![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
2
*
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269 and
Department of Health and Clinical Sciences, University of Massachusetts, Lowell, Lowell, MA 01854
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
KEY WORDS: • hamsters • guinea pigs • dietary cholesterol • casein • pectin
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, Sullivan et al. 1993
). However, the use of animal models is associated
with a number of disadvantages, including the lack of similarity to
humans in plasma lipoprotein profiles or in the activity of regulatory
enzymes of hepatic cholesterol and lipoprotein metabolism.
Pigs are well accepted models to study hypercholesterolemia and
atherosclerosis because of their similarities to humans in both of
these areas (Van Tol et al. 1991
). Yet, there is a need
for the utilization of smaller animals for more rapid studies on the
effects of diet on hyperlipidemia and atherosclerosis. The
cholesterol-fed rabbit has been used as a model of human
atherosclerosis for the rapid development of aortic lesions
(Daley et al. 1994
); nevertheless, rabbits carry most of
the cholesterol in the VLDL fraction, a situation different from humans
(Badimon et al. 1990
). The use of the rat has been in
decline because of the major differences between this animal model and
humans, including responses to diet, HDL being the major plasma
cholesterol carrier in the rat, and its resistance to atherosclerosis
(Shefer et al. 1992
).
Hamsters and guinea pigs have been extensively used for studying the
effects of diet on plasma lipid levels and the mechanisms involved
because these two animal models present characteristics that might
resemble the human situation (Fernandez and McNamara 1991
, Fernandez et al. 1993, 1994
, 1995, and
1997
, Fernandez 1995
, Nicolosi and Wilson 1997
, Spady and Dietschy 1988
,
Terpstra et al. 1991
, Woollett et al. 1992
). Guinea pigs carry the majority of cholesterol in the LDL
fraction (Fernandez and McNamara 1991
), and hamsters
develop atherosclerosis when challenged with a hypercholesterolemic
diet (Otto et al. 1995
).
The response to different hypercholesterolemic diets in general, and to
dietary cholesterol in particular, is an important area of research
that continues to be under debate. The heterogeneity of the human
response to dietary cholesterol was documented (McNamara et al. 1987
), and studies have demonstrated that saturated fat or
trans fatty acids appear to have a more pronounced effect on plasma
lipid levels than does dietary cholesterol (Hu et al. 1997
). Consumption of fruits, vegetables, and grains was
postulated as a healthy diet, which results in lowering of plasma
cholesterol (Jenkins et al. 1997
). Further, studies have
reported that lacto-ovo-vegetarians have low plasma cholesterol
concentrations and desirable lipoprotein profiles (Masarei et al. 1984
), which suggests that consuming relatively high
amounts of dietary cholesterol with high amounts of vegetable protein
and adequate amounts of dietary fiber will not result in elevated
plasma cholesterol concentrations.
The present studies were designed to compare the response to different
dietary factors, including animal protein, dietary fiber, and dietary
cholesterol, in two animals models that differ in terms of
cholesterol-carrying lipoproteins in plasma (Fernandez and McNamara 1991
, Woollett et al. 1992
). We
hypothesized 1) that dietary challenges would alter
lipoprotein distribution differently in hamsters and guinea pigs and
2) that, independent of the animal model used, the response
to dietary cholesterol would be lower with diets high in vegetable
protein and soluble fiber.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Reagents were obtained from the following sources: phosphotungstate
reagent, glucose 6-phosphate, NADP+, glucose-6-phosphate
dehydrogenase, Tween 80, triton-100, 7
-hydroxycholesterol, unlabeled
cholesterol, [4-14C] cholesterol, and triacylglycerol
enzymatic kits from Sigma (St. Louis, MO). Cholesterol enzymatic assay
kit, cholesterol oxidase, and cholesterol esterase were purchased from
Boehringer-Mannheim (Indianapolis, IN); free cholesterol and
phospholipid enzymatic assay kits were obtained from Waco Pure Chemical
Industries (Richmond, VA); halothane from Halocarbon (Hackensack, NJ);
[3-14C] HMG-CoA, [5-3H]
mevalonolactone, [1-14C ] oleoyl CoA, [4,
14C] cholesterol, and [1,2,6,7 3H]
cholesteryl oleate from Du Pont New England Nuclear (Boston,
MA). Silica gel TLC plates were from Eastman Kodak (Rochester, NY);
silica gel G TLC plates were from Analtech (Newark, DE), 7
ß-hydroxycholesterol was from Steraloids (Wilton, NH); quickseal
ultracentrifugation tubes from Beckman Instruments (Palo Alto, CA).
Diets.
Diets were prepared by Research Diets (New Brunswick, NJ). Diets for
the three experiments had the same composition except for the nutrient
under evaluation as indicated in Table 1. For study 1, the difference was the proportion of casein/soybean
protein used, which was 60/40, 20/80, or 0/100 (Table 1)
. For study 2,
the protein used was 0/100 casein/soybean protein, and the proportion
of pectin/cellulose varied: 2.7/9.8, 5.4/7.1, 10.7/1.8 g/100 g for a
total of 12.5 g/100 g dietary fiber (Table 1)
. For study 3, the diet
contained 0/100 casein/soybean protein and 10.7 g/100g pectin, while
the amount of dietary cholesterol varied 0.04, 0.08, or 0.16 g/100g
(Table 1)
, which is equivalent to 300, 600, or 1,200 mg/d in humans
(Lin et al. 1992
).
|
Male Hartley guinea pigs (Harlan Sprague Dawley, Indianapolis, IN) weighing 300–400 g were randomly assigned to each one of dietary treatments for 4 wk to achieve a metabolic steady state prior to analysis. Guinea pigs (6–8/dietary treatment) were slowly weaned onto the experimental diets by feeding them increasing amounts of the test diet over a period of 1 wk. They were housed in a room of controlled light (light 0700–1900 h) and had free access to diets and water. At the end of the experiment (4 wk), guinea pigs were anesthetized with halothane and killed by exsanguination after cardiac puncture.
Male, 10-wk-old Golden Syrian hamsters were obtained from Charles River Laboratories (Wilmington, MA). Hamsters were fed a nonpurified diet (Purina, St. Louis, MO) for 1 wk prior to the treatment period. Following this acclimation period, hamsters were randomly assigned to three groups of 15 for each study and fed the respective treatment diets for 4 wk. They were housed in individual, stainless steel hanging cages at room temperature with a 12-h light:dark cycle and had free access to food and water.
All animal experiments were conducted in accordance with US Public Health Service/US Department of Agriculture guidelines, and experimental procedures were approved by the University of Connecticut and the University of Massachusetts Institutional Animal Care and Use Committee.
Plasma lipid concentrations in hamsters.
Blood samples were taken after 4 wk from food-deprived hamsters (12
h) and collected, via the retro-orbital sinus, into heparinized
capillary tubes under ultra-pure CO2/O2
(50:50) gas (Northeast Airgas, Salem, NH) anesthesia. Plasma was
harvested after centrifugation at 1,500 x g at room
temperature for 20 min, and plasma total cholesterol
(TC)4
(Allain et al. 1974
) and triacylglycerol (TAG) concentrations
(Bucolo and David 1973
) were measured enzymatically.
Plasma VLDL and LDL cholesterol, non-HDL cholesterol (non-HDL-C),
were precipitated with phosphotungstate reagent
(Weingand and Daggy 1990
), and HDL
cholesterol (HDL-C) was measured in the supernatant. The concentration
of non-HDL-C was calculated as the difference between plasma TC and
HDL-C.
Isolation and characterization of VLDL and LDL for guinea pigs.
For the determination of plasma lipids and isolation of lipoproteins,
blood samples were taken by cardiac puncture after guinea pigs were
anesthetized with halothane. Plasma total and lipoprotein cholesterol
(Allain et al. 1974
) and TAG (Bucolo and David 1973
) were determined by enzymatic methods. Guinea pig VLDL
(d = 1.006 kg/L) and LDL (d = 1.02–1.09 kg/L) were isolated by ultracentrifugation at 120,000 x
g with the use of a Ti50 rotor. Plasma HDL cholesterol
was measured according to Warnick et al. (1982)
, with the modification
of using 2 mol/L MgCl2 to precipitate the apo
B-containing lipoproteins. The accuracy of the procedures used for
the measurements of plasma TC, HDL-C, and TAG concentrations for
hamsters and guinea pigs were checked by participation in the Lipid
Standardization Program of the Centers for Disease Control and the
National Heart, Blood and Lung Institute (Atlanta, GA).
Lipoprotein composition was determined by measuring protein
(Markwell et al. 1978
), TAG (Bucolo and David 1973
), phospholipids, and free and esterified cholesterol
(Carr et al. 1993
). The number of component molecules of
LDL was calculated assuming one apo B per particle with a molecular
weight of 412,000, as reported for guinea pigs (Chapman et al. 1975
). The molecular weight of TAG, cholesterol,
cholesteryl ester, and phospholipids were calculated as 885.4, 386.6,
664, and 734, respectively, as previously reported (Fernandez et al. 1994
).
Hepatic cholesterol and triacylglycerol determination.
Hepatic lipids were determined as described by Carr et al. (1993)
.
Briefly, for lipid extraction 1 g of liver was homogenized in
chloroform:methanol overnight. After mixing with acidified water,
phases were separated and aliquots evaporated and resuspended in
ethanol to measure total and free cholesterol and TAG by enzymatic
methods. Cholesteryl ester was calculated by subtracting free from
total hepatic cholesterol.
Hepatic HMG-CoA Reductase assay.
Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase
(EC 1.1.1.34) activity was measured as previously described
(Shapiro et al. 1969
). HMG-CoA reductase
activity was expressed as picomoles of [14C] mevalonate
produced per min per mg microsomal protein. Recoveries of
[3H] mevalonate were between 70 and 80%.
Hepatic ACAT assay.
Acyl coenzyme A cholesterol acyltransferase (ACAT) (EC 2.3.2.26)
activity was determined by pre-incubating microsomal protein
0.8–1.0 mg per assay with 84 g albumin/L, an amount of albumin
equivalent to the molar ratio of the substrate (1:1
albumin:14C-oleoyl CoA) (Smith et al. 1986
), and buffer for ACAT isolation (Fernandez et al. 1993
). Recoveries of [3H] cholesteryl
oleate were between 75 and 90%.
Hepatic cholesterol 7-hydroxylase assay.
Cholesterol 7-hydroxylase (EC 1.14.13.7) activity was assayed by the
method of Jelinek et al. (1990)
using [14C]
cholesterol as substrate, except that cholesterol was delivered as
cholesterol:phosphotidylcholine liposomes (1:8 by weight) prepared by
sonication, and an NADPH-regenerating system (glucose-6-phosphate
dehydrogenase, NADP, and glucose 6-phosphate) was included in the assay
as a source of NADPH (Fernandez 1995
).
Statistical analysis.
One-way ANOVA was used to determine differences in plasma TC, TAG,
lipoprotein composition, hepatic lipids, and hepatic enzyme activity in
hamsters or guinea pigs fed the diets in experiments 1, 2, or 3.
Newman-Keuls was used for post-hoc analysis. P
< 0.05 was considered significant. Two-way ANOVA was used to
compare the specific responses in plasma TC between hamsters and guinea
pigs consuming 60% casein, 2.7g/100 g pectin, or 0.08 g/100 g dietary
cholesterol. Linear regressions were calculated between TC for hamsters
and guinea pigs and increasing doses of diet and for ACAT and
cholesterol 7-hydroxylase activities and pectin doses in guinea
pigs.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
There were no differences in weight gain or food consumption in
hamsters and guinea pigs fed the different diets (data not shown). In
addition, both species exhibited significantly higher plasma TC
concentrations when fed 40% compared to 80 or 100% soybean protein
(Table 2
). Plasma cholesterol was 9% lower in hamsters fed 80% soybean protein
and 18% lower in those fed 100%; in guinea pigs, values were 13%
(P = 0.08) and 26% lower than in those fed 40%
soybean protein respectively. In addition, there was 7% lower
(P = 0.14) plasma TAG in hamsters fed the 100% soybean
diet than in those fed 80 or 40% soybean protein (Table 2)
. Although
guinea pigs fed 100% soybean protein had 43% lower plasma TAG than
those fed 40% soybean protein, the differences were not significant
because of the large standard deviations (P = 0.17;
Table 2
). In this and subsequent experiments, hamsters had higher
plasma TC concentrations than guinea pigs (Tables 2
3
4
).
|
|
|
). The concentrations of LDL were not determined in hamsters, and
the results are expressed as non-HDL cholesterol.
|
Increasing soybean protein resulted in significantly lower
concentrations of non-HDL and HDL cholesterol in hamsters.
Non-HDL cholesterol was 24% lower in hamsters fed the 100%
soybean protein diet compared to those fed 40% soybean protein. There
was 34% lower plasma non-HDL cholesterol in guinea pigs fed 100%
than in those fed 40% soybean protein (Fig. 1)
. No other significant
differences in plasma lipoprotein cholesterol were observed in guinea
pigs.
Guinea pigs fed the 100% soybean diet had lower hepatic total
cholesterol concentrations than those fed lower ratios of soybean
protein:casein; differences were associated with free cholesterol
concentrations (Table 5). No significant differences among treatments were observed for
esterified cholesterol or hepatic TAG.
|
Hepatic HMG-CoA reductase, cholesterol 7-hydroxylase, and ACAT
activities did not differ among the three dietary treatments. Values
were 17 ± 12, 16 ± 9 and 11 ± 5 pmol/(min·mg
protein) for HMG-CoA reductase activity; 1.70 ± 0.33, 1.33
± 0.44, and 1.72 ± 0.50 pmol/(min·mg protein) for
cholesterol 7-hydroxylase activity; and 48 ± 18, 50 ± 19, and 44 ± 5 pmol/(min·mg protein) for ACAT activity in
guinea pigs fed the 60/40, 20/80, or 0/100 casein:soybean protein
diets, respectively.
Study 2.
Plasma TC was 21 and 32% lower in hamsters and 25 and 39% lower in
guinea pigs fed the 5.4 and 10.7 g/100 g pectin diets, respectively,
compared to those fed 2.7 g/100 g pectin (Table 3
; P < 0.01). Plasma TAG were lower by an average of 40% (P
< 0.05) in the hamsters that consumed higher amounts of pectin.
Increasing the amount of soluble fiber in the diet resulted in a dose
response in the plasma cholesterol lowering in both hamsters and guinea
pigs (r = -0.985, P < 0.05).
Hamsters fed the two higher levels of pectin had lower plasma
non-HDL and HDL cholesterol concentrations than the 2.7 g/100 g
pectin group, whereas guinea pigs had similar responses only in
non-HDL cholesterol (Fig. 2
; P < 0.01). The plasma TC and non-HDL
cholesterol lowering ranged from 27 to 40% for hamsters and guinea
pigs (Fig. 2)
.
|
In both of these studies, 1 and 2, guinea pigs presented a
proportionally greater reduction in plasma non-HDL cholesterol
concentrations than hamsters when given 100% soybean protein (Fig. 1)
or 10.7 g/100 g pectin (Fig. 2)
.
Hepatic total and free cholesterol were lower in guinea pigs fed 5.4 or
10.7 g/100g pectin compared to those fed the lower dose (Table 6). No other differences were observed in hepatic lipids. As the
amount of pectin in the diet increased, there was a dose response for
some of the regulatory enzymes of hepatic cholesterol metabolism.
Hepatic cholesterol 7 -hydroxylase activity increased as the amount
or pectin in the diet increased. In contrast, ACAT activity decreased
as the amount of pectin in the diet increased (Fig. 3
). HMG-CoA reductase activity was higher in guinea pigs fed
pectin levels of 5.4 and 10.7 g/100 g, although a dose response was not
observed. Values were 8.1 ± 3.2, 15.3 ± 8.4, and 14.1
± 4.1 pmol/(min·mg protein) for guinea pigs fed 2.7, 5.4, and
10.7 g/100 g pectin, respectively.
|
|
Plasma TC concentrations increased in both hamsters and guinea pigs as
the amount of cholesterol in the diet increased (Table 4)
. Plasma
cholesterol concentrations were 9% (P = 0.27) and 50%
higher in hamsters and 20% (P = 0.09) and 94% higher
in guinea pigs fed 0.08 and 0.16 g/100 g dietary cholesterol,
respectively, compared to those fed 0.04 g/100 g dietary
cholesterol (P < 0.01). In hamsters fed 0.16 g/100 g
of cholesterol, plasma TAG concentrations were greater than in those
fed the two lower levels. However, no differences in plasma TAG were
observed in guinea pigs fed the three levels of dietary cholesterol,
indicating differences between animal models.
In hamsters, both non-HDL and HDL cholesterol concentrations
increased because of the greater intake of dietary cholesterol.
However, the proportion of non-HDL cholesterol/HDL cholesterol
changed as the concentration of dietary cholesterol increased. The
proportions were 0.64, 0.76, and 0.99, respectively, indicating that
the non-HDL-C increased more than the HDL-C. In guinea pigs, there
was a slightly greater plasma non-HDL-C concentration in those fed 0.08
g/100 g cholesterol and a 100% greater concentration in those fed the
0.16 g/100 g cholesterol diet. No differences were observed in
HDL-C (Fig. 4
).
|
Hepatic total free, and esterified cholesterol concentrations were
higher for both hamsters and guinea pigs fed 0.16 g/100 g dietary
cholesterol compared to the other dietary cholesterol doses
(Table 7). Guinea pigs had higher concentrations of free versus the
esterified cholesterol, even in the case of high dietary cholesterol,
whereas hamsters had higher concentrations of esterified cholesterol at
the highest dose of dietary cholesterol (Table 7)
.
|
). In contrast, guinea pigs fed 0.08 g/100 g had; 70% lower HMG-CoA
reductase activity than those fed 0.04 g/100 g, indicating that the
first compensatory mechanism in guinea pigs to maintain plasma
cholesterol homeostasis is the suppression of the activity of the
regulatory enzyme of cholesterol synthesis. In contrast, cholesterol
7-hydroxylase activity was not altered by increasing the amount of
dietary cholesterol fed to hamsters or guinea pigs (Table 8)
. ACAT
activity was not altered by diet in hamsters, whereas in guinea pigs
ACAT activity and the amount of free cholesterol (Table 8)
increased in
parallel with higher intakes of dietary cholesterol. These results
indicate different mechanisms to maintain cholesterol homeostasis when
challenged with high levels of dietary cholesterol exist in hamsters
and guinea pigs.
|
There was a dose response in plasma TC for both hamsters and guinea
pigs in studies 1, 2, and 3. Plasma total cholesterol decreased as the
amount of soybean protein increased in the diet (Fig. 5
upper panel). Similarly, as the amount of pectin
increased in the diet, TC decreased (Fig. 5
middle panel).
Both correlations were linear (P <0.05). Although there was
a dose response in which plasma TC increased as the amount of dietary
cholesterol increased, the best fit was obtained using a power
correlation (r = 0.95 for hamsters and r
= 0.98 for guinea pigs; Fig. 5
lower panel). Plasma TAG
concentrations were not consistently correlated with the amount of each
nutrient in diet (data not shown).
|
). Although hamsters had higher plasma TC concentrations than
guinea pigs in all three studies, guinea pigs had higher plasma
non-HDL cholesterol concentrations (Fig. 6)
. This was because
guinea pigs carry the majority of cholesterol in the LDL fraction,
whereas hamsters have different proportions of LDL and HDL cholesterol
, depending on the diet. When data were analyzed by two-way ANOVA,
diet and species effects were present. Guinea pigs had higher plasma
non-HDL cholesterol concentrations when compared to hamsters in all
three studies (P < 0.01). In addition, intake of
0.08g/100 g dietary cholesterol with 10.7 g/100 pectin and 100%
soybean protein resulted in significantly lower plasma non-HDL
cholesterol concentrations for both hamsters and guinea pigs than did
intake of 60% casein or 2.7g/100 g pectin with 0.04 g/100 g dietary
cholesterol (P < 0.05).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Hamsters and guinea pigs responses to casein/soybean and soluble fiber.
Soybean protein lowers plasma cholesterol levels in experimental
animals (Nicolosi and Wilson 1997
,
Terpstra et al. 1991
) and humans (Nagata et al. 1998
). In rats and hamsters, the hypocholesterolemic effect was
associated with increases in fecal steroid excretion (Hayashi et al. 1994
). Rabbits are very susceptible to dietary casein
because they develop endogenous hypercholesterolemia and develop
atherosclerosis with diets high in casein (Daley et al. 1994
). In this study, we have shown that hamsters had similar
hypocholesterolemic responses to soy relative to casein as previously
reported (Terpstra et al. 1991
) and that guinea pigs
also exhibit higher plasma LDL cholesterol concentrations as the amount
of dietary casein is increased. All these studies demonstrate that
there is a consistent response to casein intake across animal species,
although with different degrees of hypercholesterolemia.
Although increases of fecal steroid secretion were proposed as a
possible mechanism for the action of soybean protein (Huff and Carroll 1980
), there are many sites of cholesterol and
lipoprotein metabolism that could be affected and that have not been
studied. We found no effect from the intake of different casein/soybean
protein ratios in any of the regulatory enzymes of hepatic cholesterol
homeostasis in guinea pigs. These data suggest that other mechanisms,
not related to the synthesis or catabolism of cholesterol, are being
affected by the intake of different levels of casein and soybean
protein in this species. We did find lower hepatic cholesterol
concentrations in guinea pigs fed the 100% soybean protein diet, but
these differences were not associated with modifications in hepatic
enzyme activity.
The mechanisms associated with the hypocholesterolemic effects of
soybean are not clear. For example, because soybean protein is
deficient in some of the essential amino acids, studies in which
soybean protein is supplemented with seven essential amino acids to
make it similar to animal protein, were conducted, and no
hypercholesterolemic responses were observed (Carroll 1982
). The most consistently postulated mechanism in
animal studies appears to be related to increases in fecal steroid
secretion caused by the reduced availability of bile acids (Huff and Carroll 1980
). What is clear from studies conducted in
humans and animals is that soybean protein has a hypocholesterolemic
effect that we were able to reproduce in the present study of hamsters
and guinea pigs.
In the case of dietary soluble fiber, there were dose responses to
pectin intake in total and LDL cholesterol in guinea pigs and in total,
HDL, and non-HDL cholesterol in hamsters. Another interesting
observation was that LDL composition was altered by dietary fiber in
guinea pigs. The highest intake of pectin resulted in particles that
contained less cholesteryl ester. Higher concentrations of cholesteryl
ester induced by diet were associated with an increased risk for
atherosclerosis in African green monkeys (Carr et al. 1992
). We have also shown that smaller LDL particles resulting
from dietary interventions are removed by plasma more rapidly
(Fernandez et al. 1993
).
In guinea pigs, the plasma cholesterol lowering induced by pectin is
proportional to the dose of dietary pectin and dependent on the
depletion of hepatic cholesterol induced by the action of fiber in the
intestinal lumen (Fernandez et al. 1994
). This indicates
that pectin is possibly delaying the delivery of cholesterol to the
liver by affecting cholesterol absorption or by interrupting the
enterohepatic circulation of bile acids (Fernandez 1995
). In addition, when hepatic enzyme activities were
determined in guinea pigs fed the higher doses of dietary fiber and
compared to the lower dose, we found increases in HMG-CoA reductase
and a dose response in cholesterol 7-hydroxylase activity similar to
our previous reports (Fernandez et al. 1994
,
Fernandez 1995
). These effects of fiber on the
regulatory enzymes of cholesterol synthesis and catabolism indicate a
compensatory response to produce more cholesterol and bile acids
because of the pectin altering the enterohepatic circulation of bile
acids and increasing the demand for bile acid synthesis. In addition,
hepatic ACAT activity decreased in guinea pigs fed the higher doses of
pectin in a dose-dependent manner, indicating that ACAT activity is
related to the availability of free cholesterol in the liver, which was
altered by the intake of pectin as was observed in guinea pigs
(Fernandez et al. 1994
).
These data suggest that the alterations in hepatic cholesterol pools by dietary factors are associated with different mechanisms, which warrant further investigation. Although plasma cholesterol was decreased both by soybean protein intake and higher amounts of pectin, the hypocholesterolemic mechanisms are likely to be different.
The intake of fiber decreases the delivery of cholesterol to the liver
through the chylomicron remnants and decreases hepatic cholesterol via
increases in its catabolic pathway in hamsters and guinea pigs
(Fernandez 1995
, Horton et al. 1994
). In
guinea pigs, alterations in lipoprotein secretion, remodeling, and
catabolism by soluble fiber were also demonstrated (Fernandez et al. 1997
). Other than increases in fecal steroid secretion
(Hayashi et al. 1994
, Huff and Carroll 1980
), it is not clear how the source of dietary
protein exerts its hypocholesterolemic action.
Hamsters and guinea pig responses to dietary cholesterol.
The responses in plasma TC to dietary cholesterol were similar in
hamsters and guinea pigs. However, if we analyze the effects on the
lipoproteins, species differences were evident. In guinea pigs, there
were increases in plasma LDL cholesterol concentrations caused by
intake of increasing doses of dietary cholesterol. In hamsters, the
plasma HDL cholesterol also increased so that it accounted for 50% of
the total cholesterol at the highest dose tested (0.16 g/100g), where
the highest plasma LDL cholesterol concentrations also occurred. In
addition, in hamsters, there was a significant increase in plasma TAG
caused by increasing dietary cholesterol. In hamsters, high plasma TAG
and VLDL cholesterol caused by dietary cholesterol were observed by
other investigators (Sessions et al. 1993
,
Woollett et al. 1992
). The elevated plasma
non-HDL cholesterol fraction observed in the present study may also
be caused by elevations in VLDL cholesterol.
The observed changes in VLDL and LDL composition caused by increasing
levels of dietary cholesterol were previously reported in guinea pigs
(Fernandez et al. 1997
, Lin et al. 1994
).
Cholesteryl ester-enriched VLDL from hyperlipidemic subjects was
demonstrated to be more easily converted to intermediate-density
lipoprotein and then to LDL through the delipidation cascade
(Nestel et al. 1983
). In addition, studies in African
green monkeys have demonstrated that high-cholesterol diets lead to
the formation of cholesteryl ester-enriched VLDL that are readily
converted to LDL (Marzetta et al. 1989
). Our present
observations of guinea pigs agree with these reports because
those guinea pigs with the highest proportion of cholesteryl
esters in VLDL also had the highest plasma LDL cholesterol
concentrations.
Hepatic cholesterol concentrations were increased proportionately to
dietary cholesterol; however, significant increases were observed only
with the highest dose. Hamsters and guinea pigs have different
distributions of hepatic cholesterol in response to a cholesterol
challenge. Whereas in guinea pigs the majority of the hepatic
cholesterol exists in the free form, in hamsters most of it exists as
esterified cholesterol. In humans, the majority of the cholesterol in
liver is in the free form (Angelin et al. 1992
).
Other important differences between guinea pigs and hamsters are the responses of regulatory enzymes of cholesterol metabolism to high dietary cholesterol. In guinea pigs, HMG-CoA reductase activity was suppressed, even after concentrations of 0.08 g/100g dietary cholesterol, which is equivalent to 600 mg of dietary cholesterol per day. But, in hamsters the enzyme activity was suppressed only after feeding pharmacological doses of dietary cholesterol.
Interestingly, hepatic cholesterol 7-hydroxylase activity was not
up-regulated by high cholesterol diets in guinea pigs, and hamsters
as was reported for rats (Horton et al. 1995
). Rats have
the ability to convert excess dietary cholesterol to bile acids. The
activity of this bile acid synthesis regulatory enzyme is
extremely high, even in the absence of dietary cholesterol
(Horton et al. 1995
). The inability of hamsters and
guinea pigs to respond to excess dietary cholesterol by increasing
hepatic cholesterol catabolism may be one of the reasons why these two
animal models are more responsive than rats to dietary cholesterol.
Thus in hamsters and guinea pigs, the responses to dietary cholesterol
are more consistent with that in humans.
In guinea pigs, ACAT activity was regulated by the increases of free cholesterol concentrations in the liver. However, in hamsters, no effect on this enzyme activity was observed, even in those hamsters in which the concentration of free cholesterol in the liver was increased. Guinea pigs presented higher ACAT activity with higher levels of dietary cholesterol possibly caused by the increased substrate (free hepatic cholesterol) for this enzyme, which occurs when they were fed high cholesterol diets.
We have demonstrated previously that elevations in hepatic cholesterol
concentrations caused by high intake of dietary cholesterol result in
the suppression of hepatic apo B/E receptors in guinea pigs (Lin et al. 1994
). Suppression of LDL receptors is the main cause of
elevated plasma LDL cholesterol in this animal model. In hamsters,
decreases in mRNA levels for the apo B/E receptor that are associated
with decreases in LDL uptake were also reported with high intakes of
dietary cholesterol (Horton et al. 1993
). These reports
and our present data suggest that dietary cholesterol increases the
concentrations of hepatic cholesterol. As a compensatory mechanism, in
guinea pigs these higher concentrations of cholesterol in the liver
suppress HMG-CoA reductase activity and increase ACAT activity,
possibly because of increases in substrate availability.
However, the suppression of LDL receptors by dietary cholesterol
appears to be the major mechanism responsible for the elevated plasma
LDL cholesterol concentrations (Horton et al. 1993
,
Lin et al. 1994
).
From these studies we conclude that hamsters and guinea pigs respond to dietary treatment by lowering plasma LDL cholesterol, which is similar to humans. Based on the data presented here, we speculate that the increases in plasma cholesterol levels in response to dietary cholesterol are affected in the presence of other dietary components, such as dietary soluble fiber and the source of protein. Thus, diets containing vegetable protein or relatively high amounts of dietary fiber lessen the elevations in plasma cholesterol concentrations induced by high amounts of dietary cholesterol.
|
FOOTNOTES |
|---|
2 To whom reprint requests should be addressed.
4 Abbreviations used: ACAT, acyl coenzyme A
cholesterol acyltransferase; HDL-C, HDL-cholesterol;
HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A reductase;
non-HDL-C, non-HDL cholesterol; TAG, triacylglycerol; TC, total
cholesterol.
Manuscript received November 12, 1998. Initial review completed December 22, 1998. Revision accepted March 10, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. Allain C. C., Chan C.G.S, Poon L. C., Richard W., Fu P. C. Enzymatic determination of total serum cholesterol. Clin. Chem. 1974;20:470-475[Abstract]
2. Angelin B., Olivecrona H., Reihner E. Hepatic cholesterol metabolism in estrogen-treated man. Gastroenterology 1992;103:1657-1663[Medline]
3. Badimon J. J., Badimon L., Fuster V. Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J. Clin. Invest. 1990;85:1234-1241
4. Bucolo G., David H. Quantitative determinations of serum triglycerides by the use of enzymes. Clin. Chem. 1973;19:476-482[Abstract]
5. Carr T., Andressen C. J., Rudel L. L. Enzymatic determination of triglyceride, free cholesterol and total cholesterol in tissue lipid extracts. Clin. Chem. 1993;26:39-42
6.
Carr T. P., Parks J. S., Rudel L. L. Hepatic ACAT activity in African green monkeys is highly correlated to plasma LDL cholesteryl ester enrichment and coronary artery atherosclerosis. Arterioscler. Thromb. 1992;12:1274-1283
7. Carroll K. K. Hypercholesterolemia and atherosclerosis: effects of dietary protein. Fed. Proc. 1982;41:2792-2796[Medline]
8. Chapman J. M., Mills G. L., Ledford J. F. The distribution and partial characterization of the serum apolipoproteins in the guinea pig. Biochem. J 1975;149:423-426[Medline]
9.
Daley S. J., Klemp K. F., Guyton J. R., Rogers K. A. Cholesterol-fed and casein-fed rabbit models of atherosclerosis. Arterioscler. Thromb. 1994;14:105-114
10. Fernandez M. L. Distinct mechanisms of plasma LDL lowering by dietary fiber in the guinea pig: Specific effects of pectin, guar gum, and psyllium. J. Lipid Res. 1995;36:2394-2404[Abstract]
11. Fernandez M. L., McNamara D. J. Regulation of cholesterol and lipoprotein metabolism in guinea pigs mediated by dietary fat quality and quantity. J. Nutr. 1991;121:934-943
12.
Fernandez M. L., Abdel-Fattah G., McNamara D. J. Dietary fat saturation modifies the metabolism of LDL sub-fractions in guinea pigs. Arterioscler. Thromb. 1993;13:1418-1428
13.
Fernandez M. L., Sun D. M., Tosca M. A., McNamara D. J. Citrus pectin and cholesterol interact to regulate hepatic cholesterol homeostasis and lipoprotein metabolism: A dose-response study in guinea pigs. Am. J. Clin. Nutr. 1994;59:869-878
14.
Fernandez M. L., Vergara-Jimenez M., Conde K., Behr T., Abdel-Fattah G. Regulation of apolipoprotein B-containing lipoproteins by dietary soluble fiber in guinea pigs. Am. J. Clin. Nutr. 1997;65:814-822
15. Hayashi S., Miyazaki Y., Yamashita J., Nakagawa M., Takizawa H. Soy protein has no hypocholesterolemic action in mice because it does not stimulate fecal steroid excretion in that species. Cell. Molec. Biol. 1994;40:1021-1028[Medline]
16. Horton J. D., Cuthbert J. A., Spady D. K. Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels. J. Clin. Invest. 1993;92:743-749
17.
Horton J. D., Cuthbert J. A., Spady D. K. Regulation of hepatic 7-hydroxylase activity by dietary psyllium in the hamster. J. Clin. Invest. 1994;93:2084-2092
18.
Horton J. D., Cuthbert J. A., Spady D. K. Regulation of hepatic 7-hydroxylase expression and response to dietary cholesterol in the rat and hamster. J. Biol. Chem. 1995;270:5381-5387
19.
Hu F. B., Stramfer M. J., Manson J. Z. Dietary fat intake and the risk of coronary heart disease in women. N. Eng. J. Med. 1997;337:1491-1499
20. Huff M. W., Carroll K. K. Effects of dietary proteins in turnover, oxidation and absorption of cholesterol and on steroid excretion in rabbits. J. Lipid Res. 1980;21:546-558[Abstract]
21.
Jelinek D. E., Andersson D., Slaughter C. A., Russell D. D. Cloning and regulation of cholesterol 7-hydroxylase: The rate limiting enzyme in the bile acids biosynthesis. J. Biol. Chem. 1990;265:8190-8197
22. Jenkins D. J., Popovich D. G., Kendall C. W., Vidgen E., Tariq N., Ransom T. P., Wolever T. M., Vuksan V., Mehling C. C., Boctor D. L., Bolognesi C., Huang J., Patten R. Effect of a diet high in vegetables, fruits and nuts on serum lipids. Metabolism 1997;46:530-537[Medline]
23. Krause B. R., Newton R. S. Animal models for the evaluation of inhibitors of HMG-CoA reductase. Adv. Lipid Res. 1991;1:57-72
24. Lin E.C.K., Fernandez M. L., McNamara D. J. Dietary fat type and cholesterol quantity interact to affect cholesterol metabolism in guinea pigs. J. Nutr. 1992;122:2019-2029
25. Lin E.C.K., Fernandez M. L., Tosca M. A., McNamara D. J. Regulation of hepatic LDL metabolism in the guinea pig by dietary fat and cholesterol. J. Lipid Res. 1994;35:446-457[Abstract]
26. Markwell M.A.K., Hass S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 1978;87:206-210[Medline]
27. Marzetta C. A., Johnson F. L., Zelch L. A., Foster D. M., Rudel L. L. Metabolic behavior of hepatic VLDL and plasma LDL apo B-100 in African green monkeys. J. Lipid Res. 1989;31:1149-1159[Abstract]
28.
Masarei J.R.L., Rouse I. L., Lynch W. J., Robertson K., Vandongen R., Beilin L. J. Effects of a lactovegetarian diet on serum concentrations of cholesterol, triglyceride, HDL-C, HDL-2-C: HDL-3-C, apoprotein B and Lp(a). Am. J. Clin. Nutr. 1984;40:468-479
29. McNamara D. J., Kolb R., Parker T. S., Batwin H, Samuel P., Brown C. D., Ahrens E. H., Jr Heterogeneity of cholesterol homeostasis in man. Responses to changes in dietary fat quality and cholesterol quantity. J. Clin. Invest. 1987;79:1729-1739
30.
Nagata C, Takatsuka N., Kurisu Y., Shimizu H. Decreased serum total cholesterol concentration is associated with high intake of soy products in Japanese men and women. J. Nutr. 1998;128:209-213
31. Nestel P., Billington T., Tada N., Nugent P., Fidge N. Heterogeneity of very-low-density lipoprotein metabolism in hyperlipidemic subjects. Metabolism 1983;32:810-817[Medline]
32.
Nicolosi R. J. Dietary fat saturation effects on low density lipoprotein concentration and metabolism in various animal models. Am. J. Clin. Nutr. 1997;65:1617S-1627S
33. Nicolosi R. J., Wilson T. A. The anti-atherogenic effect of dietary soybean protein concentrate in hamsters. Nutr. Res. 1997;17:1457-1467
34. Nicolosi R. J., Wilson T. A., Krause B. R. The ACAT inhibitor, C1–1011 is effective in the prevention and regression of aortic fatty streak area in hamsters. Atherosclerosis 1998;137:77-85[Medline]
35. Otto J., Ordovas J. M., Smith D., van Dongen D., Nicolosi R. J., Schaefer E. J. Lovastatin inhibits diet induced atherosclerosis in F1B Golden Syrian hamsters. Atherosclerosis 1995;114:19-28[Medline]
36. Sessions V. A., Martin A., Gomez-Munoz A., Brindley D. N., Salter A. M. Cholesterol feeding induces hypertriglyceridaemia in hamsters and increases the activity of the Mg 2+-dependent phosphatidate phospholydrolase in liver. Biochim. Biophys. Acta 1993;1166:238-243[Medline]
37. Shapiro D. L., Imblum R. L., Rodwell V. W. Thin-layer chromatographic assay for HMG-CoA reductase and mevalonic acid. Anal. Biochem. 1969;31:483-390
38.
Shefer S. L., Nguyen B., Salen G., Ness C., Chowdhary I. R., Lerner S., Batta A. K., Tint G. S. Differing effects of cholesterol and taurocholate on steady state hepatic HMG-CoA reductase and cholesterol 7-hydroxylase activities and mRNA levels in the rat. J. Lipid Res. 1992;33:1193-1200[Abstract]
39. Smith J. L., Jersey J., Pillar S. P., Hardie I. R. Hepatic acyl-CoA cholesterol acyltransferase. Development of a standard assay and determination in patients with cholesterol gallstones. Clin. Chem Acta 1986;158:271-282[Medline]
40. Spady D. K., Dietschy J. M. Interaction of dietary cholesterol and triglycerides in the regulation of hepatic low density lipoprotein transport in the hamster. J. Clin. Invest. 1988;81:300-309
41. Sullivan M. P., Cerda J. J., Robbins F. L., Burgin C. W., Beatty R. J. The gerbil, hamster, and guinea pig as rodent models for hyperlipidemia. Lab. An. Sci. 1993;43:575-578
42. Terpstra A.H.M., Holmes J. C., Nicolosi R. J. The hypocholesterolemic effect of dietary soybean protein vs. Casein in hamsters fed cholesterol-free or cholesterol-enriched semipurified diets. J. Nutr. 1991;121:944-947
43. Van Tol A., Van Gent T., Scheek L. M. Lipoprotein structure and metabolism during progression and regression of atherosclerosis in pigs fed with fish oil-derived fatty acids. Adv. Exp. Med. Biol. 1991;285:417-421[Medline]
44.
Warnick G. R., Benderson J., Albers J. J. Dextran-sulphate-Mg2+ precipitation procedure for quantitation of high density lipoprotein cholesterol. Clin. Chem. 1982;28:1379-1388
45.
Weingand K. W., Daggy B. P. Quantification of high-density-lipoprotein cholesterol in plasma from hamsters by differential precipitation. Clin. Chem. 1990;36:575
46. Woollett L. A., Spady D. K., Dietschy J. M. Saturated and unsaturated fatty acids independently regulate low density lipoprotein receptor activity and production rate. J. Lipid Res. 1992;33:77-88[Abstract]
This article has been cited by other articles:
|
|
 |
|
  T. L. Zern, K. L. West, and M. L. Fernandez Grape Polyphenols Decrease Plasma Triglycerides and Cholesterol Accumulation in the Aorta of Ovariectomized Guinea Pigs J. Nutr., July 1, 2003; 133(7): 2268 - 2272. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. West, T. Ramjiganesh, S. Roy, B. T. Keller, and M. L. Fernandez 1-[4-[4[(4R,5R)-3,3-Dibutyl-7-(dimethylamino)-2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-5-yl]phenoxy]butyl]-4-aza-1-azoniabicyclo[2.2.2]octane Methanesulfonate (SC-435), an Ileal Apical Sodium-Codependent Bile Acid Transporter Inhibitor Alters Hepatic Cholesterol Metabolism and Lowers Plasma Low-Density Lipoprotein-Cholesterol Concentrations in Guinea Pigs J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 293 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Romero, K. L. West, T. Zern, and M. L. Fernandez The Seeds from Plantago ovata Lower Plasma Lipids by Altering Hepatic and Bile Acid Metabolism in Guinea Pigs J. Nutr., June 1, 2002; 132(6): 1194 - 1198. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Fernandez Guinea Pigs as Models for Cholesterol and Lipoprotein Metabolism J. Nutr., January 1, 2001; 131(1): 10 - 20. [Abstract] [Full Text]
|
|
|
|
|
|
S. Roy, S. Vega-Lopez, and M. L. Fernandez Gender and Hormonal Status Affect the Hypolipidemic Mechanisms of Dietary Soluble Fiber in Guinea Pigs J. Nutr., March 1, 2000; 130(3): 600 - 607. [Abstract] [Full Text]
|
|
| |||||||||||
