© 1995 American Heart Association, Inc.
Articles |
Effect of Dietary Cholesterol and Fat on the Expression of Hepatic Sterol 27-Hydroxylase and Other Hepatic Cholesterol-Responsive Genes in Baboons (Papio Species)
From the Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, San Antonio, Tex.
Correspondence to Rampratap S. Kushwaha, PhD, Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, PO Box 28147, San Antonio, TX 78228-0147. E-mail kush@darwin.sfbr.org.
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Abstract Our studies of baboons with low and high responses to dietary cholesterol and fat suggest that low-responding baboons increase the activity of hepatic sterol 27-hydroxylase, an important enzyme of bile acid synthesis, considerably more than do high-responding baboons when challenged with a high-cholesterol, high-fat (HCHF) diet. The present studies were conducted to determine whether hepatic sterol 27-hydroxylase mRNA levels and plasma 27-hydroxycholesterol concentrations also differed with dietary responsiveness. Sixteen adult male baboons with a wide range of VLDL cholesterol plus LDL cholesterol (VLDL+LDL cholesterol) response to an HCHF diet were selected. They were examined first while on a chow diet and then after 1, 3, 6, 10, 18, 26, 36, 52, 72, and 104 weeks on the HCHF diet. Plasma and lipoprotein cholesterol concentrations increased rapidly during the first 3 weeks and stabilized thereafter. On the basis of the response in VLDL+LDL cholesterol, we selected five low-responding, four medium-responding, and five high-responding baboons for more intensive study in more detail. In low responders, the major increase in serum cholesterol concentration was in HDL cholesterol, whereas in medium and high responders it was in both VLDL+LDL and HDL cholesterol. In low and medium responders, serum or VLDL+LDL cholesterol did not change after 3 weeks of consumption of the HCHF diet, whereas in high responders VLDL+LDL cholesterol declined between 78 and 104 weeks. In low and medium responders, plasma 27-hydroxycholesterol concentrations and hepatic sterol 27-hydroxylase mRNA levels increased rapidly during the first 10 weeks, declined thereafter, and stabilized at 26 weeks in low responders and at 40 weeks in medium responders. In high responders, plasma 27-hydroxycholesterol concentration did not increase. The increases in hepatic sterol 27-hydroxylase mRNA and plasma 27-hydroxycholesterol in medium responders were intermediate between those in high- and low-responding baboons. The expression of hepatic mRNA for other cholesterol-responsive genes did not differ between high- and low-responding baboons. Plasma LDL cholesterol concentrations were negatively correlated with plasma 27-hydroxycholesterol concentrations and hepatic sterol 27-hydroxylase mRNA levels when the animals had been on the HCHF diet for 10 weeks. These studies suggest that hepatic sterol 27-hydroxylase may be an important regulator of responsiveness to dietary cholesterol and fat in baboons.
Key Words: bile acids • coconut oil • LDL receptor • 27-hydroxycholesterol • HMG-CoA reductase
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Introduction |
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Individuals with high and low lipemic responses to dietary cholesterol and fat have been identified among human subjects and among individuals of several species of animals.1 2 3 4 5 6 7 8 9 In general, the high-responding individuals have a considerable increase in plasma LDL cholesterol concentration when challenged with an HCHF diet, whereas low-responding individuals have very little or no increase in LDL cholesterol.9 Studies of the metabolic and molecular mechanisms of high and low responses to dietary cholesterol and fat have not suggested a single mechanism for the variability of cholesterolemic responses to diet common to all animal species,9 but variability in absorption of dietary cholesterol and in bile acid metabolism seems to be among the important mechanisms.9 10 11 12 13 14 15
Our studies of high– and low–LDL-responding baboons suggest that in
low-responding baboons the activity of hepatic sterol 27-hydroxylase,
an important enzyme of bile acid metabolism, is increased
much more than in high-responding baboons when they are challenged with
an HCHF diet.16 These observations suggest the hypothesis
that hepatic sterol 27-hydroxylase is regulated by dietary
cholesterol and fat at the transcriptional level, and that
this regulation differs between high- and low-responding baboons. The
present study measured hepatic sterol 27-hydroxylase mRNA
concentrations before and after the high-, medium-, and low-responding
baboons were challenged with the HCHF diet. We also measured the
hepatic concentration of cholesterol 7
-hydroxylase mRNA
as an indicator of the bile acid pathway initiated by that enzyme, as
well as hepatic concentrations of LDL receptor and
3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase mRNA as
indicators of hepatic capacity to catabolize LDL and to synthesize
cholesterol, respectively. The results show that sterol
27-hydroxylase is maximally expressed in low–LDL-responding baboons
during the first 10 weeks of consumption of an HCHF diet, whereas
cholesterol 7-hydroxylase expression is not affected by
the diet in low–, medium–, or high–LDL-responding baboons.
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Methods |
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Selection of Animals
We selected 16 young male baboons (Papio species) ranging from 4 to 9 years of age from a pedigreed breeding colony. Each baboon was the offspring of a different sire. They were selected so that the sires represented a wide range of lipemic responses to an HCHF diet. Each baboon was also the offspring of a different dam, except that two (X4152 and X4197) were the offspring of one dam by different sires. Between 1 and 6 years prior to this experiment, serum and lipoprotein cholesterol levels had been measured for all baboons except one (6265) after consumption of an HCHF diet for 7 weeks. Their VLDL+LDL cholesterol levels on the chow diet before the HCHF challenge diet ranged from 0.57 to 2.04 mmol/L (1.24±0.49 mmol/L, mean±SD) and 0.52 to 4.19 mmol/L (2.12±1.01 mmol/L) after dietary challenge.
About half of the baboons had been used in dietary experiments involving various oils (olive, corn, coconut, fish) for periods of no longer than 6 months, and those experiments ended no later than 21 weeks before the first blood sample for this experiment was taken, 10 weeks before the experimental diet was begun. Thus, all the animals had been consuming the basal chow diet for 31 weeks or more before the present study's diet treatment began. The animals therefore had a wide range of lipemic responses to dietary cholesterol and fat, a broad range of genetic backgrounds, and a long period of exposure to a low-fat, cholesterol-free diet when they entered this experiment.
While the baboons were consuming the chow diet, serum and lipoprotein
cholesterol concentrations were measured at 10, 7, and 4
weeks before the experimental diet was begun. The averages of these
three observations for serum, LDL, and HDL cholesterol
concentrations while the animals were on the chow diet, as well as
other characteristics of the study baboons, are given in Table 1
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The protocol of this experiment was approved by the institutional Animal Research Committee. The Southwest Foundation for Biomedical Research is accredited by the American Association for Accreditation of Laboratory Animal Care and is registered with the US Department of Agriculture.
Diets
The animals were first maintained on a
low-cholesterol (0.08 µmol/kcal) and low-fat (10% of
total calories) monkey chow diet (Purina Co) for at least 31 weeks,
after which they were fed a high-cholesterol (3.49
µmol/kcal) and high-fat (40% of total calories from coconut oil)
diet. The ingredients of the HCHF diet (Purina monkey meal 5-5046-6,
81.4%; coconut oil, 16.5%; sodium chloride, 1.1%; retinyl acetate,
0.005%; ascorbic acid 0.2%, a vitamin mixture, 1.0%; and
cholesterol, 3.49 µmol/kcal) were mixed with water and
pelleted, and the feed was stored in a freezer. The HCHF diet with
coconut oil used in these experiments showed a maximal effect on the
LDL cholesterol response in a previous
study.17 Animals were fed once a day ad libitum and had
access to water at all times.
Experimental Protocol
All 16 baboons were studied simultaneously, first
while on the basal chow diet for 10 weeks and thereafter while on the
HCHF diet for 108 weeks. While the animals were on the chow diet, blood
and liver (punch biopsy) samples were obtained at 10, 7, and 4 weeks
before the start of the HCHF diet. While the animals were on the HCHF
diet, blood and liver samples were obtained at 1, 3, 6, 10, 18, 26, 36,
52, 78, and 104 weeks. Blood samples (20 mL each) were used for the
measurement of serum and serum lipoprotein cholesterol
(fresh serum) and plasma 27-hydroxycholesterol (frozen
plasma) concentrations. Liver samples were used for the measurements of
hepatic concentrations of sterol 27-hydroxylase,
cholesterol 7-hydroxylase, LDL receptor, HMG-CoA
reductase, and albumin mRNA.
Baboons were housed in gang cages in their social groups throughout the
study. They maintained their daily physical activities. Because they
were kept in groups, daily feed intake could not be measured. However,
baboons were weighed at the time of each bleeding. There was no loss in
body weight for any baboon. As shown in Table 1
, older baboons
maintained their body weights, whereas younger baboons increased their
body weights because of normal growth.
Blood and Liver Sampling
After an overnight fast (16 to 18 hours), baboons were
immobilized with ketamine hydrochloride (17.21
µmol/kg body weight IM) and venous blood was drawn. At each blood
sampling, four 25-mg cores of liver were obtained by punch biopsy. The
liver cores for each animal were pooled, wrapped in aluminum foil,
labeled, quick-frozen in liquid nitrogen, and stored at -70°C.
Measurement of Serum and Lipoprotein
Cholesterol
Cholesterol in serum and lipoproteins was measured
by enzymatic methods as described earlier.17 Serum HDL
cholesterol was measured after precipitation of VLDL and
LDL by heparin and MnCl2 according to the Lipid Research
Clinics procedure.18 VLDL+LDL cholesterol was
expressed as the difference between total serum and HDL
cholesterol.
Measurement of 27-Hydroxycholesterol in
Plasma
27-Hydroxycholesterol in frozen plasma was
measured by high-performance liquid
chromatography as described by Harik-Khan and
Holmes19 with slight modifications.16 A small
aliquot (0.2 mL) of plasma was saponified in a mixture of sodium
hydroxide:methanol (1:9) at 85°C under reflux for an hour. The
saponified material was extracted four times with hexane after the
addition of 5-cholesten-3ß-ol-7-one (internal standard) and water.
The mixture was centrifuged and the upper hexane layer
containing the sterols was collected, pooled, and dried under nitrogen.
The dried material was redissolved in methanol and injected onto an
8C18 Resolve Radial-Pak cartridge (8 mmx115 mm, 5-µm
particle size, Waters). The mobile phase was methanol:water (9:1), and
the flow rate was kept at 3 mL/min. Peaks were detected at a wavelength
of 210 nm, and individual peaks were identified by their retention
times compared with known standards.16 The area ratio
method was used to quantitate 27-hydroxycholesterol as
described for 7-hydroxycholesterol.20
Authentic 27-hydroxycholesterol was purchased from
Research Plus, Inc, and 5-cholesten-3ß-ol-7-one and
7-hydroxycholesterol were purchased from Steraloids,
Inc. Cholesterol was purchased from Sigma Chemical Co.
A control plasma sample was included with each set of six to eight experimental plasma samples analyzed. Control plasma was obtained from a low–LDL-responding baboon at the time of necropsy and was frozen in small aliquots (0.5 to 1.0 mL) and stored at -80°C. The coefficient of variation for 27-hydroxycholesterol in a control plasma sample measured for the whole study was 4.2%.
Measurement of Hepatic mRNA
Because only 75 to 100 mg of liver was available at each time
point, we used Northern blot analysis to measure hepatic mRNA
levels for the sterol 27-hydroxylase gene and other
cholesterol-responsive genes. Total cellular RNA was
extracted by the guanidinium thiocyanate–phenol–chloroform extraction
technique.21 Total RNA samples (15 µg each) from various
time points were fractionated on denaturing agarose gels containing
methyl mercuric hydroxide22 and transferred onto a nylon
membrane (GeneScreen Plus, Du Pont) by the capillary transfer
method23 to immobilize RNA. On each gel a
control RNA sample isolated from the liver of a baboon treated with
estrogen was also fractionated. The liver sample for the control RNA
sample was obtained at the time of necropsy, quick-frozen in liquid
nitrogen, and stored at -80°C. The 1322-bp Sma I fragment
of full-length cDNA of rabbit sterol 27-hydroxylase cloned in pGEM-4
(kindly provided by Dr David Russell, University of Texas Southwestern
Medical School, Dallas) was used as a probe to carry out Northern
blotting analysis. The probe was radiolabeled by the random
priming method.24 25 Hybridization was carried out by use
of the protocol described by Denhardt.26 The blots were
exposed to X-ray film and the autoradiograms were
scanned with a laser densitometer (LKB-Ultroscan-XL). All data are
expressed as ratios between experimental samples and the control sample
run on each gel. The blot was stripped and reprobed several times for
other genes. A 2172-bp EcoRI fragment of full-length cDNA of
rat cholesterol 7-hydroxylase cloned in pBluescript
SK-pSac-7 (kindly provided by Dr David Russell) was used as a probe for
cholesterol 7-hydroxylase. A 1000-bp Pst I
fragment of full-length cDNA of human LDL receptor cloned in pTZ18R
(ATCC, No. 79013) and a 2300-bp Sph I fragment of
full-length cDNA of human HMG-CoA reductase cloned in pBR322 (ATCC,
No. 59567) were used as probes for the LDL receptor and HMG-CoA
reductase genes. For a control, we reprobed the blot with a 2000-bp
BamHI-EcoRI fragment of pILMALB5–full-length
albumin cDNA cloned into pUC19 (ATCC, No. 61357).
Statistical Analysis
Data in tables are presented as either individual values
or mean±SD. The effects of low-response and high-response
phenotypes on plasma 27-hydroxycholesterol
levels, hepatic mRNA levels, and serum and lipoprotein
cholesterol concentrations were analyzed by ANOVA
with repeated measures. Associations between serum and VLDL+LDL
cholesterol concentrations and hepatic concentrations of
LDL receptor mRNA were calculated by univariate and
multivariate regression analysis. The level of
significance was set at P
.05, but we also report
differences at P0.1 to balance between type I and type II
statistical errors.
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Results |
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Effect of Dietary Cholesterol and Fat on Serum and Lipoprotein Cholesterol
On the basis of the level of VLDL+LDL cholesterol for the first 20 weeks of the challenge diet, we divided baboons into three categories: (1) low responders, with VLDL+LDL cholesterol concentrations of <1.94 mmol/L (seven baboons); (2) medium responders, with VLDL+LDL cholesterol concentrations of 1.94 to 3.88 mmol/L (four baboons); and (3) high responders, with VLDL+LDL cholesterol concentrations of >3.88 mmol/L (five baboons). We conducted further detailed studies on all five high-responding baboons, all four medium-responding baboons, and five low-responding baboons randomly selected from the seven. The average serum and lipoprotein cholesterol concentrations in response to the HCHF diet in these five low-, four medium-, and five high-responding baboons are shown in Fig 1A
, 1B, and 1C,
respectively.
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),
VLDL+LDL (
), and HDL (
) cholesterol
concentrations (mean±SD) in response to the HCHF diet in five
low-responding baboons (A), four medium-responding baboons (B), and
five high-responding baboons (C). Low-, medium-, and high-responding
baboons were classified as such on the basis of increase in VLDL+LDL
cholesterol concentrations in response to the HCHF diet, as
described in "Results."Serum and lipoprotein cholesterol concentrations increased in all low-, medium-, and high-responding baboons during the first 3 weeks, after which there was no further increase. After 3 weeks, serum and lipoprotein cholesterol concentrations remained stable throughout the study in low- and medium-responding baboons. However, in high-responding baboons, there was a slight but significant decrease in VLDL+LDL cholesterol concentration between 36 and 104 weeks (P=.083) of consumption of the HCHF diet. HDL cholesterol concentration was higher (P<.05) than VLDL+LDL cholesterol concentration in low-responding baboons, whereas it was lower (P<.05) than the VLDL+LDL cholesterol concentration in high-responding baboons. However, because of the slow decrease in VLDL+LDL in high-responding baboons, the HDL cholesterol concentration was similar to VLDL+LDL cholesterol concentration in these animals at 104 weeks of consumption of the HCHF diet.
Effect of Dietary Cholesterol and Fat on Plasma
27-Hydroxycholesterol Concentration
There was a significant overall effect of phenotype
(low, medium, and high responders) on plasma
27-hydroxycholesterol concentrations
(P=.020). Plasma 27-hydroxycholesterol
concentrations on the HCHF diet in low-responding baboons were higher
than those in high-responding baboons at 1 (P=.087), 3
(P=.004), 6 (P=.008), 10 (P=.002), and
104 (P=.064) weeks (Fig 1). Plasma
27-hydroxycholesterol concentration on the HCHF diet in
low-responding baboons was also higher than that in medium-responding
baboons at 3 (P=.006) and 6 (P=.045) weeks. In
addition, there was a significant overall effect of time on the HCHF
diet on plasma 27-hydroxycholesterol concentration
(P=.001) as well as a significant time-by-group interaction
(P=.005). The plasma 27-hydroxycholesterol
concentration in low-responding baboons was higher at 3
(P=.025), 6 (P=.04), and 10 (P=.035)
weeks of consumption of the HCHF diet than on the chow diet. Similarly,
plasma 27-hydroxycholesterol concentration in
medium-responding baboons was higher at 26 (P=.015) and 36
(P=.032) weeks of consumption of the HCHF diet than on the
chow diet. In high-responding baboons, plasma
27-hydroxycholesterol concentrations were higher at 26
(P=.018) and 52 (P=.021) weeks of consumption of
the HCHF diet than on the chow diet.
When the low-responding baboons began to consume the HCHF diet, plasma
27-hydroxycholesterol concentration increased rapidly,
peaked at 6 to 10 weeks, declined to about 0.05 mmol/L at 26 weeks, and
remained at that level through 104 weeks (Fig 2A).
VLDL+LDL cholesterol concentration increased only slightly
and remained stable throughout the same period. When medium-responding
baboons began the HCHF diet (Fig 2B), plasma
27-hydroxycholesterol concentration increased rapidly,
but less rapidly than in low-responding baboons, and peaked in 10 weeks
at the 0.06 mmol/L level. The increased level of plasma
27-hydroxycholesterol was maintained until 36 weeks,
after which it declined and remained stable through 104 weeks. VLDL+LDL
cholesterol concentration increased more than in
low-responding baboons. In contrast, when the high-responding baboons
began the diet (Fig 2C), plasma 27-hydroxycholesterol
concentration increased only slightly during the first 10 weeks,
although VLDL+LDL cholesterol concentration increased
sevenfold. After 6 weeks, plasma 27-hydroxycholesterol
concentration increased slowly, reached the highest observed level at
52 weeks, and declined thereafter. VLDL+LDL cholesterol
concentration also declined after 78 weeks and was only threefold
higher than baseline at 104 weeks.
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Effect of Dietary Cholesterol and Fat on Hepatic mRNA
Levels of Sterol 27-Hydroxylase
Northern blot analyses showing the effect of the
HCHF diet on hepatic levels of sterol 27-hydroxylase mRNA in two
representative baboons from each low- and
high-responding group are presented in Fig 3A.
Mean values for hepatic mRNA levels for sterol 27-hydroxylase in low,
medium, and high responders are given in Table 2. A
significant overall effect of phenotype (low, medium, and high
response) on hepatic mRNA levels (P<.001), significant
differences between means (P<.001), and time-by-group
interactions (P<.001) were observed. Hepatic mRNA levels
for sterol 27-hydroxylase in high-responding baboons were not affected
by the challenge diet, whereas they were significantly increased by the
diet in low- and medium-responding baboons (Table 2). Hepatic mRNA
levels for sterol 27-hydroxylase in low-responding baboons were higher
(P<.05) than those in medium- and high-responding baboons
on the chow diet and on the HCHF diet (Table 2). Similarly, hepatic
mRNA levels for sterol 27-hydroxylase in medium-responding baboons were
higher (P<.05) than those in high-responding baboons on the
HCHF diet (Table 2).
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Effect of Dietary Cholesterol and Fat on the
Concentrations of Hepatic mRNA for Other
Cholesterol-Responsive Genes
Hepatic mRNA levels for cholesterol
7-hydroxylase were not affected by the challenge diet in either the
low- or the high-responding baboons (Table 3, Fig 3B).
However, hepatic mRNA levels for HMG-CoA reductase were decreased on
the HCHF diet (Table 3, Fig 3D). The maximum decrease in hepatic
HMG-CoA reductase mRNA level occurred at 3 weeks on the HCHF diet, and
these reduced levels were maintained for 78 weeks. The hepatic LDL
receptor mRNA level was increased in all animals at 3 weeks of
consumption of the HCHF diet (Table 3, Fig 3C). The hepatic mRNA level
for the LDL receptor returned to baseline level or lower after 10 weeks
of consumption of the HCHF diet in both high- and low-responding
baboons and remained at this level for the rest of the experiment. The
hepatic mRNA level for the LDL receptor was negatively correlated with
VLDL+LDL cholesterol concentrations on the chow diet
(r=-.538, P=.100) and at 3 weeks on the HCHF
diet (r=-.574, P=.083) but not at 10 and 78
weeks on the HCHF diet. Hepatic mRNA levels for albumin, which
was used as a control, were not affected by the dietary
cholesterol and fat (Table 3, Fig 3E).
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Relationship of LDL Cholesterol With Plasma
27-Hydroxycholesterol and Hepatic mRNA for Sterol
27-Hydroxylase
Serum VLDL+LDL cholesterol concentration was
negatively correlated with plasma 27-hydroxycholesterol
concentration on the chow diet (r=-.537, P=.048)
and after consumption of the HCHF diet for 3 (r=-.774,
P=.001) and 10 weeks (r=-.605,
P=.022; Fig 4A). Serum VLDL+LDL
cholesterol concentration was also negatively correlated
with hepatic levels of sterol 27-hydroxylase mRNA on the chow diet and
on the HCHF diet at all time points (P<.01) studied (data
for 10 weeks are shown in Fig 4B; r=-.677,
P=.008). Plasma 27-hydroxycholesterol
concentration was positively correlated with hepatic levels of sterol
27-hydroxylase mRNA (r=.659, P=.011) at 10 weeks
of consumption of the HCHF diet.
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Discussion |
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Summary of Results
Our previous studies in low- and high-responding baboons suggested that the activity of sterol 27-hydroxylase, an important enzyme of bile acid synthesis, is increased by an HCHF diet in low-responding baboons but not in high-responding baboons.16 The present results indicate that increased activity of hepatic 27-hydroxylase in low-responding baboons is associated with increased hepatic mRNA levels, and that the increase reaches a maximum level after the diet is consumed for 3 to 10 weeks. The parallel elevations in concentrations of 27-hydroxycholesterol in plasma and of sterol 27-hydroxylase mRNA in liver in low-responding baboons suggest that the plasma 27-hydroxylase concentration reflects hepatic expression of the sterol 27-hydroxylase gene.
In contrast, neither plasma 27-hydroxycholesterol nor hepatic sterol 27-hydroxylase mRNA levels increase appreciably under the same conditions in high-responding baboons, a difference suggesting that ability to increase expression of the hepatic sterol 27-hydroxylase gene in response to a dietary challenge may be the underlying metabolic difference between the two phenotypes. Medium-responding baboons were intermediate between low- and high-responding baboons in terms of increasing plasma 27-hydroxycholesterol concentration and hepatic sterol 27-hydroxylase mRNA levels. Thus, medium-responding baboons may be heterozygous for a variant of the sterol 27-hydroxylase gene or for another gene that modulates the response of the sterol 27-hydroxylase gene to dietary cholesterol and fat.
Plasma LDL cholesterol concentrations were negatively correlated with plasma 27-hydroxycholesterol concentrations and hepatic sterol 27-hydroxylase mRNA, observations that suggest that sterol 27-hydroxylase may be associated with LDL cholesterol responsiveness to diet. Because 27-hydroxycholesterol concentration at 10 weeks was positively correlated with hepatic mRNA levels of sterol 27-hydroxylase, plasma 27-hydroxycholesterol concentration during the early phase of dietary challenge may be used as a marker for the degree of responsiveness to diet.
Role of Sterol 27-Hydroxylase and Cholesterol
7-Hydroxylase in Bile Acid Synthesis
Sterol 27-hydroxylase is a mitochondrial enzyme that initiates the
modification of the side chain of cholesterol in the bile
acid synthesis pathway.27 28 Reduced activity of sterol
27-hydroxylase in humans causes cerebrotendinous xanthomatosis, a rare
disease characterized by tendon xanthomas, premature
atherosclerosis, and cataracts.29 A number
of point mutations in the sterol 27-hydroxylase gene have been reported
in subjects with cerebrotendinous xanthomatosis.30 31 32 33
Thus, sterol 27-hydroxylase is an important enzyme of bile acid
synthesis.
Cholesterol 7-hydroxylase is a microsomal enzyme that
catalyzes the initial and rate-limiting step of the bile acid
pathway.27 28 However, the presence of both
7-hydroxycholesterol and
27-hydroxycholesterol in the plasma of baboons and
humans and in the liver of baboons suggests that both
cholesterol 7-hydroxylase and sterol 27-hydroxylase may
catalyze initial steps in the bile acid synthetic
pathway.16 19 34 Martin et al35 suggest that
there are two pathways of bile acid synthesis: one starting with
7-hydroxylation of cholesterol catalyzed by
cholesterol 7-hydroxylase and the other starting with
27-hydroxylation of cholesterol catalyzed by sterol
27-hydroxylase. They also provide evidence for another enzyme,
27-hydroxycholesterol 7-hydroxylase, that catalyzes
the 7-hydroxylation of 27-hydroxycholesterol. The
relative contribution of each pathway to bile acid synthesis in the
liver has not been established in any animal species.
Because hepatic mRNA for liver sterol 27-hydroxylase increases
threefold whereas mRNA for cholesterol 7-hydroxylase
does not change in low-responding baboons upon feeding of an HCHF diet,
the sterol 27-hydroxylase pathway may be the major pathway for bile
acid synthesis in low-responding baboons consuming this diet. The
ability of low-responding baboons to induce the sterol 27-hydroxylase
pathway upon consumption of an HCHF diet may be responsible for the
attenuated dietary response.
Regulation of Plasma
27-Hydroxycholesterol
The mRNA for sterol 27-hydroxylase is expressed in both hepatic
and extrahepatic cells, whereas the mRNA for cholesterol
7-hydroxylase is expressed only in liver.36 37 38 Thus,
plasma 27-hydroxycholesterol is derived from both
hepatic and extrahepatic cells. A rapid increase in plasma
27-hydroxycholesterol concentration paralleled
the increase in hepatic mRNA levels for sterol 27-hydroxylase in
low-responding baboons. However, after 10 weeks of the HCHF diet,
plasma 27-hydroxycholesterol concentration decreased as
quickly as it had increased in low-responding baboons. There are two
possibilities for this decrease: the enzyme induction was reversed
because of the accumulation of the reaction product or utilization
of the 27-hydroxycholesterol by another enzyme in the
bile acid pathway increased. It is not likely that enzyme induction was
reversed because (1) the hepatic mRNA concentrations for sterol
27-hydroxylase in low-responding baboons remained elevated over
baseline levels (and over levels in high-responding baboons) after 10
weeks of the HCHF diet (Table 2) and (2) sterol 27-hydroxylase activity
is positively correlated with hepatic mRNA concentrations (R.S.K., PhD,
et al, unpublished data, 1994). More likely, increased substrate
induced the next enzyme of the pathway, and
27-hydroxycholesterol was converted to bile acids.
Because hepatic mRNA for 7-hydroxylase did not change on the HCHF
diet, it is unlikely that 7-hydroxylase catalyzes this step. The
7-hydroxylation of 27-hydroxycholesterol may be
catalyzed by 27-hydroxycholesterol 7-hydroxylase, as
proposed by Martin et al.35 However, this enzyme has not
been cloned and we were unable to quantify its activity in a small
amount of liver. Further studies are needed to confirm this hypothesis
and to determine whether the decrease in plasma
27-hydroxycholesterol after 10 weeks of an HCHF diet in
low-responding baboons is associated with increased bile acid
synthesis.
Regulation of LDL Receptor and HMG-CoA Reductase Transcription by
Dietary Cholesterol and Fat
LDL receptor and HMG-CoA reductase mRNA levels were measured as
indicators of the effect of dietary cholesterol and fat on
hepatic LDL catabolism and hepatic cholesterol synthesis,
respectively.39 Hepatic LDL receptor mRNA level was
paradoxically increased at 3 weeks of consumption of the HCHF diet,
especially in low-responding baboons (Table 3). However, hepatic LDL
receptor mRNA levels were decreased after 3 weeks and did not differ
between high- and low-responding baboons. Hepatic levels of HMG-CoA
reductase mRNA also decreased rapidly on the HCHF diet in both high-
and low-responding baboons. These observations suggest that hepatic LDL
receptor mRNA concentration and hepatic cholesterol
synthesis are not responsible for the difference in dietary
response.
Adaptation to HCHF Diet in High-Responding Baboons
In several long-term experiments involving baboons fed HCHF diets,
we have observed that serum cholesterol concentrations
increase rapidly, as in this experiment; they remain stable for 1 to
1.5 years; and thereafter they decline slowly.40 41 The
decrease is largely in LDL cholesterol. In some animals
there is a decline to baseline (chow) levels by 2 years. It is common
knowledge among investigators working with diet-induced
hyperlipidemia and atherosclerosis in
other nonhuman primates and rabbits that serum cholesterol
levels decline after an HCHF diet has been consumed for more than 1 to
2 years.42 The present experiment again shows this
previously observed phenomenon of adaptation to an HCHF diet by
high-responding baboons. The mean VLDL+LDL cholesterol
concentration in the five high responders declined from a high of 4.68
mmol/L at 6 weeks to 3.51 mmol/L at 104 weeks (trend with time [36 to
104 weeks], P=.083) (Fig 1C).
Conclusions
Our results are consistent with the hypothesis that the
ability to induce hepatic sterol 27-hydroxylase at the transcriptional
level is responsible for the low–LDL responder phenotype.
However, further studies are needed to determine whether the induction
of hepatic sterol 27-hydroxylase is associated with increased bile acid
synthesis and whether the maximum increase in bile acid synthesis
occurs when plasma 27-hydroxycholesterol levels begin
to decrease after their initial rise.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-34982 and HL-41256 and contract HV-53030 from the National Heart, Lung, and Blood Institute. We thank Dr S.Q. Hasan for the measurements of 27-hydroxycholesterol concentrations and Dr Glen E. Mott for the measurement of serum and lipoprotein cholesterol concentrations.
Received January 25, 1995; accepted June 15, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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