces a variety of sequelae. With respect to the changes in lipid metabolism, the rise in intrahepatic bile acid concentration after induction of cholestasis appears to stimulate the synthesis of biliary PL, a response that likely represents a mechanism to mitigate the cytotoxic effects of bile acids. The accumulation of biliary PL in the plasma is accompanied by the increase in plasma FC. It is not known what fraction of the cholesterol accumulated in the plasma is derived from peripheral membranes. We speculate that the liver makes a significant contribution based on the enhancement of hepatic HMGR activity (suggesting stimulation of cholesterol synthesis) and depression of cyp7 activity (suggesting less efficient conversion of cholesterol into bile acids), particularly during the early stages of the response to the drug.
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ACKNOWLEDGEMENTS |
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The technical assistance of B. Stewart, D. Cikaluk, and H. Czernecka is gratefully acknowledged.
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FOOTNOTES |
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This research was supported by a grant from the Heart and Stroke Foundation of Nova Scotia, Medical Research Council of Canada Grant MT-14812, and a grant from the Alberta Heritage Foundation for Medical Research. L. B. Agellon is a Senior Scholar of the Alberta Heritage Foundation for Medical Research.
Present address of J. W. Chisholm: Dept. of Pathology/Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1040.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. B. Agellon, Lipid and Lipoprotein Research Group, 328 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 (E-mail: luis.agellon{at}ualberta.ca).
Received 22 April 1998; accepted in final form 27 January 1999.
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