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Department of Animal Sciences,
*
Department of Foods and Nutrition,
Department of Basic Medical Sciences and
**
Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 47907 and
Department of Vascular and Cardiac Diseases, Parke-Davis Pharmaceutical Research, Ann Arbor, MI 48105.
3To whom correspondence and reprint requests should be addressed
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05) by each
HMGR inhibitor; however, total liver cholesterol (mg) did not differ
among treatments. Microsomal hepatic HMGR activities were increased
one- to twofold in all HMGR inhibitor-treated groups, while HMGR mRNA levels were unaffected. Diameters of plasma VLDL
particles, the main cholesterol-carrying yolk precursor
macromolecules, were reduced (P 0.05) only in
hens fed 0.06% atorvastatin, and the particles contained 38%
less total cholesterol (P 0.05) than controls.
Plasma total cholesterol concentrations were lowered (P
0.05) by both doses of atorvastatin (-56, -63%) and
simvastatin (-36,-45%). Egg cholesterol contents were maximally
reduced by 46% (P 0.05), 7%
(P > 0.05), and 22% (P
0.05) in hens fed the 0.06% level of atorvastatin, lovastatin,
and simvastatin, respectively, while overall egg production [-19%
(P 0.05), +4% (P > 0.05),
and -3% (P > 0.05)], was much less
affected. We concluded that cholesterol per se may not be an obligatory
component for yolk formation in chickens and, as such, may be amenable
to further pharmacological manipulation
KEY WORDS: • chickens • cholesterol • egg • 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors • VLDL
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Hermier et al. 1989
), whereas in
normolipidemic humans, micro pigs, hamsters, rabbits, rats, guinea
pigs, and dogs, LDL or HDL are the main carriers of cholesterol
(Kieft et al 1991
). Moreover, the major route of
cholesterol excretion in laying fowl is via the egg, which contains
approximately two-thirds of the hen's typical daily cholesterol
production of 300 mg (Naber 1983
).
The liver and ovary are the primary sites of cholesterol biosynthesis
in laying hens (Naber 1983
). However, there is little,
if any, direct transfer of ovarian-synthesized cholesterol to
developing oocytes in vivo. Thus, the contribution of the ovaries to
egg cholesterol levels are minimal at best (Andrews et al. 1968
, Connor et al. 1965
). In contrast,
cholesterol is readily transferred from the blood across the ovarian
membranes to developing ova and therefore most, if not all, egg yolk
cholesterol originates from blood cholesterol (Andrews et al. 1968
, Connor et al. 1965
). Furthermore, Weiss et
al. (1967a
and 1967b
) demonstrated, based on the in vitro incorporation
of [14C]acetate into all lipid classes, that
the pattern of lipid biosynthesis in liver slices contrasted greatly
with that in ovarian tissue strips, but was very similar to that
observed in ovo when [14C]acetate was orally
administered to laying hens. Taken together, these studies suggest that
the liver is the origin of most of the cholesterol and lipid found in
egg yolks (Naber 1983
).
Following biosynthesis in the liver, cholesterol is primarily
incorporated into VLDL particles and secreted into the bloodstream
(Griffin 1992
, Hargis 1988
). Unlike
mammalian VLDL, circulating laying hen VLDL, because of the presence of
large amounts of apolipoprotein (apo) VLDL-II, a lipoprotein lipase
inhibitor, does not undergo any appreciable lipolysis
(MacLachlan et al. 1996
, Schneider et al. 1990
). Thus, instead of primarily giving rise to intermediate
density lipoprotein and then LDL, VLDL arrives at the oocyte plasma
membrane virtually intact. There, it is bound and internalized by the
oocyte vitellogenesis receptor
(OVR),4
a 95-kDa protein and LDL receptor supergene family member (Bujo et al. 1994
, Schneider 1996
). Binding to the OVR
is mediated by apoB (Schneider 1992
). Once internalized,
VLDL is intracellularly transformed into yolk, constituting ~60% of
its dry matter (Burley et al. 1993
) and 95% of its
cholesterol (Griffin 1992
). Vitellogenin (VTG), the
other major yolk precursor synthesized in the liver under the influence
of estrogen, also binds to and is internalized by the OVR
(Schneider 1996
). VTG comprises ~24% of the dry
matter (Burley et al. 1993
), but only 4% of the
cholesterol (Griffin 1992
), in chicken egg yolks.
Because a vast array of dietary treatments, as well as genetic
selection programs, have resulted in only slight reductions (generally
5% or less) in avian egg cholesterol content, Hargis (1988)
hypothesized that cholesterol is essential for yolk formation and that
egg production would cease when yolk cholesterol deposition was
inadequate for embryonic survival. Although it has not been possible to
either confirm or disprove this theory because of an inability to
greatly inhibit cholesterol biosynthesis while maintaining egg
production in laying hens (Griffin 1992
, Hargis, 1988
), recent work from our laboratory suggests that this
critical level hypothesis may ultimately be testable. Following the
oral administration to laying hens of
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) inhibitors
lovastatin (Elkin and Rogler 1990
) or Parke-Davis
experimental compound PD 123244–15 (Elkin et al. 1993a
), we observed reductions in egg cholesterol on the order
of 15 and 30%, respectively, with the concomitant maintenance of egg
production. HMGR inhibitors are currently the most effective and widely
used class of cholesterol-lowering drugs available for the
treatment of hypercholesterolemia in humans (Dujovne 1997
, Endo 1992
).
Although the mode of action of HMGR inhibitors is generally well
characterized in mammals (Dujovne 1997
, Huff and Burnett 1997
), the mechanisms by which these compounds function
to lower egg cholesterol levels in chickens are incompletely described.
Furthermore, it is possible that the administration to laying hens of
some of the newer, more efficacious HMGR inhibitors might result in
even greater reductions in egg yolk cholesterol levels than previously
achieved. Thus, the present study was conducted to determine the
relative responsiveness of laying hens to atorvastatin, simvastatin,
and lovastatin and to examine the physiological and biochemical
consequences of altering the unidirectional, hepatic-to-ovarian pathway
of avian cholesterol transport.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Forty 18-mo-old White Leghorn hens were obtained from the flock
maintained at the Purdue University Poultry Research Center. Each hen
was placed in an individual 30 x 35 x 45 cm slant-back
cage in an environmentally controlled room (24°C and 16 h of
light daily), and birds were assigned to one of the seven dietary
treatments (see below) on the basis of both egg production and average
egg weight during the 10 d immediately prior to the initiation of
the experiment. Ten control hens were fed a corn-soybean meal-based
layer ration (Elkin and Rogler 1990
) containing 0.06%
avicel(TM) microcrystalline cellulose (FMC, Philadelphia,
PA)(diet 1), while five hens each were fed the same ration
supplemented, at the expense of avicel, with 0.03% atorvastatin (diet
2), 0.06% atorvastatin (diet 3), 0.03% lovastatin (diet 4), 0.06%
lovastatin (diet 5), 0.03% simvastatin (diet 6), or 0.06% simvastatin
(diet 7). The two levels of each drug equated to a daily dosage of
~30 or 60 mg/hen, respectively, based on a daily feed intake of 100
g/bird (Elkin and Rogler 1990
). Hens were given free
access to feed and water throughout the 35-d experiment, and feed
intake, egg production, and egg weights were recorded daily on an
individual basis. On d 35, feed was withheld from all of the hens for
15 h, and individual blood samples were obtained by cardiac
puncture using heparinized 12-cc syringes with 18 gauge needles.
Immediately after procurement of a blood sample, each hen was killed by
cervical dislocation. Its entire liver was then removed, weighed, and
sectioned to provide samples for subsequent biochemical, enzymatic, and
Northern blot analyses. In addition, a portion of the left liver lobe
was collected from each hen and fixed in 10% buffered formalin. The
tissues were embedded in paraffin, sectioned at 5 µm, stained with
hematoxylin and eosin, and examined by light microscopy. The protocol
for this study was approved by the Purdue University Animal Care and
Use Committee.
HMGR Inhibitors.
Atorvastatin was synthesized and provided by Parke-Davis (Ann
Arbor, MI) (Roth et al. 1991
). Lovastatin (Merck Sharp &
Dohme, West Point, PA) was prepared by extracting formulated capsules
of Mevacor(TM) (Bocan et al. 1992
), while
simvastatin was provided by Merck Sharp & Dohme.
Liver and egg cholesterol analyses.
One gram of each liver sample was homogenized with 12 mL of
chloroform-methanol 2:1 (by volume) and filtered directly into a 50
mL volumetric flask using a glass microfiber filter. Following
re-homogenization and re-filtration, the liver filtrates were
diluted to a final volume of 50 mL with chloroform-methanol 2:1 (by
volume) and analyzed for cholesterol by the method of Rudel and Morris (1973)
.
One egg from each hen was collected on d 0, 7, 14, 21, 28, and 35. The
eggs were hard-cooked, and the yolks were separated, weighed, and
crumbled. A 1-g sample of each yolk was homogenized with 15 mL of
chloroform-methanol 2:1 (by volume), sonicated, and filtered as
previously described (Elkin and Rogler 1990
). Egg
homogenate filtrates were analyzed for cholesterol by the method of
Rudel and Morris (1973)
.
Hepatic microsomal HMGR and cholesterol 7
-hydroxylase assays.
Liver microsomes were isolated by ultracentrifugation (Junker and Story 1985
) and stored in liquid nitrogen until used.
Microsomal HMGR activity was determined by measuring the conversion of
14C-HMG-CoA to 14C-mevalonate (Shapiro et al. 1974
), while microsomal cholesterol 7-hydroxylase
activity was determined by measuring the incorporation of phospholipid
liposome-solubilized 14C-cholesterol into
14C-7-hydroxycholesterol (Junker and Story 1985
).
DNA probes.
The plasmid ChMTP, a full-length cDNA clone for the large subunit
of chicken microsomal triglyceride transfer protein (MTP), was a gift
from Dr. David A. Gordon (Department of Metabolic Diseases,
Bristol-Myers Squibb, Princeton, NJ). The 2.9 kb fragment cDNA was
cloned into the BamHI/BamHI site of plasmid pcDNA-3.0 (Invitrogen,
Carlsbad, CA). The plasmid CB13–1 (Parkkonen et al. 1988
), a 960 bp fragment encoding the N-terminal part of
chicken protein disulfide isomerase (PDI), was cloned into the EcoR1
site of pBR322 (Sambrook et al. 1989
) and was obtained
from Dr. Erwin Ivessa (Department of Molecular Genetics, University of
Vienna, Vienna, Austria). The plasmid HMGR #23, a 650 bp fragment
containing the catalytic domain of chicken HMGR, was cloned into the
Nco1 and Pst 1 sites of the pGEM-T vector (Promega, Madison, WI)
and was provided by Dr. Elke Hengstschläger-Ottnad (Department of
Molecular Genetics, University of Vienna). The plasmid pB2, a 1.2 kb
fragment encoding part of the C-terminal portion of chicken apoB
(Kirchgessner et al. 1987
), was cloned into the EcoRV
sites of the pBluescript KS- vector (Stratagene, La Jolla, CA) and
obtained from Dr. Madeline Douaire (Laboratoire de
Génétique, Institut National de la Recherche Agronomique,
Rennes Cedex, France). The plasmid pDF 8 containing a 1.1 kb
BamHI-EcoRI fragment corresponding to the central region of the rat
18S rRNA gene was kindly provided by Dr. Richard Torzynski (Cytoclonal
Pharmaceuticals, Dallas, TX). This clone reacts with 18S rRNA from a
number of species, including chicken, cow, and pig (S. S. Donkin,
unpublished data).
Insert cDNA was excised from the plasmids by restriction enzyme
digestion, separated by electrophoresis through low melting temperature
agarose, and purified by lithium chloride precipitation (Favre 1992
). DNA probes were labeled with 32P-labeled
dCTP using the Ready-to-Go(TM) random oligonucleotide priming kit
(Pharmacia Biotech, Piscataway, NJ) to a specific activity of
approximately 60 GBq/µg DNA.
Northern blotting.
Liver (~1 g) from each hen was homogenized in guanidinium thiocyanate
solution (4 mol guanidinium thiocyanate/L, 25 mmol sodium citrate/L
[pH 7.4], 170 mmol sarcosyl/L, and 100 mmol ß-mercaptoethanol/L)
and total RNA extracted according to the method of Chomczynski and Sacchi (1987)
. Total RNA (20 µg) was separated by electrophoresis on
either a 0.8 or a 1% agarose gel according to Sambrook et al. (1989)
,
as modified by Tsang et al. (1993)
. The integrity of the 28S and 18S
rRNA was verified after staining with ethidium bromide, and the gel was
washed twice for 15 min in 1 mol ammonium acetate/L and transferred to
a Genescreen membrane by capillary action. RNA cross-linking,
prehybridization, hybridization, and wash steps were performed as
described by Donkin et al. (1996)
. The abundance of target RNA was
visualized on Kodak X-Omat AR film (Eastman Kodak, Rochester, NY).
Membranes were reprobed following stripping in 0.1 x SSC
(15 mmol NaCl/L, 1.5 mmol Na3 citrate/L, pH 7.0) and 1%
sodium dodecyl sulfate (SDS) at 100°C for 1 h. Variations in
loading and transfer of RNA were determined using cDNA for rat 18S rRNA
(Donkin et al. 1996
).
Plasma lipid analyses.
Plasma samples were analyzed for total cholesterol according to the
method of Rudel and Morris (1973)
. Free glycerol-corrected total
plasma triglyceride concentrations were determined using a commercial
kit (Triglycerides-GB, Boehringer Mannheim Diagnostics, Indianapolis,
IN). Plasma VLDL were isolated as previously described (Elkin and Schneider 1994
), and the lipid composition of plasma VLDL
was determined based on the extraction procedure of Slayback et al. (1977)
and the HPLC method of Homan and Anderson (1998)
.
Plasma VLDL particle size determination.
A carbon-coated formvar grid was floated on a drop of each VLDL sample. If the sample was too concentrated, it was diluted (10- or 100-fold) in a buffer containing 150 mmol NaCl/L, 200 µmol EDTA/L, 1 mmol phenylmethylsulfonylfluoride (PMSF)/L and 5 µmol leupeptin/L. After the grid floated on the drop for 5–10 min, the grid was removed and excess fluid drawn off with a piece of filter paper. The grid was then touched to 23.5 mmol aqueous uranyl acetate/L and the excess stain removed with filter paper. Samples were examined in a JEOL JEM-100CX transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV. Three representative micrographs were taken of each sample at a magnification 26,000X. Photographic positives of the negatives were made at an enlargement factor of 2.5X (82,500X final magnification), and the photographs were digitized at 600 dots per inch on a Hewlett-Packard Scanjet 4C flatbed scanner (Hewlett-Packard, Palo Alto, CA). Analysis of the digitized images was performed using Optimas 5.2 software (Optimas, Edmunds, WA). The feret diameter for a minimum of 500 particles was determined. All microscopic, photographic, and image analysis procedures were conducted without the operator's knowledge of the treatments from which the samples were obtained.
Protein assay and analysis of egg yolk extracts by SDS-PAGE.
One fresh egg was collected from each hen on d 35 of the experiment.
The yolk was carefully separated from the albumen using an egg
separator, and any adhering albumen was removed by rolling the yolk on
a moist paper towel. The yolk membrane was then punctured with a
forceps, and the yolk was gently squeezed out into a graduated cylinder
and mixed with 5 volumes of an ice-cold solution containing 20 mmol
Tris/L, 150 mmol NaCl/L, 200 µmol EDTA/L, 1 mmol PMSF/L, and 5 µmol
leupeptin/L. Yolk protein was measured in the presence of 12.8 mmol
SDS/L based on the method of Lowry et al. (1951)
, as modified by Elkin and Schneider (1994)
. Egg yolk extracts were subjected to SDS-PAGE on
4.5–18% gradient gels under reducing conditions (Elkin and Schneider 1994
). Following electrophoresis, the gels were
stained with Coomassie Brilliant Blue R-250 containing 20 mmol
AlCl3/L to visualize the phosvitin bands (Elkin et al. 1995
).
Statistics.
ANOVA (Steel and Torrie 1980
) was performed on all data
using the General Linear Models procedure of the SAS(TM)
Institute (1989)
. In certain instances, single degree of freedom linear
contrasts (Steel and Torrie 1980
) were also performed.
Individual treatment differences were tested by Duncan's multiple
range test (Steel and Torrie 1980
). Differences at
P 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The hens' initial body weights ranged from 1,556–1,646 g (P > 0.05) and, during the course of the 5-wk study, the birds gained between 67 and 137 g (P > 0.05; data not shown). Feed intakes [g/(bird · d)], which did not differ (P > 0.05) among treatments, were as follows: control, 106; 0.03% atorvastatin, 99; 0.06% atorvastatin, 99; 0.03% lovastatin, 106; 0.06% lovastatin, 106; 0.03% simvastatin, 99; 0.06% simvastatin, 101. Thus, because the HMGR inhibitors were fed at dietary levels of 0.03% or 0.06%, all treated hens consumed ~30 or 60 mg of compound/d, respectively. On a body weight basis, this equaled daily doses of ~19 or 38 mg of HMGR inhibitor/kg body weight.
Liver weights, histology, cholesterol contents, and enzyme activities.
Absolute liver weights were greater than controls in birds fed either
level of atorvastatin or the low dose of lovastatin; however, when
expressed on a relative body weight basis, only the organs of birds fed
0.06% atorvastatin were significantly larger than controls
(Table 1
). Light microscopic examination of the livers of all hens in the
study revealed no evidence of necrosis, fatty change, or inflammation
(data not shown). In addition, no differences among experimental groups
were noted.
|
. Microsomal
HMGR activities were significantly elevated one- to twofold in the
livers of all HMGR inhibitor-fed hens relative to controls
(Fig. 1
), whereas none of the HMGR inhibitors had a significant effect on
hepatic microsomal cholesterol 7-hydroxylase activity [values
ranged from 1 to 2 pmol/(min·mg protein); data not shown].
|
Northern blotting was conducted to investigate the effects of HMGR
inhibitors on the abundance of liver HMGR mRNA, as well as that of
three other genes whose products play a key role in the hepatic
synthesis of VLDL: apoB (Yao and McLeod 1994
) and the
two components of the heterodimeric microsomal triglyceride transfer
protein (MTP), namely the large subunit and protein disulfide isomerase
(Gordon 1997
). In contrast to the more uniform responses
in liver cholesterol content and microsomal HMGR activity among hens
within each treatment group, there was considerable variation among
individuals with regard to hepatic HMGR mRNA abundance (Fig. 2; in particular, note 0.03% atorvastatin-fed hens 11 and 12
vs. 13–15, 0.06% atorvastatin-fed hens 16 and 17 vs. 18–20,
0.03% lovastatin-fed hens 25 vs. 21, and 0.06%
simvastatin-fed hens 36 vs. 38–40). Even more striking was the
within- and between-treatment variability in the apoB and PDI mRNA
levels, whereas MTP large subunit mRNA abundance appeared to be
unaffected by dietary treatment. However, when the signals were
normalized by the abundance of 18S rRNA, significant differences among
treatment groups were observed for only hepatic MTP and apoB mRNA
(Fig. 3
). Single degree of freedom linear contrasts revealed the following
differences (P 0.05) for MTP large subunit:
simvastatin > control, simvastatin > atorvastatin, and
lovastatin > atorvastatin; and for apoB: control > lovastatin, control > simvastatin, atorvastatin > lovastatin, and atorvastatin > simvastatin.
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Plasma VLDL particle diameters were reduced (P 0.05)
only in hens fed 0.06% atorvastatin, and the particles contained 79
and 36% less esterified- and unesterified-cholesterol,
respectively, than controls (Table 2
). Although the 0.03% dose of atorvastatin significantly lowered
VLDL-esterified cholesterol levels, neither it nor lovastatin or
simvastatin affected (P > 0.05) the relative
proportion of unesterified cholesterol, which was the major form of
this sterol present in the VLDL particles. Moreover, the relative
contents of triglyceride, phosphatidylethanolamine, sphingomyelin, and
protein were not significantly affected by any of the HMGR inhibitors.
In contrast, phosphatidylcholine was greater than in controls
(P 0.05) only in VLDL particles from hens fed 0.03%
atorvastatin.
|
) were lowered (P 0.05) by both the 0.03 and
0.06% doses of atorvastatin (-56 and -63%, respectively) and
simvastatin (-36 and -45%, respectively), while plasma triglycerides
(Fig. 5
) were depressed (P 0.05) only in hens fed
atorvastatin (-57 and -71%, respectively). Lovastatin did not
significantly affect these plasma lipid concentrations.
|
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Compared to that of control birds, overall hen-day egg production
was significantly depressed (P 0.05) only in the
atorvastatin-treated groups, with maximal reductions at each level
observed by wk 2 (Table 3
). Interestingly, the rate of lay actually increased thereafter
and, by wk 5, production rates of the low- and high-dose
atorvastatin-treated birds were 93 and 90% that of controls,
respectively. Although hens fed either level of atorvastatin or 0.06%
simvastatin laid significantly smaller eggs than controls during wk
2–5, overall egg weights were lower (P 0.05) only
in the high-dose atorvastatin-treated birds (Table 3)
. In the
latter group, maximal reductions in egg weights were noted by wk 3.
Overall yolk weights were also reduced (P 0.05) only
in birds fed 0.06% atorvastatin, with a maximal decrease observed
during wk 2 of the study (Table 3)
. Lovastatin treatment did not affect
(P > 0.05) any of the above production variables.
|
0.05) lower yolk cholesterol
contents versus controls, on both a per g of yolk and a per yolk (egg)
basis, were noted in both atorvastatin-fed groups after only 7 d of treatment (Table 4
). By wk 2, eggs from the low dose and high dose
atorvastatin-treated hens contained 27 and 45%, respectively, less
total yolk cholesterol than those of control eggs. These reductions
were maintained throughout the duration of the study such that by wk 5,
total yolk cholesterol contents of 137.2 mg (0.03% dose) and 107.9 mg
(0.06% dose) were observed. The latter figure, a 46% reduction from
the control level of 201.5 mg cholesterol/egg (wk 5), is of a magnitude
never before reported, and was accounted for by a 19% decrease in yolk
weight (Table 3)
combined with a 35% reduction in the amount of
cholesterol per g of yolk. Simvastatin also depressed yolk cholesterol
levels, but to a much lesser degree than atorvastatin. Compared to
controls, maximal yolk cholesterol reductions of 20% on a mg/g basis
and 22% on a per yolk basis were observed at wk 5 in hens fed 0.06%
simvastatin (Table 4)
. Lovastatin was the least efficacious of the
three HMGR inhibitors, as evidenced by maximal yolk cholesterol
reductions of 7% on a mg/g basis (10.8 vs. 11.6, respectively) and 7%
on a per yolk basis (186.9 vs. 201.5, respectively) in hens fed the
higher dose (Table 4)
.
|
Representative yolk protein profiles of eggs from the HMGR
inhibitor-fed birds are shown in Figure 6
(lanes 3–8) and, with the exception of some of the
minor bands between 14 and 31 kDa (phosphoproteins; Elkin et al. 1995
),
they appeared to be very similar to those of control hens (Fig. 6
,
lanes 2 and 9). However, upon examination of the gel shown
in Figure 6
plus the other four replicate gels, each of which contained
samples from different control and HMGR inhibitor-treated hens, no
consistent differences in the 14–31 kDa bands among treatments were
observed (data not shown). Taken together, these results suggested that
the proportion of VLDL relative to VTG, the other major yolk precursor
macromolecule, was not selectively reduced by HMGR inhibitor treatment
of the hens.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), whereas a 70-kg human
synthesizes ~800 mg of cholesterol/d (McNamara et al. 1987
). Thus, despite the marked differences in body weights,
feeding hens 37.5% (300/800) of the normal daily human dosage of an
HMGR inhibitor would provide an equivalent amount of compound per
amount of cholesterol synthesized daily. For example, a normal daily
human dosage of lovastatin can range from 20 to 80 mg/d (Merck Sharp & Dohme 1988
). Thus, an equivalent lovastatin dosage in a
laying hen would be between 7.5 mg/d and 30 mg/d (0.375·20 mg and
0.375 · 80 mg, respectively). Because a hen typically consumes ~100
g of feed/d, the dietary percentage of the higher dosage would be 0.030
g lovastatin/100 g feed or 0.030% of the diet. In the present study,
we chose to supplement at one and two times this amount (0.03% and
0.06%, respectively) for each HMGR inhibitor to compare them on an
equal weight basis. In previous studies, we have supplemented
lovastatin (Elkin and Rogler 1990
) and PD 123244–15,
another experimental HMGR inhibitor (Elkin et al. 1993a
), at even higher levels.
In addition to providing the initial assessment of the
responsiveness of laying hens to atorvastatin, the present work also is
the first to comparatively examine the physiological consequences of
HMGR inhibitor-mediated lipid-lowering in an oviparous vertebrate.
When orally administered to laying hens at equal weight doses for 5 wk,
atorvastatin was more efficacious than simvastatin and lovastatin in
reducing liver cholesterol contents (per g basis)(Table 1)
, the
diameter and cholesterol richness of plasma VLDL particles (Table 2)
,
total plasma cholesterol (Fig. 4)
and triglyceride (Fig. 5)
concentrations, and egg yolk cholesterol contents (Table 4)
. Similar
observations with regard the relative efficacy of these three HMGR
inhibitors on plasma nonHDL cholesterol levels have been reported in
casein-fed rabbits (Auerbach et al. 1995
), mice
(Bisgaier et al. 1997
), and humans (Dart et al. 1997
, Davidson et al. 1997
). In addition, the
temporal occurrence of the reduction in yolk cholesterol levels (i. e.,
maximal after 2 wk and sustained throughout the remainder of the study)
(Table 4)
coincided exactly with plasma lipid reductions reported in
atorvastatin-treated humans (Bakker-Arkema et al. 1996
) and mice (Bisgaier et al. 1997
). The
lesser effects of lovastatin in laying hens in the present study
compared to a previous report from our laboratory (Elkin and Rogler 1990
) may possibly have been due to differences in the
strain and age of the laying hens employed, as well as the form of
lovastatin fed (extracted compound versus ground-up tablets in
prior work).
The cause of the greater liver weights in HMGR
inhibitor-treated hens (Table 1)
is presently unknown. Despite the
lack of hepatic histopathologic lesions at the light microscopic level,
future ultrastructural studies in hens may ultimately provide an
explanation for the liver enlargement observed herein.
Serum enzymes, such as alanine aminotransferase, aspartate
aminotransferase, gamma glutamyl transferase, alkaline phosphatase, and
lactate dehydrogenase, have been used to assess hepatocellular
integrity in mammals. However, the use of serum enzymes for assessing
liver condition in birds is poorly established (Lumei 1994
). In addition, because they are also found in varying
quantities in other organs, such as the heart, kidney, skeletal muscle,
and brain, none of the above enzymes are liver-specific in avians.
Thus, we contend that the histologic method employed herein was the
most accurate way to assess hepatocellular structure and integrity in
chickens. In this regard, the lack of HMGR inhibitor-related
histopathological changes in laying hens is promising, when considering
the possible future use of these compounds by the poultry industry, and
agreed with observations in dogs fed up to 80 mg/kg atorvastatin for 12
wk (Walsh et al. 1996
) or 25 mg/kg pravastatin for 104
wk (Tarumi et al. 1989
). However, the present findings
are in contrast to reports of centrilobular necrotic hemorrhage and
degeneration in the livers of hamsters fed 10 mg simvastatin/kg for
12 d (Oms et al. 1995
). Taken together, these
observations suggest the existence of marked species differences in the
ability of HMGR inhibitors to elicit hepatic pathological changes.
Laying hens generally meet their needs for cholesterol solely by de
novo synthesis. Thus, if cholesterol homeostasis was tightly regulated
in their bodies, hepatic HMGR mRNA levels would be expected to vary
directly with the relative effectiveness of a pharmacological agent to
inhibit cholesterol biosynthesis. However, our Northern blot data
suggested otherwise, as evidenced by the lack of effect (P
> 0.05) of HMGR inhibitor treatment on the relative abundance of
hepatic HMGR mRNA (Figs. 2
and 3)
. In contrast, one- to twofold
increases in hepatic microsomal HMGR activity verses controls were
observed for each treated group (Fig. 1)
. Although immunoreactive
protein levels were not determined because of a lack of availability of
an antibody against avian HMGR, the compensatory induction of the
reductase without changes in HMGR mRNA levels may be unique to avians
and suggested that post transcriptional regulation occurred.
Interestingly, Ness et al. (1998)
recently reported that atorvastatin
caused less induction of rat hepatic HMGR mRNA than lovastatin, despite
causing a larger induction of reductase protein. They suggested that
considerable post transcriptional regulation occurred, possibly because
of stabilization of HMGR protein.
In spite of the potential role of acyl-coenzyme A:cholesterol
O-acyltransferase (ACAT) in the regulation of mammalian VLDL
secretion (Conde et al. 1996
), hepatic ACAT activities
were not determined in the present study for two reasons: 1)
most of the cholesterol in laying hen plasma VLDL (Table 2)
and egg
yolk (Griffin 1992
) is in nonesterified form, suggesting
a minimal role of ACAT in this species; and 2) we previously
demonstrated the ineffectiveness of an ACAT inhibitor, Parke Davis
experimental compound PD 132301–2, to attenuate both plasma and egg
yolk cholesterol levels in laying hens (Elkin et al. 1993b
), which contrasted with its ability to markedly lower the
plasma cholesterol concentration of nonHDL lipoproteins in rats fed a
nonpurified (chow) diet.
Although HMGR inhibitor treatment significantly affected both
apoB and MTP large subunit mRNA levels (Figs. 2
and 3)
, a relationship
between these results and the lipid-lowering efficacy of
atorvastatin, lovastatin, and simvastatin in laying hens could not be
established. In fact, in the case of apoB, the observed effects were
exactly opposite of what was expected: hepatic apoB mRNA abundance in
atorvastatin-fed birds did not differ from that of controls,
whereas hens fed lovastatin or simvastatin had markedly lower amounts
of apoB mRNA. Because the latter two compounds were less efficacious
than atorvastatin in lowering plasma and egg lipids, we conclude that
hepatic apoB mRNA levels may be normally superfluous in chickens and
that, in the case of atorvastatin, its lipid-lowering effects
occurred independently of changes in apoB mRNA levels.
Up-regulation of LDL receptors is thought to be the major mechanism of
action by which most HMGR inhibitors lower plasma LDL-cholesterol
levels in humans (Bisgaier et al. 1997
); however, recent
studies have clearly shown that these drugs also decrease the hepatic
assembly and secretion of apoB-containing lipoproteins in both
hyperlipidemic humans and animal models (Huff and Burnett 1997
). Moreover, evidence is accumulating that suggests that,
in the case of atorvastatin, the relative importance of the above two
mechanisms of action is reversed (Bakker-Arkema et al. 1996
, Bisgaier et al. 1997
, Burnett et al. 1997
, Conde et al. 1996
).
In a recent study with miniature pigs, Burnett et al. (1997)
concluded
that atorvastatin reduced circulating VLDL- and LDL-apoB
concentrations primarily by decreasing apoB secretion into the plasma
and not by an increasing hepatic LDL receptor expression. They also
suggested that the attenuation of apoB secretion may, in part, have
been due to decreased apoB mRNA abundance. Although our observations in
atorvastatin-treated laying hens (Figs. 2
and 3)
do not support the
latter concept, the marked attenuation of VLDL production and secretion
in miniature pigs by atorvastatin (Burnett et al. 1997
)
provides an explanation as to why this HMGR inhibitor is uniquely able
to greatly decrease circulating triglycerides in both
hypertriglyceridemic humans (Bakker-Arkema et al. 1996
)
and, herein, laying hens (Fig. 5)
. In atorvastatin-treated birds,
parallel reductions in the levels of circulating cholesterol (Fig. 4)
and triglycerides (Fig. 5)
, with no change in VLDL-triglyceride
contents (Table 2)
, would be in keeping with an attenuation of VLDL
secretion because VLDL is the main transporter of both lipid classes in
the plasma of adult female chickens (Elkin et al. 1993a
,
Hermier et al. 1989
).
Although the relative importance of lipoprotein receptor
up-regulation as a mechanism of HMGR inhibitor action in
egg-laying fowl is unknown, several pieces of evidence suggest that
the OVR is probably not feedback up-regulated: 1) the
receptor is reportedly present in immature oocytes in a
pre-existing pool and is most likely not subject to de novo
synthesis (Shen et al. 1993
); 2) most, if not
all, of the mRNA in vitellogenic oocytes of other oviparous species is
untranslatable (Davidson 1986
); and 3)
attempts to produce immunoprecipitable VTG/VLDL receptors following
injection of radiolabeled precursors into chicken oocytes of 10–15 mm
diameter have failed (Shen et al. 1993
). However, an
increased rate of OVR reutilization via recycling (Shen et al. 1993
) might possibly explain how atorvastatin-treated hens
were able to sustain egg production and maintain egg weights and yolk
weights at >80% of normal values (Table 3)
in the midst of marked
reductions in the levels of circulating yolk precursors (Figs. 4
and 5)
. In addition, the observation that the yolk protein profiles of eggs
from HMGR inhibitor-fed birds appeared similar to those of the
control birds (Fig. 6)
suggested that the oocytic uptake of VTG, and
possibly its hepatic synthesis and secretion, was altered in a manner
similar to that of VLDL. Because apoB and VTG are cleaved by cathepsin
D upon receptor-mediated uptake into the oocyte (Elkin et al. 1995
, Retzek et al. 1992
), a change in the
proportion of yolk apoB fragments to VTG fragments (lipovitellins and
phosvitins) would have been expected if VLDL uptake was selectively
reduced because of HMGR inhibitor effects on hepatic VLDL synthesis and
secretion. Thus, in HMGR inhibitor-fed hens, egg formation probably
occurred in a normal manner, albeit at a slower rate. This resulted in
attenuated egg production rates, as well as decreased yolk and egg
weights.
Besides the OVR, nongonadal receptors for LDL (Hayashi et al. 1989a and 1989b
) and VLDL (Bujo et al. 1995
)
have been described in chickens. However, examination of their possible
up-regulation by HMGR inhibitors is either precluded or of
questionable physiological importance. Specifically, the LDL receptor
has not been characterized at the molecular level, whereas the chicken
somatic VLDL receptor, a recently discovered splice-variant of the
OVR, is expressed at very low levels in tissues and may actually have a
physiological role that is not directly related to the transport of
lipoproteins (Bujo et al. 1995
).
The observed reduction in the average VLDL particle size in hens fed
0.06% atorvastatin (Table 2)
is of a similar nature to that previously
reported in pravastatin-treated Watanabe rabbits (Shiomi and Ito 1994
). However, in terms of being able to penetrate the
avian granulosa basal lamina of the follicle with subsequent
incorporation into egg yolk (Walzem 1996
), the
physiological importance of the atorvastatin-mediated, 1-nm
reduction in VLDL particle size (from 35.7 to 34.7 nm) is probably
minimal.
Considering the magnitude of the effects of atorvastatin on
plasma and egg yolk lipids, it is somewhat remarkable that the
processes of oocyte growth, ovulation, and oviposition were not
impaired to a greater extent. Furthermore, the ability of the
atorvastatin-treated hens to maintain a substantial reproductive
effort challenges a central dogma that large reductions in egg yolk
cholesterol cannot be achieved because a physiological control
mechanism exists that causes egg production to cease when yolk
cholesterol deposition becomes inadequate for embryonic survival
(Hargis 1988
). Moreover, because the overall rate of egg
production in hens fed 0.06% atorvastatin was 81% of that of control
birds (Table 3)
, the critical level for yolk cholesterol, if one really
exists, must be less than ~105 mg cholesterol/yolk. Interestingly,
hatchability data from this study, which will be reported elsewhere
(Elkin and Yan 1999
), suggested that yolk cholesterol content was not
the main determinant of embryonic survival and that
atorvastatin-treated hens laid a high proportion of eggs that were
unfertilized.
Studies on avian embryonic lipid metabolism also fail to support the
critical level hypothesis, albeit indirectly, by suggesting that the
amount of cholesterol normally provided in the egg yolk is considerably
in excess of the needs of the developing chick (Naber 1976
, Noble and Cocchi 1990
). Thus, yolk
cholesterol deposition is probably more a reflection of the physiology
of the hen than of the needs of the embryo (Griffin 1992
) and as such may possibly be amenable to further
pharmacological manipulation. However, the degree to which egg
cholesterol can be attenuated must have a lower limit because the
complete inhibition of cholesterol synthesis in the hen would
undoubtedly impair steroid hormone production and result in the failure
to lay eggs (Naber 1976
).
In conclusion, when orally administered to laying hens at equal weight doses for 5 wk, atorvastatin was more efficacious than simvastatin and lovastatin in reducing the cholesterol richness of plasma VLDL particles, total plasma cholesterol and triglyceride levels, and egg yolk cholesterol contents. Although our evidence is indirect, reductions in the levels of circulating cholesterol and triglycerides suggested that atorvastatin, and possibly simvastatin, decreased hepatic VLDL production and/or secretion. However, parallel changes in mRNA levels of genes regulating cholesterol synthesis and VLDL assembly were not demonstrated, indicating possible post transcriptional effects of HMGR inhibitors in laying hens. Lastly, we hypothesized that an increased rate of OVR recycling may possibly account for the remarkable ability of atorvastatin-treated hens to maintain egg production, egg weights, and yolk weights (at levels >80% of normal values) despite marked reductions in the levels of circulating yolk precursor macromolecules. Future apoB kinetic studies will help to clarify the action of atorvastatin on hepatic VLDL secretion and oocytic uptake in domestic fowl, whereas other experiments will assess potential drug residue and nutrient compositional changes in low-cholesterol eggs.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Presented in part at the 11th
International Symposium on Atherosclerosis, October 7, 1997, Paris,
France [Elkin, R. G., Yan, Z., Buhman, K. K., Story, J. A., Turek, J. J., Anderson, M., Homan, R. & Newton, R. S.
(1997) Reduction of egg yolk cholesterol content through
inhibition of hepatic cholesterol biosynthesis and alteration of plasma
VLDL composition in laying hens: Comparative effects of atorvastatin,
lovastatin, and simvastatin. Atherosclerosis 134:123 (abs.)]
4 Abbreviations used: ACAT, acyl-coenzyme
A:cholesterol O-acyltransferase; apo, apolipoprotein; HMGR,
3-hydroxy-3-methylglutaryl-coenzyme A reductase; MTP, microsomal
triglyceride transfer protein; OVR, oocyte vitellogenesis receptor;
PDI, protein disulfide isomerase; PMSF,
phenylmethylsulfonylfluoride; SSC, 150 mmol/L NaCl, 15 mmol/L
Na3citrate, pH 7.0; VTG, Vitellogenin
Manuscript received July 23, 1998. Initial review completed October 13, 1998. Revision accepted January 26, 1999.
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