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IGAN TATE UNIVER ITY

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This is to certify that the

dissertation entitled

EFFECTS OF INTERRUPTION OF ENTEROHEPATIC CIRCULATION OF
BILE SALTS ON CHOLESTEROL ME'I'AKLISVI IN CATTLE: A
I‘DVEL HYPCflHOLESTEROLEMIC MODEL

presented by

ZHOUJ I CHEN

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Animal Science
Major pleessor
Date W7y

MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771

 

 

 

 

 

LIBRARY
Mlchlgan State
Unlverslty

 

 

 

PLACE ll RETURN BOXtoromwotN-chockomm your record.
TO AVOID FINES return on or More dd. duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

usu loAn Affirmative Action/aqua Opportunity Institution
WA

EFFECTS OF INTERRUPTION OF ENTEROHEPATIC CIRCULATION
OF BILE SALTS ON CHOLESTEROL METABOLISM IN CATTLE:
A NOVEL HYPOCHOLESTEROLEMIC MODEL

Zhouji Chen

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Sciences

1994

ABSTRACT

EFFECTS OF INTERRUPTION OF ENTEROHEPATIC CIRCULATION
OF BILE SALTS ON CHOLESTEROL METABOLISM IN CATTLE:
A NOVEL HYPOCHOLESTEROLEMIC MODEL

By
Zhouji Chen

Enterohepatic circulation (EHC) of bile salts has a pivotal role in
cholesterol homeostasis in the body. Its interruption is known to cause wide-
ranging effects on cholesterol metabolism in an number of non-ruminant
species, leading to a reduction in plasma cholesterol concentration. Similar
information has not been available for ruminant animals. The objective of the
present study was to determine the effects of interruption of BBC on plasma
concentrations of cholesterol and other lipids and on hepatic cholesterol
metabolism in cattle. Five adult, nonlactating, Holstein cows were fitted with
reentrant intestinal cannula so that the portion of bile flow reaching the
intestine could be controlled. Two 15-day bile diversion periods, i.e., 50% and
22% bile diversion, were studied to examine the effects of partial bile
diversion on plasma lipids. Both bile diversion procedures caused a dramatic
reduction in plasma cholesterol concentration. After 8 days of bile diversion,
plasma cholesterol concentrations reached a low, stabilized level of about 10
mg/dl. Plasma concentrations of phospholipids and triglycerides were also
decreased dramatically in response to bile diversion. After the termination of
bile diversion, plasma concentrations of cholesterol and other lipids returned
to initial values within a week. Subsequent experiments were conducted to
determine if changes in hepatic cholesterol synthesis or uptake, during bile

diversion, could account for the response in plasma cholesterol. Cholesterol

synthesis rates, hepatic cholesterol concentrations, and hepatic low density
lipoprotein (LDL) receptor-binding activity were determined in liver biopsies
taken in precannulation control and postcannulation control periods and
taken at the end of a second 50% bile diversion period. Cholesterol synthesis
rate was barely detectable in liver tissues during the two control periods.
After 14 days of bile diversion, however, hepatic cholesterol synthesis rate
was increased by more than 10-fold. Nonetheless, the estimated total amount
of hepatic newly-synthesized cholesterol would provide no more than 5% of
the sterols depleted from the body during bile diversion (1.2 vs. 25
mmoles/day). Moreover, bile diversion almost completely depleted the liver of
cholesterol ester. This indicates that bovine liver must rely upon extrahepatic
sources of cholesterol for biliary sterol formation during this period. In
response to bile diversion, hepatic LDL receptor-binding activity was
significantly elevated, suggesting an increased uptake of plasma lipoproteins
by the liver. In conclusion, the present study, for the first time, shows that
bovine plasma concentrations of cholesterol and other lipids respond to
partial bile diversion with a dramatic reduction. The substantial
hypocholesterolemia is suggested to occur as a result of the inherently low
rate of cholesterol synthesis in the bovine liver. This study has defined a
novel animal model with which to investigate the metabolic role of plasma
cholesterol and regulation of plasma lipoprotein metabolism.

1v

TABLE OF CONTENTS

List of Tables ...................................................................................................... vii
List of Figures .................................................................................................... viii
Chapter I. Review of literature
I. Introduction ............................................................................................ 1
II. Lipoprotein cholesterol transport system ............................................ 3
A. Physicochemical properties of plasma lipoproteins .................. 3
B. Lipoprotein metabolism and cholesterol transport .................. 7
C. Regulation of HDL subpopulation distribution ...................... 13
III. Role of liver in cholesterol homeostasis ............................................ 15
A. Role of liver in whole body cholesterol synthesis .................... 15
B. Hepatic lipoprotein receptors and clearance
of plasma cholesterol ................................................................. 17
C. Biliary sterol formation and enterohepatic circulation
of bile salts ................................................................................. 23
D. Effects of interruption of EHC ................................................. 27
IV. Implications of unique features of plasma cholesterol
metabolism in dairy cows ....................................... ‘ .......................... 3 0
A. Gestation-lactation cycle related changes in
plasma cholesterol concentrations .......................................... 31
B. Possible mechanisms for the lactational
hyperalphalipoproteinemia ..................................................... 31
C. Peripartum hypocholesterolemia and its association
with cow diseases ..................................................................... 33
V. References cited ................................................................................... 37

Chapter II. Partial diversion of bile dramatically reduces plasma
cholesterol concentration in cattle

Introduction .............................................................................................. 52
Methods and materials ............................................................................ 53
Results ...................................................................................................... 57
Discussion ................................................................................................. 65
References cited ....................................................................................... 71

Chapter III. Inherently low rate of cholesterol synthesis in the liver
exaggerates the hypocholesterolemic effects of partial
bile diversion in cattle

Introduction .............................................................................................. 74
Methods and materials ............................................................................ 75
Results ...................................................................................................... 80
Discussion ................................................................................................. 89
References ................................................................................................ 96

vi

LIST OF TABLES

Table 1. Physicochemical properties of human plasma

lipoproteins classes ................................................................................ 5
Table 2. Protein composition of human plasma lipoproteins ............................. 6
Table 3. Metabolic roles of plasma apolipoproteins in lipid transport .............. 8

Table 4. Effects of partial diversion of bile flow on cholesterol
concentration and distribution among
the plasma lipoprotein fractions .......................................................... 66

vii

LIST OF FIGURES

Figure 1. Overall summary of pathways of plasma lipoprotein
metabolism and cholesterol ................................................................ 12

Figure 2. Lipoprotein receptor-mediated pathways for hepatic
clearance of plasma cholesterol and their interactions
with enterohepatic circulation of bile salts ...................................... 24

Figure 3. Mass balance of bile acids around the enterohepatic
circulation showing typical kinetic values

from healthy humans .......................................................................... 26
Figure 4. Diagram of surgical modification to the duodenum. ........................ 54
Figure 5. Diurnal changes in rates of bile flow (A)
and bile acid secretion (B) ................................................................. 59
Figure 6. Effects of controlled biliary diversion on rates
of bile flow (A) and bile acid output (B) ............................................ 60
Figure 7. Plasma concentration of cholesterol before, during
and after controlled biliary diversion ............................................... 62
Figure 8. Plasma concentration of phospholipids before, during
and after controlled biliary diversion ................................................ 63
Figure 9. Plasma concentration of triglycerides before, during
and after controlled biliary diversion ................................................ 64
Figure 10. Depletion of biliary sterols induced by chronic
diversion of 50% of bile flow ............................................................. 8 1
Figure 11. Effects of bile diversion on plasma cholesterol concentration ........ 82

Figure 12. Efi‘ects of bile diversion on hepatic cholesterol and
cholesterol ester concentrations ...................................................... 83

Figure 13. Time-course of incorporation of tritium from tritiated water
into cow and rat liver tissues and cow intestinal mucosa .............. 85

Figure 14. Effects of supplement of various substrates to MEM
on incorporation rate of tritium from tritiated water
into DPS by cow liver tissues ............................................................ 86

viii

Figure 15. Effects of bile diversion on the incorporation rate of tritium
from tritiated water into digitonin precipitable sterols (DPS)

by cow liver tissues and intestinal mucosa .................................... 87
Figure 16. Estimated quantities of cholesterol synthesized by the whole

liver and intestinal mucosa before and after bile diversion ........... 88
Figure 17. Binding of radiolabeled bovine LDL to bovine liver

homogenate as a function of 125I-LDL concentration ..................... 90
Figure 18. Effects of bile diversion on hepatic LDL

receptor binding activity ................................................................... 91

CHAPTERI

REVIEWOF LITERATURE

 

I. Introduction

II. Lipoprotein cholesterol transport system
A. Physicochemical properties of plasma lipoproteins
B. Lipoprotein metabolism and cholesterol transport
C. Regulation of HDL subpopulation distribution

III. Role of liver in cholesterol homeostasis
A. Role of liver in whole body cholesterol synthesis
B. Hepatic lipoprotein receptors and clearance
of plasma cholesterol
C. Biliary sterol formation and enterohepatic
circulation of bile salts
D. Effects of interruption of EHC

IV. Implications of unique features of plasma cholesterol

metabolism in dairy cows

A. Gestation-lactation cycle related changes in plasma
cholesterol concentrations

B. Possible mechanisms for the lactational
hyperalphalipoproteinemia

C. Peripartum hypocholesterolemia and its association
with cow diseases

V. References cited

 

I. INTRODUCTION

Cholesterol is an essential constituent of living tissues. It plays a
critical role in the body as an indispensable component of biomembranes
and as an obligatory precursor for the biosynthesis of steroid hormones and

bile acids. Cholesterol is also required by the liver and intestine for the

2

synthesis and secretion of lipoproteins. In order to fulfill the cholesterol
demands of various cells, a continuous supply of cholesterol is required. All
mammalian cells can synthesize cholesterol to meet their requirement, but
this does not usually occur. In fact, a significant amount of cholesterol used
by the peripheral tissues is usually derived from plasma lipoproteins (1, 2).
This is especially true for the steroidogenic tissues such as adrenals and
corpora lutea of the ovary, which are dependent upon the uptake of
circulating lipoprotein-carried cholesterol to maintain physiological levels
of steroid hormone production (3-5). Therefore, an adequate supply of
plasma cholesterol to the tissues is important for maintaining cellular and
endocrine functions in the body.

Plasma cholesterol concentration in most animal species is
maintained within a narrow range throughout different physiological
stages, but this does not appear to be true for dairy cattle. For example,
large fluctuations in blood cholesterol concentrations through the gestation-
lactation cycle have been well documented in dairy cows (6). The
mechanisms regulating these large changes in plasma cholesterol are not
known. Nonetheless, the degree of peripartum reduction in plasma
cholesterol concentration has been shown to be associated with metabolic
diseases of the cow, including fatty liver and infertility (7-10). This sheds
light on the necessity for understanding the physiological role and the
regulation of plasma cholesterol concentrations in dairy cattle. To date,
however, studies of the cholesterol transport system in cattle have been
primarily focused on qualitative and quantitative aspects of the plasma
lipoprotein profile. A major emphasis has been on the physical and
chemical characteristics of the major lipoprotein particles and their
apolipoproteins. Very little information is available regarding the pathways

3

of lipoprotein metabolism and their regulation in the system. In contrast to
this, numerous investigations have been directed towards elucidation of the
mechanism(s) of control of plasma cholesterol metabolism in other species,
especially humans and experimental animals such as rats. The purpose of
this chapter is to review current knowledge on the major pathways of
plasma cholesterol metabolism and their regulation in these species, with
emphasis on the role of liver. Extrapolation of this knowledge to cattle will
then be made based on available information on physicochemical

characteristics of bovine plasma lipoproteins.

II. THE LIPOPROTEIN CHOLESTEROL TRANSPORT SYSTEM

Nearly all cholesterol in plasma is carried by plasma lipoproteins,
which in turn act as vehicles for transport of cholesterol through the
aqueous medium of plasma. An abnormal plasma cholesterol
concentration usually reflects an underlying change in the concentration of
a specific lipoprotein. In order to understand plasma cholesterol
metabolism, therefore, it is necessary to consider the nature and origin of
plasma lipoproteins, their interconversions within the plasma and their

extravascular metabolism.

A. Physicochemical properties of plasma lipoproteins

Lipoproteins are macromolecular complexes of protein,
phospholipid, cholesterol, cholesterol ester and triglyceride. The
hydrophobic components, i.e., triglycerides and cholesterol esters, form the
core of the lipoprotein particles while polar phospolipids and unesterified
cholesterol, together with apoproteins, form the surface monolayer coafl 11).

4

Traditionally, lipoproteins are classified according to their hydrated
densities into four major classes: very low density lipoproteins (VLDL) and
chylomicrons, <1.006 g/ml; low density lipoproteins (LDL), 1.006 to 1.062
g/ml; and high density lipoproteins (HDL), 1.063 to 1.21 g/ml. In addition,
based on the difference in their surface charge, lipoproteins can also be
separated into four electrophoretic classes on media such as paper or
agarose (12). Due to their large size, the chylomicrons remain at the origin
while the three other classes migrate with difi‘erent mobilities in an electric
field. HDL and LDL are referred to as a- and B-lipoproteins because their
mobilities are identical to a- and B-globulins, respectively. VLDL is
pre-B-lipoprotein, so-called because its mobility is slightly greater than
B-lipoproteins. The physicochemical characteristics of lipoproteins vary
according to the lipoprotein density classifications. Table 1 is a summary of
some major physicochemical properties of various classes of lipoproteins of
human plasma. The density of lipoprotein particles is inversely related to
their size, reflecting the relative amounts of low density, non-polar core
lipid and high density, surface protein present. Chylomicrons and VLDL
are large particles and are highly enriched in triglyceride, but they are low
in cholesterol content. In contrast, LDL and HDL are small, relatively
dense particles which are rich in cholesterol esters, but low in triglyceride
content. It should be noted that HDL are polymorphic and consist of several
more or less discrete subspecies. For example, there are two major
subclasses of HDL, i.e., HDL2 and HDL3, in human plasma. The core
diameter of HDLz is about 50% larger than that of HDL3, resulting in a core
volume which is 3.5 larger. Hence, HDL2 contains 34 fold more cholesterol
ester and triglyceride molecules than HDL3 (14). Similar subpopulations of
HDL particles can also be found in other species (15, 16).

Table l. Physicochemical Properties of Human Plasma Lipoprotein Classesa

 

 

Lipoprotein Density Diameter Electrophoretic Protein Major
(g/ml) (nm) mobility (% weight) lipid
Chylomicrons <0.93 75-1200 Remains at origin 1.5-2.5 TGc
VLDLb 0.93-1.006 30-80 Pre-B-lipoproteins 5-10 TC
1131. 1.006-1.019 25-35 Slow pre-B- 15-20 TG/CE
lipoproteins
LDL 1019-1063 18-25 B—Lipoproteins 20-25 CE
HDL .
HDL2 1.063-1.120 9-12 a-Lipoproteins 40-45 CE/PL
I-IDL3 1.120-1.210 5-9 a-Lipoproteins 50-55 CE/Pl.

 

a Compiled from Myant (13) and Gotto et a1 (1 1).

b-0 Abbreviations: very low density lipoprotein, VLDL; intermediate density
lipoprotein, IDL; low density lipoprotein, LDL; high density lipoprotein, HDL;
triglyceride, TG; cholesterol ester, CE; and phospholipid, PL.

The fate of lipoproteins is largely determined by the amount and the
type of protein present on the surface. The protein composition of various
lipoprotein classes is shown in Table 2. In human lipoproteins, apoA-I and
apoA-II are the two major proteins of HDL, whereas apoB is the major
protein of LDL and also comprises about half the protein of VLDL. The
apoCs (i.e., 0-1, 0-11 and C-III) are present in chylomicrons and VLDL
and, to a minor extent, in HDL (13, 17). Likewise, apoE is a constituent of
triglyceride-rich lipoproteins and is also detectable in HDL (17), especially
the large HDL particles such as HDL1, which is present in low

concentrations in normal human plasma.

6

Table 2. Protein composition of human plasma lipoproteins*

 

 

 

Lipoprotein Major Minor Trace

Chylomicrons ApoB and apoC apoB apoA-I and
apoA-II

VLDL ApoB and apoC apoB apoA-I and
apoA-II

IDL ApoB and apoB apoC --

IDL ApoB -- apoC

HDL2 ApoA-I and apoA-II apoC apoB and apoD

HDL3 ApoA-I and apoA-II apoC apoE and apoD

"' Adapted from Myant (13).

Bovine plasma lipoproteins are also commonly separated into VLDL,
LDL and HDL fractions according to their hydrated densities, although this
might not be an appropriate procedure because there is an overlap in the
density of the various functional types of lipoproteins in this species (6). As
with most other mammals, HDL is the predominant lipoprotein class in
cattle, accounting for up to 90% of the total plasma lipoproteins (6, 16).
Bovine HDL is characterized by its high concentration of cholesterol esters
and by the presence of large HDL particles (18-20). These large HDL
particles normally account for half of total bovine plasma HDL in lactating
cows (18-20). They are distributed over the density interval of 1.06 to 1.12
g/ml and range in particle size from 12 to 16 nm in diameter (6, 18, 21).
Such HDL particles are larger than human HDL2 and are normally absent
from human plasma. However, the large bovine HDL appear to posses
physicochemical characteristics similar to those of HDL1 from normal rat
plasma (14, 16) or HDLc isolated from animals such as pigs when they are
fed a high-cholesterol diet (22). Unlike HDL1 or HDLc, however, the bovine

7

particles do not appear to contain apoE (6, 10, 23). While the majority of
bovine a-lipoproteins can be isolated in HDL fraction, some have a density
beyond the lower density limit of the HDL and are usually partitioned into
LDL fraction (6, 19). Thus, unlike LDL from other species, bovine LDL
contains (It-lipoproteins as well as B-lipoprotein fraction (6, 19). These
physicochemical properties of bovine HDL/a-lipoproteins reflect the unique

features of plasma cholesterol transport in the cattle.

B. Lipoprotein metabolism and cholesterol transport

The liver and small intestine are the primary organs for lipoprotein
synthesis and secretion. In plasma, the lipoproteins secreted from these
organs undergo extensive metabolic transformation. Through interactions
with enzymes and cell surface receptors, the apoproteins direct each
lipoprotein to its site of metabolism. Many of the functions associated with
apoproteins are summarized in Table 3 while the major pathways for the
transfer of cholesterol through lipoprotein conversion are depicted in
Figure 1.

Input of cholesterol into the plasma can come either from
endogenous synthesis or from exogenous sources, i.e., dietary cholesterol.
However, due to the low content of cholesterol in ruminant diets, the
exogenous supply of cholesterol to ruminants must be very limited. Dietary
cholesterol supply to this species may be further limited by the action of
ruminal microflora which hydrogenate cholesterol in the rumen (24).
Thus, ruminant animals must rely on endogenous cholesterol synthesis for
cholesterol input. Cholesterol synthesized by the small intestine is exported
to the plasma pool mainly as a component of chylomicrons. Upon entering

the general circulation, the triglyceride core of chylomicrons is rapidly

8

hydrolyzed by lipoprotein lipase (LPL) on the endothelium of peripheral
capillaries, with apoC-II acting as activator for the enzyme (11). During
this process, chylomicrons are reduced in mass and the core becomes
enriched with cholesterol esters. In addition, the chylomicrons acquire
additional apoproteins, including apoE, leading to the formation of
chylomicron remnant particles. As the size of the chylomicrons
diminishes, the surface components, including unesterified cholesterol,
are transferred to HDL (11, 14). The chylomicron remnants are removed

from the plasma by the liver via hepatic receptors specific for apoE (25).

Table 3. Metabolic Roles of Plasma Apolipoproteins in Lipid Transport“

 

 

Function Apolipoprotein
Lipoprotein biosynthesis/secretion B-48 (intestine)
B-100 (liver)
Enzyme activation
Lipoprotein lipase C-II
Lecithin-cholesterol acyltransferase A-I
C-I
A-IV
Interaction of lipoproteins with cellular receptors
LDL receptor recognition (B/E receptor) B- 100
VLDL/remnant receptor (E receptor) E
HDL receptor A-I: A-II
Inhibition of interaction with hepatic receptors C-l
C-II
C-III

 

"' Adapted from Gotto et a1 (11).

9

Ruminant animals also possess the capability to produce
chylomicrons from the intestine (6, 26) and the resulting particles also
appear to be removed by the liver (27). However, due to the low lipid content
of ruminant diets, production of chylomicrons by ruminant intestine
normally might occur at a very low rate. Moreover, the ingesta in the
ruminant digestive tract are released from the rumen-reticulum in a
continuous manner (28). The low intake of dietary fat plus this digestion
and absorption pattern means a small continuous supply of dietary lipids to
the small intestine of the cattle. This has been suggested to result in
production of VLDL-like particles by the ruminant intestine, which have
lower triglyceride content and a higher hydrated-density than typical
chylomicrons (6, 29). Thus, plasma concentrations of typical chylomicrons
are usually low in ruminants (6, 29). Nevertheless, cholesterol molecules
required for intestinal triglyceride-rich lipoprotein production in
ruminants are mainly synthesized within individual enterocytes, where
the rate of cholesterol synthesis appears to be determined by the amount of
triglycerides synthesized and secreted by these cells (30).

The liver exports cholesterol to the circulation as a component of
VLDL (Figure 1). The VLDL particles are, like chylomicrons, also rich in
triglyceride but they contain a larger amount of cholesterol ester. The
major apoproteins in VLDL are apoB-100 and apoE (11, 17 ). After secretion
from the liver, much of the core triglyceride of VLDL is removed in the
capillary beds in a manner analogous to chylomicron metabolism (11).
Concomitantly, some surface constituents, especially free cholesterol, are
transferred to HDL. The remaining surface components and core lipids
form IDL (11, 17). A significant fraction of IDL is taken up by liver, while
the remainder undergoes further lipolysis of triglyceride and phospholipid

10

to give rise to LDL. The precise mechanism for this lipolysis process is
unclear, but it appears to involve the action of hepatic lipase (31). During
the conversion of VLDL to LDL, apoE is lost from the particle and
only apoB-100 is retained (31). LDL is greatly enriched in cholesterol ester,
with apoB- 100 being the only significant protein component. LDL delivers
cholesterol to peripheral tissues for utilization as well as to the liver for
catabolism. Its uptake is mediated by the well-characterized LDL (or
apoB/E) receptor, which is present on most cells (32). The apoprotein profile
of ruminant VLDL appears to be similar to that of nonruminant species (6,
10, 23), suggesting that the above pathways for plasma VLDL metabolism
might be applicable to ruminant animals. Nonetheless, ruminant liver is
known to have very low capacity for VLDL secretion (10, 26). Thus, the
hepatic contribution to the plasma cholesterol pool through VLDL secretion
might be very small in this species.

HDL is formed in the circulation from precursors secreted by the
liver and intestine (Figure 1). The major apolipoproteins (i.e., apoA-I and
apoA-II) of HDL are synthesized in both the liver and small intestine. A
significant portion of HDL apolipoproteins are transferred from
triglyceride-rich lipoproteins during their lipolysis. The initial particle
secreted into the circulation is a discoidal lipoprotein composed of
phospholipids, apolipoproteins, and small amounts of free cholesterol (14,
33). Because of the low lipid content of nascent HDL particles, input of
cholesterol to the circulation as these HDL particles must be very limited. It
has also been proposed that some apoA-I are secreted as the pure protein
fi'om the liver and that even the discoidal particles can be formed in the
circulation (34, 35). The discoidal HDL is transformed into the mature,
spherical HDL through the action of lecithin-cholesterol acyltransferase

ll

(LCAT) (33), which hydrolyzes C18:2 fatty acid from surface lecithin and
reesterifies it to free cholesterol (36). HDL can be viewed as the site of
plasma cholesterol esterification. This is especially true for animal species
such as ruminants, which have very low hepatic acyl-CoAzcholesterol
acyltransferase activity (37). This enzyme is responsible for the cholesterol
esterification inside the cell. In fact, almost all of the cholesterol esters in
ruminant plasma are generated through the LCAT reaction (37, 38). The
free cholesterol substrate for LCAT may come from other lipoproteins or
from cell membranes, whereas the HDL phospholipid consumed by the this
reaction appears to be mainly derived from the surface coat of triglyceride-
rich lipoproteins during remnant formation (14). It is apparent that the
majority of substrates for HDL formation come from the redundant surface
lipids and proteins generated during the lipolysis of the triglyceride-
transporting lipoproteins, i.e., VLDL and chylomicrons. Thus, HDL might
be an essential product of triglyceride transport. .

As with the cholesterol associated with other lipoprotein classes,
HDL-carried cholesterol is also delivered to the liver and other tissues for
metabolism. A portion of HDLcholesterol is transported directly with HDL
particles, whereas other portions may be transferred to other lipoprotein
classes and then delivered to the cells along with these lipoproteins. A
detailed discussion of the pathways for HDL cholesterol uptake will be
presented in section III B. The major apoproteins of HDL, apoA-I and
apoA-II, are catabolized slowly in vivo . The circulating half-time of HDL
protein in humans is 3-6 days (39). Liver and kidney are the major sites of
catabolism of apoA-I (14, 39).

12

 

 

 

        

    
 

 

 

 

  
  
  

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

FA 3 5
TG ‘°
Small LP hylomlcron
Intestine 4% C + CE Remnants
Liver
‘ and/or
surface mats] Peripheral
[0, PL, protein Tissues
. _| E
Liver w
Surface coats:
C, PL, protein TP
B10:) E 3100 E B100
LPL HL
LDL
20% C+CE o 44% C+C
TG
FA -

 

 

 

Figure 1: Overall summary of pathways of plasma lipoprotein metabolism and cholesterol
transport. Lipoprotein components include unesterified cholesterol (C), cholesterol esters
(CE), phospholipids (PL), triglyceride (TG) and fatty acids (FA). The letters projecting
from each particle refer to the apolipoproteins: A-I, B and E, which mediate lipoprotein
cholesterol metabolism. The important regulators of lipoprotein metabolism include
lipoprotein lipase (LPL), lecithin-cholesterol acyltransferase (LCAT), hepatic lipase (HL),
and cholesterol ester transfer protein (CETP).

l 3
C. Regulation of HDL subpopulation distribution

As mentioned earlier, there are several subpopulations of HDL
particles in the plasma. These HDL subspecies undergo dynamic
interconversions in the circulation and many of them are remodeled by
acquisition of surface components of triglyceride-rich lipoproteins. It
appears that the large HDL particles are formed during active lipolysis of
triglyceride-rich lipoproteins when large amounts of surface lipids are
transferred to small HDL particles such as HDL3 (14). Thus, the
concentration of HDL2 in human plasma appears to be proportional to the
rate of triglyceride transport in the circulation (40). Lipoprotein lipase is the
key enzyme required for the hydrolysis of triglyceride-rich particles. The
activity of this enzyme has been shown to be positively correlated with
plasma HDL cholesterol concentrations in human populations (41). The
elevated concentration in HDL cholesterol observed in human subjects with
higher levels of lipoprotein lipase activity is primarily due to an increase in
HDL2 associated cholesterol (41, 42). Likewise, transgenic mice expressing
human lipoprotein lipase have higher plasma concentrations of HDL
cholesterol than their non-transgenic litter mates (43, 44). Conversely, a
genetic defect in lipoprotein lipase in humans is known to result in reduced
HDL2 cholesterol concentration with a concomitant increase in VLDL- and
IDL-cholesterol content (45).

The large HDL particles can be converted back into smaller particles
in the plasma. The underlying mechanism for this conversion is still
unclear. However, in species such as rabbits and humans, which have
high levels of plasma cholesterol ester transfer activity (46), considerable
quantities of cholesterol esters carried by the large HDL particles are

transferred to other lipoprotein classes such as VLDL and LDL in an

14

exchange of triglycerides (47). The triglyceride which is transferred into the
HDL particles can be rapidly hydrolyzed by hepatic lipase (14, 18). Moreover,
since hepatic lipase also possesses potent phospholipid hydrolysis activity, it
could partially deplete the HDL particles of surface phospholipid molecules
(48). The hydrolysis of phospholipid has been suggested to be a critical step
in the remodeling of HDL particles (48, 49). Genetic absence of either
cholesterol ester transfer protein (47, 50) or hepatic lipase (51) has been
shown to cause a remarkable increase in HDLz cholesterol concentration in
humans. Furthermore, comparison of the plasma cholesterol ester
transfer activity and lipoprotein profile among different animal species
reveals a clear association between low levels of this activity in the plasma
and the prominence of a subclass of large HDL, i.e., HDL1. For example,
dogs, rats, and mice, with low levels of plasma cholesterol ester transfer
activity (46), have high plasma concentration of HDL1 (16). Conversely,
injection of cholesterol ester transfer protein into the rat results in
reduction of the large HDL1 species and formation of smaller HDL (52).
This provides direct evidence for the dependence of the presence of
prominent HDL1 on low plasma cholesterol ester transfer activity. Taken
together, there is convincing evidence that cholesterol ester transfer protein
and hepatic lipase' play a pivotal role in the conversion and metabolism of
the large HDL particles. Interestingly, neither of these two proteins
appears to be expressed at a discernible level in ruminants (46, 53-55).
Concomitantly, bovine lipoprotein contains in high proportion a subclass of
HDLl-like large HDL particles (6). Thus, the accumulation of such HDL
particles in bovine plasma might be due to the apparent absence of plasma

cholesterol ester transfer activity and hepatic lipase in this species.

15

HI. ROLE OF LIVER IN MAINTENANCE OF CHOLESTEROL
HOMEOSTASIS

As the major site of both the synthesis and catabolism of plasma
lipoproteins, liver has a central role in cholesterol metabolism and in the
regulation of lipoprotein cholesterol concentration in the circulation. The
major pathways concerning cholesterol metabolism in the liver are
depicted in Figure 2. The control of the hepatic pathways is complex and
interrelated. Presumably, perturbation of this system produces wide-

ranging effects on lipoprotein cholesterol metabolism.

A. The role of liver in whole body cholesterol synthesis

Like all other mammalian cells, liver cells are capable of
synthesizing cholesterol from acetyl CoA. Cholesterol biosynthesis occurs
chiefly in the liver cell microsomes and the primary rate-limiting step is
the conversion of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) into
mevalonic acid, which’is catalyzed by HMG CoA reductase (56). The activity
of this enzyme is under tight control by feedback inhibition from cholesterol,
or more specifically, oxysterol (56, 57). Liver is commonly considered to be
the most important site for whole body cholesterol synthesis. This concept
arose from early studies (58-60) where assays of rates of cholesterol
synthesis were performed in vitro using various l4C-labeled substrates
such as 1‘lC-acetate. However, it became clear that the ability of different
tissues to take up and utilize these 1‘1'C-labeled substrates varies
considerably and most of these substrates were poorly utilized in extra
hepatic tissues (61). Moreover, the specific activity of the 1‘lC-acetyl CoA pool
that is the immediate precursor for cholesterol biosynthesis appears to be

l6

disproportionately (relative to the liver) diluted in many tissues by the
unlabeled, endogenous acetyl-00A (61). These observations raise the
possibility that the role of the liver in whole body cholesterol synthesis might
have been overestimated in some species. More recently, Dietschy and his
colleagues have developed a more reliable system for measurement of in
vivo or in vitro cholesterol synthesis rates based on measurements of the
incorporation of 3H from 3H20 into digitonin-precipitable sterols in tissues
(61, 62). Using this method, these investigators have built up a detailed
picture of cholesterol synthesis in the tissues of a number of animal species
(2, 63). These recent studies have indeed demonstrated that the contribution
of liver cholesterol synthesis to whole body synthesis has been overestimated
in some species. Although in species such as the rat and monkey, the liver
contributes more than 50% of the whole body cholesterol synthesis, the liver
of some other species, including rabbits and guinea pigs, has relatively low
activity for cholesterol synthesis. In these latter species, the contribution of
the liver to whole body synthesis is less than 20% (1, 2, 63). Thus, it is
probable that the quantitative importance of liver in whole body cholesterol
synthesis varies considerably among animal species. .

In contrast to the experimental animals, information on cholesterol
synthesis rates by ruminant tissues is very limited. Only two studies have
been reported on the measurement of cholesterol synthesis rates by several
tissues from small ruminants, including goats (64) and sheep (30). These
studies showed that the liver of these animals has a very low capacity for
cholesterol synthesis, whereas the intestine and adipose tissues have high
cholesterol synthesis rates (30, 64). Thus, it has been suggested that the
liver is a minor site of body cholesterol synthesis in ruminants. However,

these early studies used l“C-labeled substrates, including 1‘lc-acetate and

17

1"*0 glucose, to determine tissue cholesterol synthesis rates. It is known
that these substrates are poorly utilized by ruminant liver (65 ). Thus, the
role of ruminant liver in cholesterol synthesis remains to be clarified.
Nonetheless, because the ruminant liver does not normally produce
significant amounts of VLDL (26), the hepatic contribution to cholesterol in
the ruminant plasma pool might be very limited. The intestine of
ruminants might be the major contributor of blood cholesterol. This is not
only because this organ appears to have high cholesterol-synthesis activity
(30, 64), but also because it is the predominant site of triglyceride-rich
lipoprotein production (6, 26). Thus, feed intake, or any other dietary factors
which potentially affect intestinal lipoprotein production might have a large

influence on plasma cholesterol concentrations in ruminants.

B. Hepatic lipoprotein receptors and hepatic clearance
of plasma cholesterol

While the liver may play only a minor role in whole body cholesterol
synthesis in some species, it nevertheless remains the key organ for the
regulation of cholesterol balance within the intact animal. The terminal
catabolism of most lipoproteins occurs mainly in the liver by means of
receptor-mediated pathways (Figure 2). The best characterized. pathway for
cellular lipoprotein uptake is LDL receptor-mediated endocytosis. The LDL
receptor, discovered by Brown and Goldstein in cultured human fibroblasts
(66), is now recognized to be mainly operative in vivo on the surface of
hepatocytes (32). This receptor is a cell surface glycoprotein, and it binds
specifically to apoB- 100, the major apoprotein in LDL. It also binds to apoE
which is found in several lipoproteins including VLDL remnants and a

subspecies of HDL in most of the mammals (67). Thus, this receptor

18

mediates the removal from the blood of most of these apoB/apoB containing
lipoprotein particles. After binding to the receptor, the lipoprotein particles
are internalized and then digested in lysosomes (32). The cholesterol esters
are hydrolyzed to yield free cholesterol, which may be catabolized into bile
acids by the hepatocytes and then secreted into the intestine for their
removal from the body (Figure 2). The importance of the LDL receptor in
mammalian cholesterol metabolism is best manifested by the examples of
genetic defect in this receptor occurring either naturally or experimentally.
The genetic deficiency of LDL receptors in humans (32), rabbits (68), and
rhesus monkeys (69) causes severe hypercholesterolemia. Likewise,
experimental disruption of the LDL receptor gene in mice results in a
several-fold increase in plasma cholesterol concentration and the
hypercholesterolemia is reversed when expression of the receptor protein is
restored in these mice (70). Expression of the LDL receptor is regulated by
the cellular cholesterol status (32). The LDL receptor gene is always
partially suppressed. This suppression is part of a feedback regulatory
mechanism designed to maintain constant intracellular levels of
cholesterol in liver and other cells (32). When the intracellular cholesterol
level falls, the LDL receptor gene is activated as the cell attempts to obtain
more cholesterol. Regulation of the hepatic LDL receptor is a major factor
controlling the plasma concentration of atherogenic lipoprotein classes in
human. Hence, much interest has been centered on this receptor in
hepatocytes and on its dietary, hormonal and pharmacological regulation
in physiological and pathological states. The LDL receptor has also been
identified in bovine tissues including liver (7 1). However, the physiological
significance of this receptor in regulation of bovine plasma cholesterol

metabolism is yet to be determined. In view of the low concentration of LDL

19

in bovine plasma and the lack of apoE in bovine HDL particles (6, 10, 23), it
appears that this receptor might have a less significant role in cattle than
other species such as humans or rats, which have either high plasma LDL
concentration or large proportion of apoB-rich HDL particles.

The liver also takes up cholesterol of intestinal origin, including
dietary cholesterol, which is contained in chylomicron remnants (Figure
2). This uptake occurs by way of a high-velocity, saturable transport system
that probably depends on the presence of receptors on the hepatocytes that
interact with the apoE of the remnants (72). Since the Km for this transport
is very low, the plasma is cleared completely of the particle within minutes
(72, 73). The identity of the chylomicron remnant receptor has not been fully
established, but a vast body of evidence suggests that the recently cloned
LDL-related protein/a-2-macroglobulin receptor is a candidate for this
receptor (72-74). Several recent studies (75, 76) have also suggested that
hepatic lipase plays an important role in the receptor-mediated endocytosis
of the remnant particles by the liver cell. Unlike the LDL receptor, the
chylomicron remnant receptor does not seem to be tightly regulated by
cellular cholesterol balance (73). However, endocytosis of the remnant
particles, like that mediated by the LDL receptor, leads to the same
sequence of processing within hepatocytes. It is apoE of the surface of
remnant particles that directs these particles towards the liver for
clearance. Thus, ablation of the apoE gene in mice causes massive
accumulation of remnant particle-associated cholesterol in the blood,
especially after the ingestion of a high-fat or high-cholesterol meal (77, 78).
Although the expression of the remnant receptor in the ruminant liver has
not been examined, liver has been shown to be the major site of removal of
chylomicron cholesterol in sheep ( 27). Moreover, apoE has been identified in

20

the bovine triglyceride rich-lipoprotein particles (6, 23). Thus, it is likely
that the cholesterol carried in chylomicrons in bovine plasma is also
cleared by the liver.

The liver is an important site for the catabolism of HDL. Because
HDL can serve as an acceptor for excessive cellular cholesterol from the
extrahepatic tissues (39), the HDL particles deliver to the liver for
catabolism not only the cholesterol originating from liver and intestine, but
also cholesterol removed from a variety of peripheral tissues (Figure 2).
This is the so-called reverse cholesterol transport system and is believed to
play an important role in maintenance of cholesterol homeostasis in the
body. The mechanisms that are responsible for the removal of HDL, or of
the cholesterol carried in HDL, from the plasma are complex and still
poorly understood. Four pathways have been proposed to play a role in this
process (Figure 2). The first pathway depends on plasma cholesterol ester
transfer activity. As noted earlier, this activity mediates the net transfer of
HDL cholesterol ester to VLDL or LDL, apparently in exchange for
triglyceride (47). The transferred cholesterol esters are then cleared from
the circulation via the LDL receptor-mediated pathway. The cholesterol
ester transfer activity in plasma is high in some species such as humans
and rabbits (46). This pathway has been suggested to play the most
important role in the disposition of HDL-cholesterol in humans (47, 73). A
genetic defect in this protein causes a marked increase in HDL and in
plasma total cholesterol concentrations (50, 79). However, plasma
cholesterol ester transfer activity is very low in most mammals including
ruminants (46), hence, this pathway might not be operative in cattle.

Another pathway for HDL cholesterol removal involves the

interaction of LDL receptors with apoE contained in some HDL particles,

21

leading to the LDL receptor-mediated endocytosis of these particles by
hepatocytes. ApoE-rich HDL particles can be found in the plasma of most
mammals. In species such as rats, up to 60% of total HDL cholesterol is
carried in apoE-rich HDL particles (15, 67). Presumably, this pathway
might play an important role in the hepatic HDL cholesterol uptake in this
species. In contrast, no apoE nor apoE-like protein has been found in bovine
HDL or a-lipoproteins (6, 23). This suggests that the LDL receptor-mediated
pathway plays a minimal role in hepatic uptake of bovine HDL particles.

A third pathway for HDL cholesterol removal is dependent on the
action of hepatic lipase. Owing to its phospholipase A activity (39, 80), it
appears that this enzyme is able to hydrolyze HDL phosphatidylcholine at
the surface of hepatocytes or endothelial cells, leading to the production of
lysophosphatidyl choline (80). This in turn causes fusion of the surface of
HDL to the external leaflet of the plasma membrane, thereby facilitating
transfer of cholesterol esters into the cell (39, 80). In this case, the enzyme
would exert a "receptor" function. This process leads to the selective uptake
of cholesterol esters from HDL without the latter being internalized. The
role of hepatic lipase in promoting HDL cholesterol ester transfer into cells
has been well documented in cell culture (39) and rat liver (81, 82), though
the quantitative significance of this pathway is poorly understood.
Nonetheless, because the ruminant liver is almost devoid of hepatic lipase
activity (53-55), it is obvious that this pathway might not be operating in
ruminant animals.

The final pathway for hepatic HDL cholesterol uptake is mediated by
a specific, saturable process in which the hepatocytes remove cholesterol
esters from the apoE-free HDL particles and release the apoproteins
without the latter necessarily being degraded (39, 83, 84). This process leads

22

to a greater fractional rate of the uptake of cholesterol esters than that of
HDL apolipoproteins. When HDL containing 125I-tyramine cellobiose-
labeled apo A-I and [3H]-cholesterol ethers (non-hydrolyzable tracers for
apoprotein and cholesterol ester, respectively) was injected into rats, it was
observed that hepatic uptake of the cholesterol ester tracer was at least two-
fold more rapid than that of the apoprotein tracer (85). The underlying
mechanism(s) of this pathway is still poorly defined. The binding of HDL
particles to the surface of the cell appears to be the initial event in this
pathway and thereafter, the cholesterol esters could be delivered to the cell
through either of the following two separate mechanisms. One is by the
internalization and lysosomal degradation of the whole HDL particle (14,
39, 84), while the other is by selective uptake of cholesterol esters without the
internalization of the HDL particles (83, 86) after which the cholesterol
esters appear to be hydrolyzed extralysosomally (87) by a neutral cholesterol
ester hydrolase (88). The former mechanism might be mediated by a
specific receptor. In fact, a high-affinity HDL-binding site has been
identified on hepatocytes (14, 39, 89) and on liver membranes from various
species including cattle (90, 91). Furthermore, several membrane-bound
HDL binding proteins have also been purified from hepatic tissues (92, 93).
These observations support the existence of a putative HDL receptor on the
cell surface. The role of such a receptor in selective uptake is yet to be
clarified. Regardless of the underlying mechanism, this pathway has been
shown to play a quantitatively important role in the clearance of HDL
cholesterol esters by the liver. It is the dominant pathway for the hepatic
uptake of HDL cholesterol esters in the rat (94). Even in species such as
rabbits, which have about four times the plasma cholesterol ester transfer
activity of humans (46), a significant amount of HDL cholesterol ester is

2 3
taken up by the liver through this pathway (95, 96). Because the other three

pathways, as discussed above, do not appear to be operative in cattle, it
could be readily imagined that this pathway plays a particularly important
role in the catabolism of HDL cholesterol in the bovine species. It is
intriguing to speculate that this species might serve as a unique animal
model for the study of hepatic HDL cholesterol uptake through this
pathway.

C. Biliary sterol formation and enterohepatic circulation of bile salts

The lipoprotein cholesterol reaching the liver can be reutilized either
for biogenesis of biomembranes or for synthesis and secretion of
lipoproteins. It can also be re-esterified and stored as cholesterol esters in
the cell. Alternatively, cholesterol can be secreted into the biliary
system either as intact cholesterol or, after conversion, as bile acids (Figure
2). The hepatic conversion of cholesterol to bile acids is a major mechanism
whereby cholesterol is eliminated from the body (13, 63).

The first and rate-limiting step of this process is hydroxylation at the
7 a position of the sterol nucleus, catalyzed by a microsomal P450-dependent
enzyme, cholesterol 70t-hydroxylase (97). Bile acid synthesis appears to be
under feedback regulation by bile acids returning to the liver (63, 97, 98).
There is also some evidence suggesting that bile salt synthesis is regulated
by free cholesterol availability in some specific precursor pool (99, 100). The
cholesterol substrate for bile acid synthesis can come either from
cholesterol synthesized de novo in the liver, or from the uptake of
lipoprotein cholesterol. However, under normal physiological conditions,
the majority of cholesterol utilized for bile acid synthesis is derived from the
uptake of plasma lipoproteins (63, 101-103). Furthermore, in rats, it has

24

been shown that the uptake of esterified cholesterol from HDL rather than
from LDL is more efficiently coupled to the formation of bile acids (101, 104,
105). It appears that free cholesterol from HDL2 is preferentially utilized for
bile acid synthesis (63, 101, 102, 104). Free cholesterol from chylomicron
remnants is also a preferred substrate for bile acid synthesis (63, 101).

 

 

   
  

  

  

 

 

 

 

 

 

 

 

 

 

Extra-hepatic Liver Intestine
“fissues
Dietary lipids
"—“ d:
W'Hcrlgn 8'”
V .7... ->
. Cholesterol-belt
Chol -*-> HDL BA
/ ACAT
(E; Portal veln
T ' r
P cm:
tfife (39 (are
VLDL
J
Remnant‘ Chylomlcron '
\ Feces
LDL BA/Chol

 

 

 

Figure 2: Lipoprotein receptor-mediated pathways for hepatic clearance of plasma
cholesterol and their interactions with enterohepatic circulation of bile salts. The meanings
of the abbreviations used in this schematic are as follows: HMGR, I-lMG-CoA reductase;
C7aH, cholesterol 7 or-hydroxylase; ACAT, acyl-CoAzcholesterol acyltransferase; CE,
cholesterol ester; R-R, remnant receptor; L-R, LDL receptor; H-R, HDL receptor; HL,
hepatic lipase; CETP, cholesterol ester transfer protein; Chol, cholesterol; BA, bile acids:
PC, phosphatidylcholine; TG, triglyceride; MG, monoglyceride; and FA, fatty acids.

25

Like the cholesterol pool utilized for bile acid synthesis, preformed
cholesterol is the preferred source of cholesterol secreted into the bile (63,
102, 103). Free cholesterol carried in the HDL fraction, especially HDL3, is
preferentially used for the formation of biliary cholesterol (102, 106). The
rate of cholesterol secretion, however, is determined by the species and rate
of bile acid secretion, as well as the rate of biliary phospholipid secretion
(63, 107). The lipoproteins, presumably HDL, are also the major source of
biliary phospholipids (108).

In human bile, bile salts comprise more than 60% of total solute
weight, with phospholipids accounting for about 20% and free cholesterol,
about 5% (109). Bile also contains bile pigments and a mixture of proteins
and electrolytes. After secretion from the liver, the bile is stored in the
gallbladder until this organ, under the influence of gastrointestinal
hormones, discharges it into the intestine (109). Bile salts in the duodenum
and ileum aid in the digestion and absorption of dietary fats. Small
quantities of bile salts can be absorbed by passive difl'usion along the length
of the intestine, whereas the major portion is absorbed by an active
transport system in the terminal ileum (109, 110). As shown in Figure 2,
after entering the portal vein, the absorbed bile salts are bound to albumin
and then transported rapidly to the liver, cleared eficiently, and resecreted
(109, 111). This enterohepatic circulation (EHC) of bile salts is a highly
efficient process and more than 95% of the bile salts are reabsorbed into the
circulation (109-111). Thus, to maintain EHC, the liver normally only has to
synthesize a small amount of bile salts to replace the fecal loss. The details
of EHC have been worked out in a number of animal species (Ill-115). For
example, the quantitative aspect of this circulation in humans is depicted
in Figure 3. In contrast to other species, very little information on EHC is

26

available for ruminants. Based on some early studies, it appears that one of
the characteristics of EHC in ruminants is the pattern of continuous
secretion and the lack of a clear diurnal change in bile salt secretion (116,
117). This might occur as a result of thecontinuous flow of digest from the
rumen into the lower digestive tract (28).

 

Enterohepatic

   
   

1 2-36 g/day

Blllary secretlon = pool x cycles "“93th

(1 2-36 g/day) (3 s) x(4-1 2/day) Reabsorptlon
> 95%

 

 

 

 

L , J
1 1 .s-35.4 g/day k
OXCI'OUOII Fecal OXOMIOI'I
<05 mg/day 0.2-0.6 glday

Circulation

 

 

 

Figure 3: Mass balance of bile acids around the enterohepatic circulation showing typical
kinetic values from healthy humans. The pool size of bile acids in human EHC is about
3 g. This small pool of bile acids recirculatcs through the intestinal tract 4-12 times per day,
resulting in a large daily flux of bile acids. Due to the high efficiency of reabsorption of bile
salts by the intestine and uptake of bile acids by the liver, however, only a very small
proportion of bile acids is lost from the EHC. Thus, the liver is only required to synthesize
a small amount of bile acids to maintain a normal EHC. The data presented in this diagram
were taken from Carey et al. (109).

2 7

D. Effects of interruption of EH C

The maintenance of a normal EHC relies on the efficient
reabsorption of bile salts by the intestine. Interruption of EHC can be
achieved either by surgical procedures such as ileal resection and biliary
diversion, or by pharmacological means such as bile acid sequestrant (e.g.,
cholestyramine) treatment. These procedures exaggerate the fecal loss of
bile sterols and diminish the amount of bile salts returning to the liver.
This results in the release of feedback inhibition of bile acid formation in the
liver and hence, the rate of bile acid synthesis increases (63, 107, 112, 118).
Due to the large daily flux of biliary sterols through the intestinal tract,
however, a slight increase in the percentage of loss requires a large
proportional increase in synthesis to maintain the EHC. If the interruption
is only partial, the increase in bile acid synthesis may be sufficient to
maintain a normal rate of secretion of bile salts into the intestine and the
size of bile acid pool remains unchanged. For example, it has been shown
that rhesus monkeys can compensate for up to a 20% biliary loss and
maintain a normal bile acid pool size by increasing their production rate of
the acids (112). If, however, the rate of fecal loss of bile salts exceeds the
maximal capacity of the liver for synthesizing bile acids, the bile salt pool
decreases and a new steady state is achieved in which the rate of secretion
of bile salts is subnormal or greatly reduced. Total diversion of bile produces
a rapid and dramatic drop in bile secretion rate while the rate of bile acid
synthesis is maintained at the maximal level (109, 112, 113, 115).

The augmented bile acid synthesis rate resulting from the
interruption of EHC in turn largely enhances the demand for cholesterol in
the liver cell. In response to this, the liver increases its capacity for de novo

cholesterol synthesis. In rats, a five to ten-fold increase in the rate of

28

hepatic cholesterol synthesis can be observed after bile diversion or
cholestyramine treatment (101, 107, 118 ). Similar results have also been
reported in a number of other species (63, 101, 119). The increased demand
for cholesterol can also cause the mobilization of hepatic cholesterol stores,
i.e., the esterified cholesterol in the liver (101, 119). Under most
circumstances, however, the local cholesterol supply is not sufficient to
meet the increased cholesterol requirement. Hence, the liver has to
increase its recruitment of lipoprotein cholesterol from the circulation. In
response to the interruption of EHC, increased expression of LDL receptor
on the hepatocytes is usually induced (32, 63, 120-122). This may serve as an
important mechanism for the enhanced uptake of lipoprotein cholesterol by
the liver. Yet, the efi'ect of interruption of EHC on other hepatic pathways of
lipoprotein uptake is unclear.

As a result of the enhanced hepatic uptake of lipoprotein cholesterol,
however, plasma cholesterol concentration may be reduced. The
hypocholesterolemic effects of ileal bypass surgery or bile resin therapy
have been well documented in humans (101, 120, 123) as well as in
laboratory animals such as rabbits (121, 124) and hamsters (119, 125). These
treatments cause a reduction of 25-35% in plasma total cholesterol
concentrations in these species and the reduction occurs almost exclusively
in LDL cholesterol. Furthermore, because these treatments fail to reduce
plasma cholesterol in patients with homozygous hypercholesterolemia
(126), who lack functional LDL receptors (32), their hypocholesterolemic
effects appear to be entirely dependent on the ability of the liver to increase
its uptake of plasma cholesterol through the LDL receptor-mediated
pathway. In contrast to LDL, plasma VLDL concentration may increase in

response to these treatments in humans (101, 123, 127, 128) and rhesus

29

monkeys (129). This has been shown to occur as a result of an increased
VLDL production rate (127-129), although the mechanism is still poorly
understood. Likewise, HDL cholesterol concentration is not affected or is
slightly increased during bile resin therapy (63, 101) or following ileal
bypass surgery in humans (123). Nonetheless, there appears to be a
significant alteration in distribution of cholesterol among HDL
subfractions. These treatments have been shown to increase the plasma
concentration of HDL2 cholesterol with minimal effects on HDL3 (101, 123).
While the effect of treatment on HDL fractional rate of clearance is not
known, the production rate of HDL might be expected to increase as the
turnover rate of VLDL is known to be increased (127, 128). In addition, it
has been shown that intestinal production of nascent HDL particles is
enhanced during biliary diversion in the rat (130). Furthermore, the
esterification of cholesterol through the LCAT system also appears to be
induced by cholestyramine treatment in humans (101), leading to the
accumulation of cholesterol esters in HDL2.

Most of the previous studies on the effects of interruption of EHC were
focused on its effectiveness in lowering LDL cholesterol, the atherogenic
cholesterol in humans. Because of this focus, these studies have been on
species such as humans, rabbits and hamsters, in which LDL serves as the
major carrier of plasma cholesterol (16). Consequently, the understanding
of such effects on species with HDL as the predominant plasma lipoprotein
is very limited. However, it is known that in one of these species, i.e., the
rat, induction of fecal loss of biliary sterols does not result in appreciable
reductions in plasma cholesterol concentration (113, 118, 131). The reason
for this difference between the rat and the other species is not known.

However, the explanation might be found in the higher hepatic cholesterol

30

synthesis rate of the rat, compared to most other mammals (1, 2). This
would allow the rat to alter its rate of cholesterol synthesis over a very wide
range to adapt to changes in fecal loss of sterols (118). It is not known
whether interruption of EHC could reduce plasma cholesterol
concentration in other "HDL" mammalian species such as cattle. As noted
earlier, however, unlike the rat, cattle appear to have low rates of hepatic
cholesterol synthesis. Thus, it would be interesting to examine the efl'ects of
interrupting EHC on hepatic cholesterol metabolism and on plasma

cholesterol concentration in bovine species.

IV. IMPLICATIONS OF UNIQUE FEATURES OF PLASMA
CHOLESTEROL METABOLISM IN DAIRY COWS

Most comparative lipoprotein studies have been undertaken with the
objective of increasing our understanding of human hyperlipoproteinemia
and atherosclerotic disease processes. Research on bovine lipoprotein
metabolism, on the other hand, has been conducted primarily with the
objective of developing ways to improve productivity of ruminant animals.
With respect to dairy cattle, the major goals of lipoprotein investigations
have been related to either animal health or milk fat synthesis and
secretion. Although the understanding of bovine lipoprotein metabolism is
still incomplete, these previous studies have revealed some interesting
phenomena in bovine plasma lipoprotein cholesterol metabolism.
Knowledge of the underlying mechanisms for these phenomena would
provide insight into the role and regulation of plasma cholesterol in dairy

COWS.

31

A. Gestation-lactation cycle related changes in plasma
cholesterol concentration

The large fluctuation in plasma cholesterol concentrations that
occurs through the gestation-lactation cycle is a well documented
phenomenon in dairy cattle. As early as in 1931, Maynard and co-workers
(132) demonstrated that blood lipids decrease prior to parturition and then
begin to increase again during lactation. Of all the lipid classes, the
greatest changes and the highest concentrations were in serum total
cholesterol. Since then, this finding has been corroborated frequently by
other investigators (8-10, 133-137). Typically, the maximum cholesterol
concentration occurs about midway during lactation and the minimum
concentration at or near parturition, with a two-three fold difference
between these two values. The decline in plasma cholesterol concentration
during the peripartum period is not peculiar to dairy cows since some other
species, including guinea pigs (138), non-human primates (139) and rabbits
(140) also undergo a marked reduction in serum cholesterol concentration
in the later stages of pregnancy. However, the large increase in plasma

cholesterol during lactation appears to be unique to cattle.

B. Possible mechanisms for the lactational hyperalphalipoproteinemia

The lactational hypercholesterolemia of cattle is due to a marked
increase in HDL, occurring mainly in the cholesterol ester-enriched, large
HDL-particle fi'action (6, 19, 134). Thus, it can be generally considered as
hyperalphalipoproteinemia. The underlying mechanism(s) has not been
examined. However, as discussed earlier, the formation of large HDL
particles in other animal species is known to be related to triglyceride
transport activity. Plasma triglyceride-rich lipoproteins are important

32

sources of triglyceride for milk fat synthesis (29, 141). During lactation, the
mammary lipoprotein lipase activity is elevated (142), leading to extensive
lipolysis of triglyceride-rich lipoproteins by the mammary tissue. Moreover,
the production rate of triglyceride-rich lipoproteins by the intestine might
also be largely increased during midlactation when feed intake has peaked.
When combined, the increased rates of lipolysis and triglyceride-rich
lipoprotein secretion would result in an increased rate of lipoprotein
turnover. Surface remnants of these lipoproteins would provide cholesterol
substrate for the LCAT system, as well as other components for HDL
formation. In addition, as the adipose tissue is depleted during early
lactation, efflux of cholesterol from depot stores into the blood provides
additional cholesterol for HDL cholesterol ester synthesis through the
LCAT reaction.

While the rate of formation of the cholesterol ester-enriched large
HDL particles is increased during lactation, cattle appear to lack the ability
to efficiently metabolize these particles. This is because plasma cholesterol
ester transfer activity and hepatic lipase are almost absent in the bovine
species (46, 53, 54). As noted earlier, both of these two factors play a critical
role in the conversion of large HDL particles into smaller ones for
metabolism. Moreover, it has been shown in humans (143) and other
species (14) that the fractional catabolic rate of HDL-apoproteins is inversely
correlated with particle size and the ratio of cholesterol esters/(apo Al + apo
AII), implicating that the removal rate of the large bovine HDL particles
might be very slow. Taken together, the marked increase in HDL-
cholesterol during lactation appears to be the result of an active lipolysis
process combined with the relative low capacity of the ruminant liver to

dispose of remnant particles and their metabolites. Nonetheless, the

33

accumulation of these large HDL particles might serve as a reservoir of
apoCII for activating lipoprotein lipase and also of essential fatty acids. In
ruminants, essential fatty acids are stored in plasma mainly in form of

phospholipids and cholesterol esters (65).

C. Peripartum hypocholesterolemia and its association with
cow diseases

The mechanism(s) responsible for the fall of plasma cholesterol
during the peripartum period is still poorly understood. Feed intake is
usually reduced shortly before parturition and during early lactation.
Presumably, the intestinal production of triglyceride-rich lipoproteins and
thus, cholesterol synthesis is reduced in association with the depressed feed
intake. This in turn results in a diminished entry rate of cholesterol into
the plasma pool as the intestine is the major contributor of plasma
triglyceride-rich lipoproteins in cattle (26). On the other hand, the removal
of blood cholesterol might be increased as a result of the formation of
colostrum. Plasma cholesterol has been shown to be an important source of
milk cholesterol in several species (144, 145) including ruminants (146).
Bovine colostrum is enriched with cholesterol, phospholipids and
B-carotene but not with triglycerides (137, 147), as compared to mature
milk, suggesting the possible mammary uptake of LDL or/and HDL
particles. Although this does not appear to have been experimentally
proven, it is unlikely that mammary tissues lack the lipoprotein receptors
as both LDL receptor and HDL receptor appear to be ubiquitously distributed
among mammalian cells.

Concomitant with the peripartum fall in plasma cholesterol

concentration, there is a sharp increase in the plasma concentration of

34

non-esterified fatty acids. A number of studies (148- 150) have demonstrated
that plasma cholesterol ester transfer activity can be activated in vitro by
free fatty acid. Although this activity is low in normal bovine plasma (46), it
has yet to be determined whether the activity is increased by non-esterified
fatty acids during the peripartum period. If this is the case, this activity
might mediate the transfer of cholesterol esters from HDL to LDL, leading
to an enhanced catabolic rate of HDL cholesterol via the LDL receptor-
mediated pathway.

While the mechanism for the regulation of bovine plasma cholesterol
concentration has yet to be clarified, previous studies have indicated that
the degree of reduction in plasma cholesterol concentration during the
peripartum period is associated with morbidity and mortality in dairy cattle
(7, 151, 152). The nature of the association between blood cholesterol and
disease incidence is not clear, but blood cholesterol concentration and
hepatic fat concentration are inversely related in peripartum cows (7 -10,
152). It is generally assumed that fatty liver has a direct adverse efl'ect on
health and that hypocholesterolemia occurs secondary to reduced hepatic
lipoprotein secretion (10). It is possible, however, that there may be
additional, direct effects of hypocholesterolemia on -cow health. For
example, it has been shown that lipoproteins can exert a modulating effect
on immune system components (153-155). While any direct effect of
hypocholesterolemia on immunity in dairy cows is speculative, it is known
in cows that both the phagocytic and lymphocytic arms of the immune
system are suppressed during the peripartum period, the same period
during which blood cholesterol concentrations are at their lowest.
Moreover, it is very clear that life-threatening disease in dairy cows is also

most prevalent in the peripartum, hypocholesterolemic period (156). Thus,

35

it appears that a clearer understanding of cholesterol metabolism in dairy
cows may lead to a better appreciation of the mechanisms of some
peripartum diseases.

In addition to possible effects on disease incidence, it has also been
suggested that reduced plasma cholesterol concentration might impair the
reproductive performance of the cow (7-9). This may be due to a reduction in
the synthesis of sex steroid hormones such as progesterone from the corpus
luteum of the ovary. Plasma lipoprotein-bound cholesterol is an important
source of cholesterol for cells in general and specifically for steroidogenic
tissues. Cholesterol is the precursor for all steroid hormones, including
progesterone. Although steroidogenic cells are capable of de novo
cholesterol synthesis, they rely on the uptake of lip0protein cholesterol to
maintain physiological levels of steroid hormone production. This has been
demonstrated by numerous in vitro studies using luteal or granulosa cells
from bovine (157-159) or other souraas (3, 5, 160). Similarly, an impairment
in the supply of plasma cholesterol to steroidogenic tissues in vivo has been
shown to result in a lower production of steroid hormone in rats (5) and in
humans (4). Thus, the reduced peripartum plasma cholesterol
concentration in the cow might limit the supply of cholesterol for
progesterone synthesis, leading to impaired reproductive performance.
Moreover, recent studies have also suggested that plasma lipoprotein
supply is involved in embryo development (161-163). A reduced
concentration of serum cholesterol appears to be associated with low
embryo recovery from superovulated cows (162, 163). Thus, in addition to its
role in steroidogenesis, plasma cholesterol might also play some other

important roles in the reproductive system of the animal.

36

It is apparent that a more complete understanding of the role of
plasma cholesterol in ruminant metabolism will help us develop strategies
to improve productivity of the cow. An experimental technique of
modulating blood cholesterol is critical to the design of in viva experiments
with which to study the role of plasma cholesterol in ruminant metabolism.
However, such a technique is not available at the present time. Since
interruption of EHC has been shown to produce reversible reduction in
blood cholesterol concentration in a number of animal species, it was the
first objective of the following studies to examine the possibility of using this
method to modify blood cholesterol levels in dairy cows. The second objective
was to determine the effects of interruption of EHC on bovine hepatic
cholesterol metabolism. The elucidation of these effects might provide
valuable information for understanding of the role of ruminant liver in

regulating plasma lipoprotein cholesterol metabolism.

3 7
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135. Kweon, O. H., H. Ono, K. Osasa, M. Onda, K. Oboshi, H. Uchisugi, S.
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138. Li, R. R., L. K. Bale, M. T. R. Subbiah. 1978. Bile acid and cholesterol
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139. Martin, D. E., R. C. Wolf, and R. K. Meyer. 1971. Plasma lipid during
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141. Emery, R. S. 1973. Biosynthesis of milk fat. J. Dairy Sci. 56:1187-1195.

142. Liesman, J. S., R. S. Emery, R. M. Akers, and H. A. Tucker. 1988.
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143. Brinton, E. A., S. Eisenberg, and J. L. Breslow. 1994. Human HDL
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147. Bitman, J ., and D. L. Wood. 1990. Changes in milk fat phospholipids
during lactation. J. Dairy Sci. 73:1208-1216.

50

148. Lagrost, L., and P. J. Bater. 1991. Effects of various non-esterified fatty
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by the cholesteryl ester transfer protein. Biochim. Biophys. Acta.
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149. Lagrost, L., and P. J. Bater. 1991. Effects of various non-esterified fatty
acids on the particle size redistribution of high density lipoproteins

induced by the human cholesteryl ester transfer protein. Biochim.
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150. Sammett D., and A. R. Tall. 1985. Mechanisms of enhancement of
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151. Kweon, O. K., H. Ono, T. Seta, M. Onda, K. Oboshi, and H. Kanagawa.
1985. Relationship between serum total cholesterol levels before
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152. Van Dijk, 8., Th. Wensing, and G. H. Jornna. 1989. Hepatic lipidosis in
dairy cows related to health and fertility. Proceeding, 7th
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153. Edgington, T. S., and L. K. Curtiss. 1981. Plasma lipoproteins with
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154. Cuthbert, J. A., and P. E. Lipsky. 1987. Provision of cholesterol to
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155. Jurgens, G., Q. B. Xu, L. A. Huber, G. Block, H. Howanietz, G. Wick,
and K. N. Traill. 1989. Promotion of lymphocyte growth by high
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156. Erle, H. N., R. D. Smith, R. B. Hillman, P. A. Powers, M. C. Smith, M.
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Res. 45:333-335.

157. Savion, N ., R. Laherty, D. Cohen, G. M. Lui, and D. Gospodarowicz.
1982. Role of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme
A reductase in progesterone production by cultured bovine
granulosa cells. Endocrinology. 110:13-22.

51

158. O'Shaughnessy, P. J ., and D. C. Wathes. 1985. Role of lipoproteins and
de novo cholesterol synthesis in progesterone production by cultured
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production in newly isolated cells and during cell culture. J.
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160. Azhar, S., M. Menon, and K. M. J. Menon. 1981. Receptor-mediated
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665:362-375.

161. Henault, M.A., and G.J. Killian. 1993. Composition and morphology of
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466-474.

162. Schafer, M., T. T. Hien, S. Paarmann, and G. Kramer. 1990.
Relationship of metabolic status with embryo recovery in high-
yielding cows. (Beziehungen zwischen Stoffwechselstatus und
Ergebnis der Embryogewinnung beiHochleistungskuhen) Archiv
fur Experimentelle Veterinarmedizin. 44 : 157-162.

163. Kweon, O. K., H. Kanagawa, and H. Yamashina. 1987 . Relationship
between serum total cholesterol level and the number of transferable
embryos in relation to milk yield in superovulated cows. Jpn. J. Vet.
Res. 35: 73-78.

CHAPTERII

PARTIAL DIVERSION OF BILE DRAMATICALLY REDUCES PLASMA
CHOLESTEROL CONCENTRATION IN CATTLE

INTRODUCTION

Hepatobiliary excretion of cholesterol and its major metabolites, the
bile acids, is the key route for the elimination of cholesterol from the body.
Such excretion thereby plays an important role in regulating body
cholesterol metabolism. Under normal conditions, more than 95% of the
bile salts secreted by the liver are reabsorbed in the ileum by an active
transport mechanism (1) and return to the liver via the hepatic portal vein
(2, 3). To maintain a steady state, therefore, the liver is normally required to
synthesize only small amounts of bile salts to replace the fecal loss (4).
However, when the enterohepatic circulation (EHC) is interrupted, either
by direct biliary drainage (5,6), or by inducing fecal loss of bile sterols by
means of bile acid sequestrants (7, 8) or ileal bypass (7, 9), the rate of bile
acid synthesis is augmented (5, 7, 10). The increase in bile acid synthesis in
turn leads to an enhanced uptake and catabolism of plasma lipoprotein
cholesterol by the liver (7, 10, 11). As a result of this, plasma cholesterol
concentration is usually reduced (6-12). The impact of interruption of EHC
on cholesterol metabolism has been investigated in a number of species (6-
12), yet similar information has not been available for ruminant animals,
including cattle.

Like most of the mammals, the majority of the circulating cholesterol
in cattle is carried by high-density lipoprotein (HDL) (13, 14). However,
unlike other species, in which circulating cholesterol levels are normally

52

53

maintained within a relative narrow range through different physiological
stages, plasma cholesterol concentration in cattle undergoes considerable
fluctuations during the gestation-lactation cycle (14-16). The mechanisms
regulating these large changes in plasma cholesterol are not completely
understood. Nonetheless, the degree of peripartum reduction in plasma
cholesterol concentration has been shown to be associated with metabolic
diseases of the cow (17-19). To understand the role of plasma cholesterol in
ruminant metabolism, we attempted to use interruption of EHC as a tool to
study the metabolic effect of reduced plasma cholesterol concentration in
cattle. .

The objective of this study was to determine and characterize the
effects of controlled biliary diversion and subsequent restoration of EHC on
plasma concentrations of cholesterol and other lipids in cows.
Interestingly, we found that partial diversion of bile caused a reduction of
more than 90% in plasma concentrations of cholesterol and other lipids.
This study presents a unique animal model with which to investigate the
metabolic role of plasma lipoprotein cholesterol.

MATERIALS AND METHODS

Animals and diet

Five healthy adult, nonpregnant, nonlactating Holstein cows were
used. They were kept under environmentally controlled conditions
(temperature maintained at 20 C) in individual pens. Throughout the entire
experimental period, the animals had free access to alfalfa hay, water and
trace-mineralized salt. The diet was supplemented daily with four pounds
of concentrate containing ground corn, oats, and vitamins. The animals

were weighed weekly.

5 4

mm

The cows were surgically prepared and fitted with reentrant
intestinal cannula using a modification of the procedure of Symonds et a1
(20). Surgical anesthesia was induced by intravenous administration of
guaifenesin and sodium thioamylal and maintained by inhalation of
halothane. Under general anesthesia and using aseptic surgical technique,
a 20-cm section of duodenum that included the insertion of the common bile
duct was transected at each end. The ends were then closed to form an
isolated segment. The remaining portions of the duodenum were then
joined to reestablish the patency of the intestine. A reentrant intestinal
cannula ( Figure 4 ) was placed with one arm in the isolated intestinal
segment and the other in the intact duodenum. Through this cannula
flowed all biliary secretions. When required, controlled portions of bile flaw
could be diverted from the body ( Figure 4 ).

 

r N

"T" Coyector ,7” Body wall
,1

Bile duct

Isolated segment
of duodenum

 

 

 

 

 

 

 

 

Figure 4: Diagram of surgical modification to the duodenum.

5 5

During the first 5 days following the surgery, to minimize the
internal pressure in the isolated segment of intestine, the reentrant
cannula was interrupted and all of the bile flow was diverted from the
isolated segment into a reservoir flask. Bile was infused to the cow, as it
was collected, through the cannula arm connected to the intact duodenum
using a peristaltic pump ( Model 1210; Harvard Apparatus Inc., South
Natick, MA ). Infusion rates were adjusted occasionally so that only a
small amount of bile accumulated in the flask. Cows were given
oxytetracycline ( 3 gm ) through the intestinal cannula twice a day during
this period. Thereafter, the reentrant intestinal cannula was reestablished
and biliary secretions flowed directly into the intact duodenum via the
cannula. A one-way valve ( Figure 4 ) was used to prevent back-flow of
ingesta into the cannula and isolated intestinal segment. Bile flow through
the cannula was checked at least four times a day and the cannula flushed
‘ once a day with physiological saline. Cows were allowed at least a 4-week
recovery period before any experiments were started.

After the recovery period, to obtain baseline values of bile flow and
total bile acid concentration and to examine the diurnal variations of these
values, rates of bile flow were measured and bile samples collected. For this
purpose, the reentrant cannula was closed and all bile collected in a flask.
At 30-min intervals, bile volume was measured and 3-ml aliquots were
taken. Collected bile was then infused into the duodenum at approximately
the same rate as it was collected. Bile flow measurements were carried out
for a period of 24 hours.

Twenty-four hours following the completion of bile flow rate

measurement, bile diversion experiments commenced. Two bile diversion

56

periods were studied. Initially, 50% of bile flow was diverted for 15 days.
This was followed by a 21-day period of no bile diversion and completed by a
15-day period of 22% bile diversion. During the periods of bile diversion, the
reentrant cannula was interrupted and all bile collected into a flask. Bile
was aspirated from the collection flask into two tubings, each connected to
the same peristaltic pump ( Model 1210; Harvard Apparatus Inc., South
Natick, MA ). Thus, the same rate of peristaltic motion was applied to each
tubing. One tube was connected to the duodenal arm of the cannula and the
other to a discard flask. During periods of 50% bile diversion, tubings of
equal diameter were used, while during periods of 22% bile diversion, a
discard tubing with one third the cross-sectional area of the reinfusion
tubing was selected. The pumping rate was adjusted so that there was
always a small amount of bile in the collection flask.

The volume of diverted bile was measured twice a day. A fresh bile
sample ( 2-3 ml ) was collected daily and stored at -20 C until analyzed. A
coccygeal-vessel blood sample was collected into an EDTA-treated tube
between 10-12:00 am daily throughout the experiment. Plasma was
immediately collected by centrifugation and stored at -20 C. When required,
a large volume ( 30 ml ) of plasma was collected and stored at 4 C for
lipoprotein isolation.

Biochemicalanahses

Total cholesterol was measured enzymatically using a reagent kit
(Sigma, St. Louis, MO). Plasma concentrations of phospholipids were
determined by an enzymatic method ( WAKO Chemical Ltd., Dallas, TX ).
Plasma triglycerides were measured by a colorimetric procedure using a

triglyceride reagent kit ( Sigma, St. Louis, MO ). Lipoproteins were isolated

57

by discontinuous density-gradient ultracentrifugation in a vertical-tube
rotor as described previously (21). Total bile acids in bile were enzymatically
determined with a reagent kit ( Sigma, St. Louis, MO ).

Calculations and statistics

Bile flow rates were expressed in milliliters per 100 kg body weight
per h. Output rates of total bile acids were determined by multiplying bile
flow by bile acid concentration. Plasma lipid values were expressed in
milligrams per deciliter. Values are presented as means :1; S.E.M. The data
were analyzed by analysis of variance and the significance of difference

determined by appropriate t test, at a P<0.05 level of significance.

RESULTS

lil'l' [IE . llllll
The animals were healthy and maintained constant body weights

throughout the entire study. Plasma albumin and bilirubin concentrations
were normal, indicating that hepatic protein synthesis and excretion
functions were not impaired. During the first four days after the surgery,
blood cholesterol concentrations were decreased by 20%, compared to
samples collected before surgery, but they returned to near the pre-surgical
values by the end of the first post-surgical week. When no bile was diverted,
the rates of bile flow and total bile acid secretion were nearly constant
throughout a 24 h period ( Figure 5 ). This is consistent with the pattern of
continuous bile secretion which is typical of the ruminant animal (20, 22).

The average bile flow rate was 132 ml/100 kg body weight/h, which is

58

comparable to the values previously reported on adult cattle (20). These
results suggested that a normal EHC was maintained in these cows.

In addition, partial bile diversion did not appear to have any negative
effect on feed intake and general health of the animals.

i' .- . 0101.10.31 03"501 u ' ' Lo. on o.‘ 1.! ~- 1' or

To examine the effects of interruption of EHC on plasma lipids, we
initially diverted 50% of bile flow from the animals for a period of 15 days. In
response to this bile diversion treatment, both bile flow and biliary bile acid
secretion dropped sharply during the first 24 h and then stabilized at 30%
and 8%, respectively, of their initial values. The values normalized after the
termination of bile diversion ( Figure 6 ). Total bile acid concentrations in
the bile were also reduced by several fold during bile diversion (data not
shown), thus the dramatic decline in bile acid output rate occurred as a
result of the combined effects of a reduced bile flow rate and the lowered
concentration of bile acids.

It has been previously reported that the rhesus monkey can increase
its bile acid synthesis rate to compensate for the loss of biliary sterols when
up to 24 % of the bile is diverted (5). To compare cattle to this monogastric
species, we have also examined the effects of 22% bile diversion on bile flow
and bile acid secretion in cows. As shown by Figure 6, diversion of 22% bile
flow caused a remarkable decrease in the rates of bile flow and bile acid
output in a manner comparable to 50% bile diversion. The reduction in bile
and bile acid secretion rates was only slightly greater when 50% of bile was
diverted, as compared to 22% diversion. These data suggest that bovine liver
has a smaller capacity than the rhesus monkey to increase its bile acid

synthesis in response to the induced loss of biliary sterols.

59

A130; A
“160;

ml/ 00

Bile flow

 

 

Blle acids (mmoIe/100 kg per h)

 

 

I I I I l 1 T U l I I I U r fitfir I

0 4 s 12 1 116 ' 20 24
Secretion period (h)

Figure 5: Diurnal changes in rates of bile flow (A) and bile acid secretion (B). Values
represent means 1"- SEM (n=5).

6O

lzuummonl intact EHC j

c
H
E
m
m

 

 

 

 

 

 

 

 

 

 

 

 

Experimental days

Figure 6: Effects of controlled biliary diversion on rates of bile flow (A) and bile acid

output (B). Values represent means 1’ SEM (n=5).

61
E55 5 ll ”I l. . l [.1

As a result of bile diversion, plasma total cholesterol concentration
decreased dramatically, reaching a stable level 8 days after bile diversion
(Figure 7). Based on the stabilized levels, diversion of 50% bile flow caused a
significantly greater reduction than 22% bile diversion ( 91% vs. 78%;
P<0.001, n=5 ). When the bile diversion protocols were terminated, however,
plasma cholesterol concentrations returned towards the control values
rapidly. In no other species has interruption of EHC been shown to result in
such a dramatic reduction in plasma cholesterol.

In addition to the decrease in blood cholesterol, partial bile diversion
also resulted in a dramatic decline in plasma phospholipids and
triglycerides. The changes in plasma phospholipid concentration followed a
pattern similar to that of cholesterol ( Figure 8 ). However, the difi'erences
in plasma phospholipid concentrations between the 50% and 22% bile
diversion periods were not significant ( P>0.05 ). In addition, it appeared
that concentration of plasma phospholipids decreased more rapidly than
that of plasma cholesterol.

Plasma triglyceride concentrations dropped sharply after the
initiation of bile diversion and stabilized within 24 h thereafter ( Figure 9 ).
This is in contrast to plasma cholesterol and phospholipid concentrations
which did not drop so rapidly. When steady state was reached, both bile
diversion procedures caused a 70% reduction in triglycerides compared to
pre-diversion values. The rapid declines in plasma triglycerides and in bile
acid secretion appeared to occur in a coordinated manner. This suggests
that the dramatic drop in plasma triglycerides observed in this study might
be due to the reduced availability of bile acids for lipid absorption by the
small intestine.

62

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To further characterize the effects of partial bile diversion on plasma
cholesterol metabolism, we determined the cholesterol concentrations in
various plasma lipoprotein fractions. Plasma samples were collected at 0 h,
12 h, and 14 days of bile diversion, and lipoproteins were isolated and
cholesterol concentrations in the lipoprotein fractions were determined.
The results are shown in Table 4. At 12 hours after bile diversion, VLDL
cholesterol and LDL cholesterol were significantly ( P<0.01 ) reduced by
about 50% for both bile diversion procedures, whereas there were no
significant changes in HDL cholesterol ( Table 4 ). These data demonstrate
that, in response to bile diversion, HDL cholesterol declined less rapidly
than VLDL- and LDL-cholesterol, possibly due to the low turnover rate of
HDL, compared to other lipoproteins. By day 14 of bile diversion, however,
HDL cholesterol was reduced by 90% and 85% for 50% diversion and 22%
diversion, respectively. The corresponding LDL cholesterol and VLDL
cholesterol concentrations were reduced by an extent comparable to that of
HDL. Thus, distribution of cholesterol among different lipoproteins on day
14 of bile diversion was not different from initial values ( Table 4 ).

DISCUSSION

The role of EHC in determining cholesterol homeostasis and the
effects of its interruption on plasma cholesterol metabolism have been well
documented in humans and a number of other animal species (6-12). No
similar study has been reported on ruminant animals. The present study
has characterized the changes in plasma cholesterol and other lipids
during chronic diversion of bile in cattle. This study demonstrates that

66

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67

interruption of EHC dramatically influences plasma cholesterol
concentration in this species.

The effects of EHC interruption on plasma cholesterol observed in
this study are unprecedented. A greater than 90% reduction in plasma
cholesterol concentration was achieved in our animal model by partial bile
diversion, whereas only moderate reductions have been reported in other
species (5, 6, 8, 12), including human (7 , 9), in response to either complete
or partial interruption of EHC. The dramatic change in blood lipids was not
due to the surgical modification of the animals per se since the blood
cholesterol levels and other lipid concentrations normalized when the
animals recovered from the surgery. Furthermore, these animals
maintained a normal bile secretion pattern and bile acid secretion rate
during the control periods after surgery, implicating that EHC was
functional. While both 22% and 50% bile diversion caused a dramatic drop
in plasma cholesterol, diversion of 50% bile produced a significantly greater
reduction in blood cholesterol than 22% bile diversion. This indicates that
the response in plasma cholesterol concentration is dependent on the
amount of bile diverted. In addition to plasma cholesterol concentration,
plasma concentrations of triglycerides and phospholipids were also
reduced greatly after bile diversion. This also appears to be unique for the
ruminant animal since no significant reduction in these two plasma lipids
has been observed in other species. In contrast, it has been shown in a
number of monogastric species that interruption of EHC causes an
increase in plasma triglyceride concentration (8,11). All of these changes in
blood cholesterol, phospholipids, and triglycerides were reversible since
plasma concentrations of these lipids returned to normal values after the

termination of bile diversion.

68

Because HDL-cholesterol accounts for more than 80% of plasma
cholesterol in cattle, the dramatic decrease in total cholesterol
concentration mainly depends on the reduction in HDL cholesterol. Thus,
although VLDL- and LDL-cholesterol were reduced by more than 50%
during the first 12 h of bile diversion, only a minor decline was observed in
plasma total cholesterol because HDL-cholesterol did not change
significantly during this period. Plasma concentration of total cholesterol
did not reach a new steady-state level until a week after the initiation of bile
diVersion, suggesting a low clearance rate of HDL. This might be due to at
least in part the low level of plasma cholesterol ester transfer protein in this
species (23), which plays an important role in the transport of HDL
cholesterol to the liver ( 24). A recent study has shown that a genetic defect
in this transport protein in human causes remarkably delayed catabolism
of HDL without affecting its production rate (25). Furthermore, apoprotein
E, which is present in more than 50% of the HDL particles in other species
including rats (26), does not appear to exist in bovine HDL (14). As HDL-
associated apoE functions to mediate the rapid clearance of HDL cholesterol
by the liver (26), the lack of this apolipoprotein in HDL would be expected to
further delay hepatic clearance of HDL-cholesterol in bovine plasma.

While partial bile diversion remarkably reduced HDL-cholesterol in
our animal model, a similar observation has not been reported for other
species. In species such as humans, in which LDL is the major carrier of
plasma cholesterol (13), interruption of EHC mainly reduces LDL-
cholesterol while concomitantly causing a slight increase in HDL-
cholesterol (9, 11). On the other hand, in the high HDL-cholesterol species
such as rats (13), bile diversion only produces a small drop in plasma total
cholesterol (6, 10), suggesting that HDL-cholesterol was not affected

69

significantly. It is not known whether the dramatic decline in HDL-
cholesterol in our model is due to an increase in HDL clearance rate or a
decrease in HDL synthesis or both. Nonetheless, it has been shown in the
rat that biliary diversion does not affect intestinal HDL secretion (27).
However, a full understanding of HDL-cholesterol metabolism in our
animal model requires detailed kinetic studies.

In addition to the changes in plasma lipids, both 22% and 50% bile
diversion sharply decreased bile flow rate and bile acid secretion. The
decrease in the availability of bile acids for the absorption of dietary lipids by
the small intestine might be the primary mechanism for the rapid decline
in plasma triglyceride concentration in response to bile diversion. This is
because triglycerides which are absorbed by the small intestine and
secreted into the blood as chylomicrons are the major source of circulating
triglycerides in ruminant animals (28, 29). In fact, previous studies have
shown that bile diversion greatly impaired the secretion of chylomicrons in
rats (27, 30). The reduced intestinal secretion of triglyceride-rich lipoprotein
may lead to decreased intestinal cholesterol synthesis and secretion. This
would also contribute to the hypocholesterolemic effect of bile diversion.

The exact mechanism responsible for the unprecedented
hypocholesterolemic effects of interruption of EHC in cattle observed in this
study remains to be elucidated. Nonetheless, because the dramatic
reduction in plasma cholesterol is reversible and because the degree of
reduction appears to be proportional to the amount of bile diverted from the
animal, this animal model might be of important value for the study of the
role of plasma cholesterol in ruminant metabolism. In addition, no special
drug treatment was required for this experimental technique, further
signifying the usefulness of this animal model. Although bile acid

70

sequestrants can be used in a number of monogastric animals to provide an
alternative means for the surgical approach to interrupt EHC by inducing
fecal loss of bile sterols (7, 8, 11), these agents do not seem to be effective in
ruminant animals. A previous study (31) has shown that feeding of a bile
acid sequestrant to cows failed to produce any influences on fecal excretion
of lipid and on blood cholesterol and other blood lipid concentrations. The
intestinal concentration of the sequestrant in cattle might be too low for
effective action, due to the large mass of ingesta in the ruminant digestive
tract compared to monogastric animals. Thus, it appears that surgical
intervention is a necessary approach to interrupt the EHC in ruminants.

In summary, the present study, for the first time, shows that plasma
concentrations of cholesterol and other lipids in cattle responded to partial
bile diversion with a dramatic reduction. Since no pharmacological
treatments were involved and the animals maintained a healthy condition
during bile diversion, this animal model provides excellent opportunities to
further understanding of the regulation of plasma lipoprotein
concentrations, as well as the metabolic role of circulating cholesterol in

animals.

7 1
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lipid Res. 9: 297-309, 1968.

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Med. 21: 501-534, 1976.

. Small, D. M., R. H. Dowling, and R. N. Redinger. The enterohepatic
circulation of bile salts. Arch. Intern. Med. 130:552-573, 1972.

. Carey M. C., and M. J. Cahalane. Enterohepatic circulation. In: Arias I.
M., W. B. Jakoby, H. Popper, G. Schacter, and D. A. Shafritz (eds)

The liver: biology and pathobiology. Raven Press, New York, pp573-
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. Dowling, R. H., E. Mack, D'. M. Small, and J. Picott. Effects of controlled
interruption of the enterohepatic circulation of bile salts by biliary

diversion and by ileal resection on bile salt secretion, synthesis and
pool size in the rhesus monkey. J. Clin. Invest. 49:232-242, 1970.

. Kuipers, F., R. Havinga, H. Bosschieter, G. P. Toorop, F. R. Hindriks,
and R. J. Vonk. Enterohepatic circulation in the rat.
Gastroenterology 88:403-11, 1985.

. Grundy, S. M., E. H. Ahrens, Jr, and G. Salen. Interruption of the
enterohepatic circulation of bile acids in man; comparative effects of
cholestyramine and ileal exclusion on cholesterol metabolism. J.
Lab. Clin. Med. 78:94-121, 1971.

. Parkinson, T. M., K. Gunderson, and N. A. Nelson. Effects of Colestipol
(U-26597 A), a new bile acid sequestrant, on serum lipids in
experimental animals and man. Atherosclerosis 11:531-537, 1970.

. Moore, R. B., I. D. Frantz, Jr., and H. Buchwald. Changes in cholesterol
pool size, turnover rate, and fecal bile acid and sterol excretion after
partial ileal bypass in hypercholesterolemic patients. Surgery 65:98-
108, 1969.

10. Smit, M. J., A. M. Temmerman, R. Havinga, F. Kuipers, and R. J.

Vonk. Short— and long-term effects of biliary drainage on hepatic
cholesterol metabolism in the rat. Biochem. J. 269:7 81-788, 1990.

11. Packard, C. J ., and J. Shepherd. The hepatobiliary axis and lipoprotein

metabolism: efl‘ects of bile acid sequestrants and ileal bypass surgery.
J. Lipid Res. 23:1081—1098, 1982.

12. Trezzi, E., G. Maione, U. Fox, and A. L. Catapano. Effects of partial

ileal bypass on plasma clearance and binding of lipoproteins to liver

13.

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membrane in the rabbit. Atherosclerosis 46:269-273, 1983.

Champman, M. J. Comparative analysis of mammalian plasma
lipoproteins. Plasma lipoproteins: preparation, structure and
molecular biology. Methods Enzymol. 128:70— 143, 1986.

Puppione, D. L. Bovine lipoproteins. In: L. A. Lewis and H. K. Naito
(eds) CRC Handbook of Electrophoresis. Vol. IV. Lipoprotein studies
of non-human species. CRC Press Inc, Boca Raton, FL. pp185-202,
1983.

Raphael, B. C., P. S. Dimick, and D. L. Puppione. Lipid characterization
of bovine serum lipoproteins throughout gestation and lactation. J.
Dairy Sci. 56: 1025- 1032, 1973.

Schweigert, F. J. Effects of gestation and lactation on lipoprotein pattern
and composition in dairy cows. J. Anim. Physiol. a. Anim. Nutr.
63:75-83, 1990.

Gerloff, B. J ., T. H. Herdt, and R. S. Emery. Relationship of hepatic
lipidosis to health and performance in dairy cattle. J. Am. Vet. Med.
Assoc. 188:845-850, 1986.

Kappel, L. C., R. H. Ingraham, E. B. Morgan, L. Zeringue, D. Wilson,
D. K. Babcock, and M. A. Stat. Relationship between fertility and
blood glucose and cholesterol concentrations in Holstein cows. Am.
J. Vet. Res. 45:2607-2612, 1984.

Ruegg, P. L., W. J. Goodger, C. A. Holmberg, L. D. Weaver, and E. M.
Huffman. Relation among body condition score, serum urea nitrogen
and cholesterol concentrations, and reproductive performance in
high-producing Holstein diary cows in early lactation. Am. J. Vet.
Res. 53:10-14, 1992.

Symonds, H. W., D. L. Matter, and E. D. Hall. Surgical procedure for
modifying the duodenum in cattle to measure bile flow and the

diurnal variation in biliary manganese, iron, copper and zinc
excretion. Res. Vet. Sci. 32:6-11, 1982.

Cheung, B. H., T. Wilkinson, J. C. Geer, and J. P. Segrest. Preparative
and quantitative isolation of plasma lipoproteins: rapid, single
discontinuous density gradient ultracentrifugation in a vertical
rotor. J. Lipid Res. 121:284-290, 1980.

. Harrison, F. A. Bile secretion in sheep. J. Physiol. 162:212-224, 1962.

Ha, Y. C., and P. J. Barter. Differences in plasma cholesterol ester
transfer activity in sixteen vertebrate species. Comp. Bioch. Physiol.
71 B:265-269, 1982.

24

25

26

27

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. Tall, A. R. Plasma lipid transfer proteins. J. Lipid Res. 27:361-367, 1986.

. Ikewaki, K., D. J. Rader, T. Sakamoto, M. Nishiwaki, N. Wakimoto, J.
R. Schaefer, T. Ishiwaki, T. Fairwell, L. A. Zech, H. Nakamura, M.
Nagano, and H. B. Brewer, Jr. Delayed catabolism of high density
lipoprotein apolipoproteins A-1 and A-II in human cholesterol ester
transfer protein deficiency. J. Clin. Invest. 92:1650- 1658, 1993.

. Mahley, R. W., T. L. Innerarity, S. C. Rall, Jr., and K. H. Weisgraber.
Plasma lipoproteins: apolipoproteins structure and function. J. Lipid
Res. 25:1277-1294, 1984.

. Beamot, H. R., R. M. Glickman, L. Weinberg, and P. H. R. Green.
Efi'ects of biliary diversion on rat mesenteric lymph apolipoprotein-I
and high density lipoprotein. J. Clin. Invest. 69:210-217, 1982.

. Pullen, D. L., J. S. Liesman, and R. S. Emery. A species comparison of
liver slice synthesis and secretion of triacylglycerol from
nonesterified fatty acids in media. J. Anim. Sci. 68:1395-1399, 1990.

. Emery, R. S., J. S. Liesman, and T. H. Herdt. Metabolism of long chain
fatty acids by ruminant liver. J. Nutr. 122:832-837, 1992.

. Davidson, N 0., M. E. Kollmer, and R. M. Glikman. Apolipoprotein B
synthesis in rat small intestine. Regulation by dietary triglyceride
and biliary lipid. J. Lipid Res. 27:30-39, 1986.

. Wrenn, T. R., J. Bitman, J. R. Weyant, R. W. Miller, and L. P. Dryden.
Failure of DEAE Sephadex feeding to lower blood and milk lipids of
cows. J. Diary Sci. 55:1597-1601, 1972.

CHAPTERIII

INHERENTLY LOW RATE OF CHOLESTEROL SYNTHESIS IN THE
LIVER EXAGGERATES THE HYPOCHOLESTEROLEMIC EFFECTS OF
PARTIAL BILE DIVERSION IN CA'I'I‘LE

INTRODUCTION

The liver plays a central role in the regulation of plasma cholesterol
concentration. Input of cholesterol into the plasma pool can come from the
secretion of hepatically derived plasma lipoproteins, especially very-low-
density lipoprotein (VLDL) (1). Cholesterol for lipoproteins can be derived
from its de novo synthesis by the liver (1, 2), and, in most species, the liver is
the major site of cholesterol production (3, 4). On the other hand, the liver is
also the organ primarily responsible for the clearance of plasma
lipoproteins and the catabolism of cholesterol to bile acids (5, 6). Biliary
excretion of cholesterol and bile acids provides the major pathway
determining cholesterol homeostasis in the body.

The role of ruminant liver in regulating cholesterol metabolism is
poorly understood.and might be expected to differ from many monogastric
animals. Unlike many other species, ruminant liver appears to be a minor
site for body cholesterol synthesis (7, 8). The ruminant liver also has a very
limited capacity to synthesize and secrete VLDL (9, 10). Therefore, the
contribution of ruminant liver to plasma cholesterol might be minimal.
With respect to cholesterol removal, however, the ruminant liver possesses
mechanisms for the clearance of plasma cholesterol similar to those of

other species. For example, the liver is the primary organ for the uptake of

74

75

chylomicron cholesterol in sheep (11). Furthermore, low-density lipoprotein
(LDL) receptor has been demonstrated in liver tissues from cattle and has
been suggested to play a role in regulating bovine serum cholesterol
concentration (12).

Recently, we have reported that partial diversion of bile produces a
90% reduction in plasma cholesterol concentration in cattle (13). In no other
species has interruption of enterohepatic circulation (EHC) of bile been
shown to cause such a dramatic reduction in plasma cholesterol. The
objective of the present study was to determine if changes in hepatic
cholesterol synthesis or uptake, during bile diversion, could account for the

previously observed response in plasma cholesterol.

MATERIALS AND METHODS

Materials

[3H]-water (100 mCi/mmole) and Sodium [1251] Iodide (carrier free,
pH 7-11) were purchased from New England Nuclear Corp. (Boston, MA).
Minimum essential media (MEM 12000) was purchased from GIBCO
(Grand Island, NY). Durapore filters (0.45 am, 25 mm in diameter,
catalogue number HVLP 02500) were obtained from Millipore Corp.
(Bedford, MA). Bovine serum albumin (fraction V), newborn calf serum,
and heparin (grade I; from porcine intestinal mucosa) were purchased
from Sigma Chemical Co. (St Louis, MO). All other chemicals used were of
analytical grade obtained from commercial sources.

76
Animalsanddiet

Five adult, non-pregnant, non-lactating, Holstein cows were used.
The cows were kept in an environmentally controlled facility in individual

pens and fed an alfalfa hay-based diet as described previously (13).

Experimentalprocedums
The cows were surgically altered as described previously (13) and

fitted with cannula for the collection of bile. Following surgery, the animals
were initially used to study the effects of bile diversion on plasma lipid
concentrations (13). After completion of the initial study, the animals were
allowed a 14oday adjustment period without bile diversion. At the end of this
period, a liver biopsy was taken as described in the following section. The
cows were then allowed a further 10-day adjustment period. Plasma
cholesterol concentrations before and after the liver biopsy had restabilized
at about 85 mg/dl, which is in the range of normal values for nonlactating
cows. Following the adjustment period, 50% of the total bile flow was
diverted from the animals according to the procedure described previously
(13) for an experimental period of 14 days. The volume of the diverted bile
was measured twice a day and 3-ml aliquots of fresh bile were taken daily
and stored at -20 C until analyzed. Coccygeal-vessel blood samples were
taken daily and plasma collected as described (13) for the measurement of
plasma cholesterol concentration.

Three liver biopsies and one intestinal mucosal biopsy were taken
during the course of the experiment. The first liver biopsy was taken during
the initial surgery (pre-cannulation control). Subsequent liver biopsies were
carried out under local anesthesia. The second liver biopsy was taken after

the adjustment period and before the experimental period (post-cannulation

77

control) while the third was taken at the completion of the experimental
period. Also at the end of the experiment, under regional anesthesia,
a 20-cm segment of jejunum was removed, immediately flushed with
ice-cold phosphate-buffered saline and the mucosa scraped for
measurement of cholesterol synthesis rate. Immediately following
collection of the terminal samples, the animals were euthanized by
administration of an over-dose of sodium pentobarbital. The liver and whole
small intestine were dissected from the animals to determine the liver
mass and total mass of small intestinal mucosa.

For comparison, small intestine biopsies were obtained from four
adult, non-pregnant, non-lactating, noncannulated, Holstein cows using

the procedure described above.

Deta'mimtionof Cholesterol synthesis rate

Cholesterol. synthesis rate was determined in liver tissues and
intestinal mucosal samples by measuring the incorporation rate of [3H]-
H20 into digitonin-precipitable sterols (DPS) as described by Andersen et al
(14). Immediately after collection, intestinal mucosal scrapings or thin
liver slices (approximate 150 mg) were placed in screw-capped 20-ml glass
vials containing 5 mCi of [3H]-H20 in 1 ml of MEM. medium under an
atmosphere of 95% Oz - 5% C02. The vials were then incubated for 2 h
at 37 C in a metabolic shaker or at 0 C in an ice bath. Following incubation,
3 ml of 1.4 N KOH in 80% ethanol was added to the incubation mixture and
incubated at 85 C for 3-4 hr to saponify the lipids. Non-saponifiable lipids of
the reactants were extracted into hexane and treated with digitonin to
precipitate DPS (15). Following precipitation, the resultant digitonides were
washed with ethanol and diethyl ether and then split with pyridine.

78

Thereafter, the free sterols were extracted into diethyl ether (15), dried and
assayed for radioactivity. According to thin layer chromatographic
analysis, about 90% of the radioactivity in DPS was associated with
cholesterol. Rat liver slices were used as a positive control for the assays.
Rat liver was obtained from adult, male Sprague-Dawley rats fed a
commercial diet. All assays were performed in triplicate incubations.

The incorporation rate of tritium into DPS was corrected for bound
[3H]-H20 (incorporated at 0 C) and expressed as nmole of 3H incorporated
per gram tissue per hr (nmole/ g.hr). To account for dilution with tissue
water, the specific activity of the media water was determined after
incubation by direct assay of the media. Absolute rates of cholesterol
synthesis were estimated from the incorporation of 3H assuming that 23 3H
atoms are incorporated into each molecule of cholesterol (14, 16). Thus, the
nmole cholesterol synthesized/gram tissue per day = (nmole 3H

incorporated/g.hr /23) x 24 hours.

Isolation and labelingofLDL

Low density lipoproteins were isolated from the serum of a healthy
lactating cow by ultracentrifugation and affinity chromatography on
heparin-Sepharose as described by Cordle et al. (17). The LDL preparation
was further purified by gel filtration chromatography using Bio-Gel A5-M
(18) to remove VLDL particles. LDL was radiolabeled with 1251 according to
a modified McFarlane procedure (19) as described (20). The specific
activities of 126I-LDL ranged from 350 to 520 cpm/ng protein. More than 95%
of the 1251 radioactivity was precipitable by tricholoroacetic acid. The labeled
LDL were sterilized through a 0.45 pm Durapore filter, stored at 4 C, and
used within 2 weeks.

79

Hepatic LDL receptorbindingassay

Within 1 h following liver biopsy, a portion ( about 2.5 grams ) of the
liver sample was homogenized in 10 ml of buffer A ( 50 mM NaCl / 1.0 mM
CaClzl 20 mM Tris, pH 7.5). The homogenates were flushed through a 19-
gauge needle 10 times and thereafter stored at -70 C until assayed.

LDL receptor binding activities of the tissues were determined by
measuring heparin sensitive binding of 125I-LDL to the liver homogenate as
described by Rudling et al (21). Fifty milligrams of homogenate protein were
incubated on ice for 2 h in 150 ul of buffer B ( NaCl, 100 mM; CaClz, 1 mM;
BSA 20 mg/ml; Tris-HCl, 50 mM, pH 7.5 ) containing a fixed amount of 1251-
labeled LDL (50 pg protein /ml ). After incubation, aliquots (60 ul ) of the
binding reaction mixture were applied onto Durapore filters which were
mounted on a sampling manifold (catalogue number: XX 2702550; Millipore
Corp., Bedford, MA). The filters were washed eight times with 3 ml of
ice-cold bufl'er C ( 100 mM NaCl, 0.5 mM CaClz, BSA 0.1 mg/ml, 50 mM Tis-
HCl, pH 7.5) by applying suction to the filters. After the final wash, the
filters were incubated at 4 C for 20 min with 2 ml of buffer D ( 50 mM
NaCl/10 mM Hepes, pH 7.5 ) alone (for total binding) or with 10 mg/ml of
heparin (for heparin-resistant binding). After incubation, suction was
applied and the filters were washed twice with 3 ml of bufl‘er C. 1251 activity
in the filters was determined in a gamma-counter. Heparin-sensitive
binding of 125I-LDL was calculated by subtracting the heparin-resistant
binding from total binding. All binding assays were performed in triplicate.

80
Miscellaneousmdures

Protein contents of liver homogenate and LDL were determined as
described by Lowry et al (22). Biliary bile acid concentration was assayed by
an enzymatic method (Sigma, St. Louis, MO). Plasma cholesterol was
determined enzymatically using a reagent kit from Sigma (St. Louis, MO).
Cholesterol in bile was measured enzymatically after treatment of bile with
alcoholic KOH to remove the bile pigments and extraction of the biliary
cholesterol with hexane. The concentrations of cholesterol and cholesterol
ester in the liver were determined enzymatically after lipid extraction (23)
and isolation of unesterified cholesterol and cholesterol ester by thin layer
chromatography using silica gel HL Uniplates (Analtech Inc., Newark,
DE) and a solvent system of hexane-diethyl ether-acetic acid 85:15:1 (v/v/v).

The data are presented as means 1; S.E.M. and were analyzed using

analysis of variance and appropriate t test.

RESULTS

 

Diversion of 50% of bile flow led to the loss of approximately 180
mmole biliary sterols during the first 24 h ( Figure 10 ). Thereafter, the
amount of diverted sterols sharply declined and stabilized at a steady-state
level of about 25 mole per day. The majority of diverted biliary sterols were
bile acids, with biliary cholesterol accounting for less than 5% of the total
biliary sterols ( Figure 10 ).

As shown in Figure 11, 50% bile diversion was followed by a dramatic
decline in plasma cholesterol concentration. After 8 days, plasma

cholesterol concentration reached a steady-state level of 8 mg/dl, less than

81

10% of the original values. The decrease of plasma cholesterol
concentration was in a pattern similar to that observed in our previous

study (13).

 

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o n J n I 1 l n J n l n I n l o

0 2 4 6 8 1O 12 14
Days relative to partlal blle dlverslon

 

Figure 10: Depletion of biliary sterols induced by chronic diversion of 50% of bile flow.
Bile diversion was carried out as described in Experimental Procedure. The amount of bile
diverted was measured every 24 h and sampled. Total bile acids and biliary cholesterol
concentrations were determined by enzymatic methods. Each data point represents
the mean i SEM; n=5.

82

 

. h

Plasma cholesterol (mg/dl)
'13

 

 

 

l I l T

l l T l l l l 1
-1o -8 -6 -4 -2 o 2 4 6 8 1o 12 14
Days relative to partial bile diversion

Figure 11: Effects of bile diversion on plasma cholesterol concentration. Plasma samples
were obtained every other day and cholesterol concentrations were determined by
enzymatic methods. Each value represents the means i SEM (n=5).

83

In response to bile diversion, liver content of esterified cholesterol
was reduced by more than 90% (p<0.0001) by day 14, as compared to the pre-
cannulation or post-cannulation controls, whereas the unesterified
cholesterol content remained unchanged (p>0.05) (Figure 12). These data
demonstrate that 50% bile diversion depleted the liver of cholesterol stores.

 

Pro-cannulation

Post-cannulation

Bile diversion

 

 

 

Liver Cholesterol (rig/mg protein)

 

Free Chol. Esteriiled Chol. Free/Esterltled

Figure 12: Effects of bile diversion on hepatic cholesterol and cholesterol ester
concentrations. Lipids were extracted from liver tissue samples and free cholesterol and
esterified cholesterol were separated by thin layer chromatography, reextracted with ether,
and then quantified by enzymatic methods. Each value represents the means i SEM (n=5).

84

Incorporation rate of [3H]-H20 into DPS was barely detectable in the
bovine liver tissues, only about 10% of that of the bovine intestinal mucosa,
or 5% of the rat liver tissues (Figure 13). Nevertheless, as with intestinal
mucosa and the rat liver tissues, incorporation of [3H]-H20 into DPS by the
bovine liver tissues was essentially linear with respect to time over a 2.3—h
incubation period (Figure 13). Furthermore, incorporation was also
proportional to the amount of the bovine liver tissues incubated per vial, at
least from 50 to 200 mg (data not shown). To examine whether the low
incorporation rate was due to a limitation of substrate in the MEM
medium, incorporation rates of [3H]-H20 into DPS by bovine liver slices
incubated in MEM media supplemented with various substrates were
determined (Figure 14). The results showed that inclusion of additional
substrate into MEM had no effects on the incorporation rates (Figure 14).
This further confirms an inherently low rate of cholesterol synthesis in
bovine liver.

After 14 days of bile diversion, however, the incorporation rate of
[3H]-H20 into DPS in the liver was increased by more than 10 fold (30.0001)
(Figure 15), while there was no significant difference in the incorporation
rates between the two controls (Figure 15). This indicates that the hepatic
cholesterol synthesis rate was greatly increased in response to bile
diversion. In contrast to this, the [3H]-HzO incorporation rate into DPS of
the intestinal mucosa of these cows following bile diversion was only
slightly greater than that of the intestinal mucosa from the control animals
(35% ; p<0.05; Figure 15).

85

 

 

‘ -I- Bet liver
- + Bovine intestinal mucosa

+ Bovine liver

 

 

 

Incorporation of tritium into DPS (nmole/g)
.5

 

 

 

 

 

 

zoo.
150-
100-
50..
o . ‘ fl
I I l I I I
o 30 60 90 120 150
incubation time (min)

Figure 13: Time-course of incorporation of tritium from tritiated water into cow and rat
liver tissues and cow intestinal mucosa,. The tissue explants (150 mg) were incubated
in 1 ml MEM containing 5 mCi tritiated water at 37 C for the indicated time periods. After
incubation, the digitonin precipitable sterols (DPS) were isolated from the incubation
mixture and the radioactivities of the DPS were determined. Incubations were performed in
triplicate. Each data point represents the means i SEM of three separate experiments.

86

 

Incorporation rate of tritium into DPS (nmole/g. hr)
0|
1

 

 

 

 

 

Control Acetate Propionate Lactate Glucose Octanoic acid

Figure 14: Effects of supplement of various substrates to MEM on incorporation rate of
tritium from tritiated water into DPS by cow liver tissues. About 150 mg of cow liver
explants were incubated in 1 ml of MEM containing 5 mCi of tritiated water alone or with
15 moles of acetate, propionic acid, lactate, glucose, or octanoic acid. After incubation,
the digitonin precipitable sterols (DPS) were isolated from the incubation mixture and the
radioactivities of the DPS were determined. Each data point represents the means i SE of
three incubations.

87

 

E Pre-cannulation
200- [J] Post-cannulation
l Biliary dlverslon

 

 

 

160‘ P<0.001

P<0.05

d
N

O
l

Incorporation rate of 3H Into DPS
(nmole/hr. g)

 

 

 

 

0 m1

Bovine liver Bovine Intestinal
Mucosa

Figure 15: Effects of bile diversion on the incorporation rate of tritium from tritiated water
into digitonin precipitable sterols (DPS) by cow liver tissues and intestinal mucosa.
Incorporation rates of tritium into DPS were determined as described in Materials and

Methods. Each point represents the mean 1; SEM; n=5 except for control intestinal
mucosa (n=4).

 

Based on the incorporation rate of [3H]-H20 into tissue DPS and the
weights of liver and small intestinal mucosa, the amounts of cholesterol
synthesized by the liver and intestinal mucosa were estimated. As shown in
Figure 16, in the normal state, the estimated rate of small intestinal
cholesterol synthesis was five times greater than that of liver. After bile

8 8
diversion, as a result of the dramatic increase in the hepatic cholesterol
synthesis rate, the apparent hepatic contribution to cholesterol synthesis
was twice that of the small intestinal mucosa (Figure 16). The estimated
rate of hepatic cholesterol synthesis was approximately 1.2 mmoles / day
(Figure 16). This amount of cholesterol, however, would provide no more

than 5% of the sterols lost during bile diversion ( 1.2 vs. 25 mmoles/day ).

 

1400

 

' P<0.001 Control

1200-
Blle diversion

 

 

 

 

$3

Amount of cholesterol synthesized
(nmole/day)
l .

 

 

   

 

Liver Intestinal mucosa

Figure 16: Estimated quantities of cholesterol synthesized by the whole liver and intestinal
mucosa before and after bile diversion. The absolute amount of newly synthesized
cholesterol was estimated based on the incorporation rate of tritium into DPS and the total
mass of the liver and intestinal mucosa as described in Materials and Methods. Each point
represents the mean i SEM; n=5 except for control intestinal mucosa (n=4).

89
EL . LDL l. l. ..

To further understand the possible changes in bovine hepatic
cholesterol metabolism in response to bile diversion, we have also
determined the response in liver LDL receptor-binding activity . The
binding of 125I-labeled bovine LDL to liver homogenate was in a saturable
manner (Figure 17). Scatchard plot analysis (24) of the binding data
revealed a linear plot with an equilibrium dissociation constant (Kd) of
16 ug LDL protein /ml (Figure 17), reflecting a single class of LDL binding
sites in this tissue. The LDL-receptor binding activity of the liver tissue
samples was then determined by measuring the heparin- sensitive binding
of 125I-LDL to liver homogenate under a fixed concentration of 125I-LDL
which is approximately three times of the Kd. The results (Figure 18) show
that, by day 14 of bile diversion, liver LDL receptor-binding activity was
increased by 40% (P<0.001) as compared to pre-cannulation and post-
cannulation control samples, while there was no change in the binding
activity between samples taken at the two control times (Figure 18). These
data implicate an enhanced uptake of LDL during bile diversion.

DISCUSSION

We have previously shown that bovine plasma cholesterol
concentration responds to interruption of EHC of bile salts with a much
more dramatic decrease than that previously reported in other species (13).
To understand the underlying mechanism for this observation, in the
present study, we have determined the responses in hepatic and intestinal
cholesterol metabolism induced by bile diversion.

90

20

 

 
 
 
 

.5
?)

 

i

  

d
N
I

 
 

i3

 
 
 

‘

   

Bound/free
O O
'0 b

   

P
.

  
 

if;

  
 

125l-LDL bound (ng/mg protein)

 

 

 

   

ll 5 10 15 20
mum. bound (ng/mg)

 

 

 

I 1 fi I
l l I l

o 20 40 so so 100
125I-LDL concentrations (pg/ml)

Figure 17: Binding of radiolabeled bovine LDL to bovine liver homogenate as a function of
12SI-LDL concentration. Liver homogenates (3 mg proteins) were incubated in 150 ill of
buffer B with the indicated concentrations of 12SI-LDL for 2 h on ice. Specific bindings
were determined by measuring the heparin-sensitive bindings. Each data point represents
the means i SE of three incubations. Inset: Scatchard plot of the specific binding.
Bound/free represents the amount of specifically bound LDL (rig/mg) divided by the
concentration of unbound LDL in the reaction mixture (pg/ml).

 

l

N
0|
J

P&.m1

l

l

 

1

3
l

l l l 1

Vi
11111411:

SN

 

 

 

 

 

 

 

A

Pre-cannulatlon Post-cannulation Bile dlverslon

 

0

Figure 18: Effects of bile diversion on hepatic LDL receptor binding activity. LDL receptor
binding assays were carried out as described in Experimental Procedures. Each value
represents the mean 3; SEM of five cows.

Two control periods were employed in this study in order to ascertain
the effects of the cannulation procedure per as on hepatic cholesterol
synthesis rates and the expression of hepatic LDL receptors. It was
assumed that the samples taken at the time of the cannula placement
(pm-cannulation controls) represented the completely natural state, while
the samples taken after post-surgical stabilization, but prior to bile
diversion (post-cannulation controls) would reveal direct effects due to the

presence of the cannulas. The lack of differences in either cholesterol

92

synthesis rate or LDL receptor-binding activity between these two control
periods indicates that there was no detectable effect of the cannulation
procedure on the variables of interest.

The importance of ruminant liver to total body cholesterol production
has not been known with certainty. Previous studies (7, 8) have suggested
that ruminant liver was not a major site of body cholesterol synthesis, but
this judgment was based on experiments with 1‘ivC-labeled acetate, a
substrate which is poorly utilized by ruminant liver (25). In the present
study, by using 3H-water as labeled substrate, we confirmed the
conclusions from these earlier studies (7, 8). We found that the normal
cholesterol synthesis rate in the bovine liver was barely detectable, while the
small intestinal cholesterol synthesis rate was relatively high. We further
estimated that, at the organ level, the small intestinal mucosa synthesizes
five times as much cholesterol as does the liver in the adult, bovine female.
This demonstrates that, in this species, the small intestine is a much more
important site for cholesterol production than the liver.

An important finding in this study was that the inherently low rate of
cholesterol synthesis in the bovine liver can be augmented by bile diversion,
indicating that the bovine liver has the capacity to increase cholesterol
synthesis when cellular cholesterol is depleted. This is consistent with
results of studies with other species (6, 26). However, since the basal rate of
bovine hepatic cholesterol synthesis is so low, the absolute magnitude of
this increase is negligible. At the new steady-state, during bile diversion,
the bovine hepatic cholesterol synthesis rate was increased by more
than 10-fold, with a total estimated synthesis rate of 1.2 mmoles
cholesterol/day. This amount of cholesterol was far less than the cholesterol
required to account for the sterols lost due to bile diversion, which was at

93

least 25 mmoles/day. Furthermore, the hepatic cholesterol store was almost
completely depleted at this time period. Taken together, these data suggest
that bovine liver primarily relies on extrahepatic tissues to provide
cholesterol as substrate for increased bile acid synthesis during bile
diversion. Alternatively, it might be possible that, in our study, the in vivo
cholesterol synthesis rates in these, animals might have been
underestimated by the in vitro measurements. However, based on extensive
studies of a variety of tissues from several species, a previous study (26) has
reported that the in vitro cholesterol synthesis rate obtained by using 3H-
water as labeled substrate represented 10 to 40% of the in vivo rate.
Therefore, it is unlikely that the discrepancy between the estimated and the
actual in vivo cholesterol synthesis rate could explain the large difi'erence
between the rates of sterol loss and the hepatic cholesterol synthesis during
bile diversion.

It appears that, due to the low capacity of ruminant liver to increase
its cholesterol synthesis rate, interruption of EHC in ruminants produces
an effect analogous to the combined administration of an HMG-CoA
reductase inhibitor drug and bile acid sequestrants to humans or other
species. In each case, there is a net increase in sterol elimination from the
body combined with relatively low rate of cholesterol synthesis in the liver.
The combination of these effects is known to cause a large reduction in
plasma cholesterol concentration (6, 27). This reduction in plasma
cholesterol appears to occur as a result of increased LDL receptor
expression in the liver, which in turn leads to an increase in the clearance
of circulating LDL cholesterol (27 ).

The increased hepatic LDL receptor-binding activity observed in this
study further supports the enhanced catabolism of lipoprotein as a

94

mechanism of plasma cholesterol reduction during bile diversion in cattle.
This finding also provides evidence that bovine hepatic LDL receptor
expression is regulated metabolically. Hepatic LDL receptor plays a key role
in regulating plasma cholesterol catabolism in the body (5, 28). A
significant negative relationship between liver LDL receptor-binding
activity and plasma cholesterol concentration in cattle has also been
observed by other investigators (12), suggesting a possible physiological role
of the hepatic LDL receptor in ruminants. However, the LDL receptor-
dependent pathway might only play a relatively minor role in plasma
lipOprotein catabolism in our animal model. This is because that LDL
cholesterol only accounts for a very minor fraction of total plasma
cholesterol in cattle (29) and hence, an increase in the hepatic uptake of
LDL alone might not cause a significant change in plasma cholesterol
concentration. In fact, we have previously shown that even a 50% reduction
in both VLDL and LDL cholesterol did not result in any appreciable
decrease in plasma cholesterol in our animal model (13). Furthermore,
high density lipoprotein (HDL) particles of ruminant animals do not
appears to contain apoE (30, 31), which is a ligand for hepatic lipoprotein
receptors, including the LDL receptor (32). In addition, bovine plasma
possesses very low levels of cholesterol ester transfer activity (33), which
mediates the transfer of HDL cholesterol to LDL and for clearance by the
liver (34). Thus, it appears that LDL receptor does not participate in the
hepatic clearance of HDL cholesterol in cattle.

In view of the large reduction in HDL cholesterol observed in this
study, it is intriguing to speculate that mechanisms of hepatic uptake of
HDL cholesterol might not involve the LDL receptor. In addition to the LDL
receptor, HDL cholesterol can be taken up by the hepatocytes through two

95

other pathways, which are independent‘of the LDL receptor-mediated
process. The first pathway involves the uptake and internalization of the
whole HDL particle and is known to be mediated by the cell surface HDL
receptor (35, 36), whereas the second one appears to involve selective uptake
of cholesterol esters from HDL by the cell without the HDL particle being
internalized (35, 37). It is not clear whether I-IDL receptor is also involved in
the latter pathway, but the role of hepatic lipase has been proposed (38, 39).
However, previous studies (40-42) have shown that ruminant liver is almost
devoid of hepatic lipase. This suggest that the pathway of selective uptake of
cholesterol ester might not be. fully functional in bovine liver. Presumably,
the HDL receptor-mediated pathway might play an important role in the
uptake of HDL cholesterol by the liver. In fact, high affinity binding sites for
HDL have been identified in bovine liver plasma membrane (43). Because
the preponderance of bovine plasma cholesterol is in the HDL fraction,
understanding HDL metabolism is important for the complete
understanding of our animal model.

In conclusion, the present study shows that bovine liver responds to
interruption of EHC with augmented cholesterol synthesis and enhanced
capacity for lipoprotein uptake. However, due to the inherently low rate of
cholesterol synthesis, bovine liver must rely on uptake of lipoprotein
cholesterol for bile acid synthesis during bile diversion. Thus, bile diversion

causes a dramatic decline in plasma cholesterol in this species.

 

96

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