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Departments of Medicine and Biochemistry and The John P. Robarts Research Institute (J.R.B., L.J.W., D.E.T., S.J.K., M.W.H.), University of Western Ontario, London, Ontario, N6A 5K8 Canada; Department of Medicine (P.H.R.B.), University of Western Australia, Perth, Western Australia, 6001 Australia; and Parke-Davis Pharmaceutical Research (R.S.N.), Warner Lambert Company, Ann Arbor, Michigan 48105
Address all correspondence and requests for reprints to: Murray W. Huff, The John P. Robarts Research Institute, 4–16, University of Western Ontario, 100 Perth Drive, London, Ontario N6A 5K8, Canada. E-mail: mhuff{at}julian.uwo.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ApoB kinetic studies have demonstrated that HMG-CoA reductase inhibitors decrease LDL apoB production rates (PRs) (10, 11, 12). Furthermore, there is increasing evidence in both experimental animals and humans that HMG-CoA reductase inhibitors lower apoB concentrations by decreasing hepatic VLDL apoB secretion into plasma (10, 11, 12). However, not all studies have been consistent with this concept (10, 11, 12). Although the HMG-CoA reductase inhibitors have similar chemical structures, they differ in their chemical derivation, solubility, and pharmacokinetic behavior (4, 5, 6, 7, 8, 9, 13, 14). Therefore, the differences in apoB secretion observed in vivo among reported studies may, in part, pertain to the type and dose of HMG-CoA reductase inhibitor administered. However, other factors, including subject variability and study design, methods, and analysis, could contribute to the inconsistent findings.
Although, atorvastatin and simvastatin are both lipophilic compounds, they differ significantly in their elimination half-lives (t1/2). Atorvastatin is a synthetic HMG-CoA reductase inhibitor with a mean plasma t1/2 of approximately 14 h (8). Moreover, due to active metabolites, the t1/2 of HMG-CoA reductase inhibitor equivalents with atorvastatin treatment extends to 20–30 h. In contrast, simvastatin is an inactive prodrug that is activated to the open acid form in the liver. The plasma t1/2 for simvastatin is 2 h for its active ß-hydroxyacid metabolite (5). Clinical studies, using a reduction in LDL cholesterol concentration as an endpoint, have demonstrated an increased dose efficacy of atorvastatin over simvastatin (15), and other statins (16) within their approved dose ranges in subjects with hypercholesterolemia.
Watts et al. (17) found a direct correlation between cholesterol synthesis and hepatic secretion of VLDL apoB in normolipidemic subjects. However, no significant correlation was observed between plasma LDL cholesterol concentrations and hepatic apoB secretion. Naoumova et al. (18) in subjects heterozygous for familial hypercholesterolemia (FH) treated with HMG-CoA reductase inhibitors (including atorvastatin and simvastatin) proposed that the higher the baseline level of plasma mevalonate, the greater the response in terms of LDL cholesterol lowering. However, no significant relationship was observed between LDL cholesterol and plasma mevalonate. Recently, the same investigators (19) demonstrated in heterozygous FH subjects that atorvastatin and simvastatin initially reduce cholesterol synthesis to a similar extent; however, the suppression is extended with atorvastatin treatment. They proposed that the greater efficacy of atorvastatin was a result of more prolonged inhibition of HMG-CoA reductase, an effect related to its hepatic selectivity and longer duration of action in the liver.
Plasma lipoprotein distribution, composition, and apoB metabolism in pigs and humans are similar (20, 21). A large portion of LDL, the major cholesterol-containing lipoprotein, is synthesized by the direct pathway in pigs (22), a pathway also observed in humans (23). We have previously shown using apoB kinetic studies in the miniature pig that lovastatin treatment reduced LDL apoB production, primarily direct LDL synthesis (24, 25). No significant changes in VLDL apoB metabolic parameters were observed. Furthermore, in the same animal model, atorvastatin treatment significantly decreased hepatic VLDL apoB secretion into plasma in addition to reducing LDL apoB PRs (26). Although the dose of inhibitor and diet differed between the studies, we postulated that the effect on VLDL apoB secretion may relate to a more sustained inhibition of HMG-CoA reductase with atorvastatin compared with lovastatin treatment.
Hepatic apoB containing lipoprotein secretion into plasma can be regulated by lipid availability (10, 27, 28, 29, 30). HMG-CoA reductase inhibition should limit the availability of free cholesterol and/or cholesteryl ester for incorporation into VLDL and hence reduce VLDL secretion. In the present studies, two potent HMG-CoA reductase inhibitors, atorvastatin and simvastatin, at high dose, were used as probes to test the hypothesis in vivo, that the magnitude of decrease in hepatic VLDL apoB secretion is determined by the extent of HMG-CoA reductase inhibition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Atorvastatin or simvastatin was placed in gelatin capsules and, to ensure ingestion, was administered by hand before the daily feeding. The HMG-CoA reductase inhibitors were given at 0900 h each day. During the control phase pigs received a placebo capsule. Each animal received a 590-g ration of a diet containing fat (34% of calories; polyunsaturated-monounsaturated-saturated fatty acid ratio of 1:1:1) and cholesterol (400 mg/day; 0.1%; 0.2 mg/kcal) (26). Control and treatment lipid and lipoprotein concentrations were measured as described below.
Lipoprotein turnover studies
Lipoprotein turnover studies were performed essentially as
described previously (22, 24, 31) with minor modifications (26). In
brief, VLDL [density (d) < 1.006 g/ml]
and LDL (d = 1.019–1.063 g/ml) were isolated by
sequential ultracentrifugation from plasma (100–150 mL) obtained after
a 24-h fast and subsequently radiolabeled with 131I and
125I, respectively (24). All labeled lipoproteins were
autologous, i.e. each pig was reinjected with its own
lipoproteins that were isolated during the control or treatment phase,
respectively. Radiolabeling was performed using the iodine monochloride
technique (22). Of the total VLDL radioactivity, less than 2% was free
iodine, 10–44% was bound to lipid, and 30–57% of the protein-bound
label was bound to apoB. Of the total LDL radioactivity, less than 1%
was free iodine, 11–31% was bound to lipid, and 77–93% of the
protein-bound label was bound to apoB. In the control study, after a
24-h fast, each animal received 20 µCi [131I]VLDL apoB
and 15 µCi [125I]LDL apoB given as a bolus by the
indwelling catheter. In the treatment study, after a 24-h fast, each
animal received 20 µCi [131I]VLDL apoB, 15 µCi
[125I]LDL apoB, and 2.5 mCi
L-[4,5-3H]leucine (Amersham Pharmacia Biotech
Canada Ltd., Oakville, Ontario, Canada; specific activity 155 Ci/mmol)
given as a bolus by the indwelling catheter. Blood sample collection,
administration of diet and drugs during the turnover study, lipoprotein
isolation, and plasma apoB and plasma leucine determinations were as
described previously (26). VLDL, IDL, and LDL apoB concentrations were
constant over the sampling time period (24).
Kinetic analysis
The turnover data were analyzed by using the multicompartmental
modeling program SAAM II (SAAM Institute, Seattle, WA) running
on a Pentium- based personal computer. The model structure, the
assumptions made in developing the model, and the constraints applied
to the model were essentially those as previously reported (26, 31).
This model was simultaneously fit to the sets of tracer data for all
lipoprotein fractions. This approach permitted the integration of all
tracer data into a single model. Based on the lipoprotein distribution
of radioactivity in plasma samples spiked with
[131I]VLDL, the initial conditions (the initial amount of
radioactivity in each fraction) were incorporated into the
compartmental model (26).
Hepatic total and microsomal lipids
Total liver lipids were extracted from 1.0-g sections of liver
obtained at killing that had been stored at -80 C (26). Due to the
study design we were unable to obtain control liver tissue from the
experimental animals. However, control liver tissue was removed from 10
pigs obtained from the same supplier and maintained on the same diet
for the study period. Microsomes were isolated from liver homogenates
(31), and lipids were extracted from microsomes (1 mg protein) using
the method of Folch et al. (32). Total cholesterol, free
cholesterol, and triglyceride concentrations were quantitated in
hepatic total and microsomal lipid extracts (26).
Hepatic HMG-CoA reductase activity
Liver samples obtained at killing were immediately frozen in
liquid N2 and stored at -80 C until analyzed. Microsomes
were isolated from tissue homogenates (31), and HMG-CoA reductase
activities were determined using a modification of the method of
Shapiro et al. (33). In brief, microsomes (35 µg
protein/25 µl) were preincubated at 37 C for 30 min (50 mmol/liter
KPO4; 10 mmol/liter dithiothreitol; 1 mmol/liter
EDTA-Na2; pH 7.4) before incubation with 28 µl of the
reaction mixture [final concentrations: 0.037 µCi
DL-[3-14C]HMG-CoA, NEN Life Science Products (Mandel, Scientific, Guelph, Ontario, Canada), specific
activity, 57.9 mCi/mmol; 16 mmol/liter glucose-6-phosphate; 0.175 U
glucose-6-phosphate dehydrogenase; 2.5 mmol/liter NADP; 130
µmol/liter HMG-CoA; 100 mmol/liter KPO4; 5 mmol/liter
dithiothreitol; 0.5 mmol/liter EDTA-Na2; pH 7.4]. After 30
min, the reaction was stopped with 24 µl of a HCl/mevalonolactone
mixture [25 ml 6 N HCl; 90 µCi
RS-[5-3H(N)]mevalonolactone, NEN Life Science Products, 33 Ci/mmol; 800 µg mevalonolactone, Sigma Chemical Co. (St. Louis, MO); 25 ml deionized H2O].
[3H]mevalonolactone was used as an internal standard to
assess recovery. Samples were incubated at 37 C for 30 min to convert
the reaction product mevalonate to mevalonolactone and then
deproteinized by centrifugation (5 min) in a microfuge
(Eppendorf, Brinkman Instruments, Mississauga, Ontario,
Canada). Fifty microliters of the supernatant were applied to
Fisherbrand silica gel G TLC plates (Fisher Scientific,
Nepean, Ontario, Canada) and developed in acetone-toluene-acetic acid
(75:25:1, vol/vol). The plates were dried in air and the spots
visualized by I2 vapor. The region containing the
lactonized mevalonate (Rf = 0.6–0.9) was scraped from
the TLC plates and counted in Aquasol 2 (DuPont NEN,
Canberra-Packard, Mississauga, Ontario, Canada) using a liquid
scintillation counter (LS 3801, Beckman Instruments (Can) Inc., Mississauga, Ontario, Canada). HMG-CoA reductase activity
was expressed as picomoles of [14C]mevalonate
produced/min/mg of microsomal protein.
Oleate incorporation into hepatic cholesteryl ester
Liver samples obtained at killing were immediately frozen in
liquid N2 and stored at -80 C until analyzed. The activity
of hepatic acyl coenzyme A: cholesterol acyltransferase (ACAT) in crude
homogenates was determined by the rate of incorporation of
[1-14C]oleic acid (Amersham Pharmacia Biotech) into cholesteryl ester, essentially as described by
Gallo et al. (34). ACAT activity was determined in liver
homogenates, rather than microsomes, to avoid the loss of inhibitors
during microsome preparation.
Ribonuclease (RNase) protection assay for abundance of liver and
intestine apoB, LDL receptor, and HMG-CoA reductase mRNA
Liver and small intestine samples obtained at killing were
immediately frozen in liquid N2 and stored at -80 C until
analysis (26). Total RNA was isolated, its integrity verified, and
content determined. Pig specific cDNAs for apoB and LDL receptor,
cloned into Bluescript plasmid (kindly provided by Dr. Alan D. Attie,
University of Wisconsin-Madison, Madison, WI), and a
HindIII/PstI fragment of human HMG-CoA reductase
(ATCC, Manassas, VA), subcloned into Bluescript plasmid
served as templates to synthesize antisense RNA probes. These
riboprobes were then used to measure abundandance of hepatic and
intestinal apoB, LDL receptor, and HMG-CoA reductase mRNA using an
RNase protection solution hybridization assay (26).
Analyses
Fasting blood samples (20 ml) were taken on day 1, 4, and 6 of
the turnover study, and plasma concentrations of total cholesterol and
triglyceride, VLDL cholesterol and triglyceride, and high density
lipoprotein (HDL) cholesterol were measured (24). VLDL was obtained
after ultracentrifugation at d < 1.006 g/ml, and HDL
was obtained after precipitation of other lipoproteins by dextran
sulfate-magnesium chloride (24). LDL cholesterol was calculated by
difference. The concentrations of total cholesterol, triglyceride, free
cholesterol, esterified cholesterol, phospholipid, and protein were
measured in the plasma and various lipoprotein fractions as described
previously (24). To determine whether either statin altered the size
distribution of the lipoproteins and/or if the distribution of
cholesterol among the lipoprotein classes was affected by statin
treatment, plasma was separated by the high performance gel
chromatographic method of Kieft et al. (35). On-line
cholesterol content of each fraction was determined, and elution peak
retention times were used to compare the relative sizes of the
lipoproteins. Tests for statistical significance of differences between
control and statin-treated animals were compared by paired t
test (36). P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Atorvastatin treatment significantly
reduced total plasma and VLDL triglyceride concentrations by 46%
(P = 0.014) and 50% (P = 0.019),
respectively. Total plasma cholesterol, VLDL cholesterol, LDL
cholesterol, and HDL cholesterol were significantly decreased by 31%
(P = 0.0008), 30% (P = 0.047), 42%
(P = 0.008), and 19% (P = 0.003),
respectively. VLDL apoB and LDL apoB were significantly decreased by
49% (P = 0.00007) and 39% (P =
0.015), respectively. In contrast, total plasma and VLDL triglyceride
concentrations were nonsignificantly reduced by simvastatin treatment.
Total plasma, LDL cholesterol, and LDL apoB concentrations were
significantly reduced by 20% (P = 0.021), 24%
(P = 0.021), and 26% (P = 0.001),
respectively. VLDL cholesterol, HDL cholesterol, and VLDL apoB
concentrations were nonsignificantly decreased.
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and 3. A
fit of the model to the apoB-specific radioactivity curves for
[131I]VLDL, [131I]IDL, and
[131I]LDL for one representative animal is shown in Fig. 1.
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). This was primarily due to a
significant 50% (P = 0.0007) reduction in the VLDL
apoB PR as the FCR was unchanged. The amount of VLDL apoB converted to
LDL apoB was decreased by 22%, and, although not statistically
significant (P = 0.061), conversion was reduced in all
five animals studied. The flux of apoB cleared directly, without
conversion to IDL or LDL, was significantly reduced by 51%
(P = 0.0005). Neither the percentage of VLDL apoB flux
converted to LDL, nor the percent cleared directly, was affected by
atorvastatin treatment. Similarly, simvastatin treatment resulted in an
apparent decrease (24%) in VLDL apoB pool size (P =
0.166). This was primarily due to a significant 33%
(P = 0.024) reduction in the VLDL apoB PR, as the FCR
was unchanged. The amount of VLDL apoB converted to LDL apoB was not
affected by simvastatin treatment. The flux of apoB cleared without
conversion to IDL or LDL was significantly reduced by 35%
(P = 0.042). Neither the percentage of VLDL apoB flux
converted to LDL, nor the percent cleared directly, was affected by
simvastatin treatment. The FCRs for VLDL apoB direct catabolism
[k (0, 1), k (0, 2) and k
(0, 3)] and for IDL direct catabolism [k (0, 4) and
k (0, 5)] were not significantly affected by either
inhibitor (data not shown). No significant changes were observed in the
production or flux of VLDL apoB that was converted to LDL without being
transported through the plasma IDL pool, or in the amount of VLDL apoB
converted to LDL via the IDL fraction (data not shown).
Atorvastatin treatment decreased the LDL apoB pool size by 38%
(P = 0.015) (Table 3). This was primarily due to a
significant 30% (P = 0.037) reduction in the LDL apoB
PR, which was accounted for by the combination of a 22% decrease in
LDL apoB derived from VLDL apoB catabolism (P = 0.061)
and a significant 32% decrease in LDL apoB direct synthesis
(P = 0.039). Total apoB production into plasma,
calculated as the sum of VLDL apoB production plus LDL apoB direct
production, decreased significantly by 48% (P =
0.0003). Atorvastatin treatment resulted in a significant 15% increase
in the LDL apoB FCR (P = 0.008). By comparison,
simvastatin reduced the LDL apoB pool size to a lesser extent (26%;
P = 0.001) (Table 3). This was due to a significant
21% (P = 0.002) reduction in the LDL apoB PR, which
was accounted for by a 23% decrease in LDL apoB direct synthesis
(P = 0.007). Total apoB production into plasma
decreased significantly by 32% (P = 0.018). The LDL
apoB FCR was unchanged by simvastatin treatment.
VLDL and LDL were analyzed for lipid and protein composition; however, the percent composition was not altered for any of the parameters measured with either atorvastatin or simvastatin treatment (data not shown). Moreover, plasma cholesterol distribution among the lipoprotein classes, as assessed by high performance gel chromatography, showed no major changes with either HMG-CoA reductase inhibitor. The elution peak retention times for VLDL, LDL, and HDL were unchanged (data not shown) suggesting no significant effect of statin treatment on particle size.
Approximately 24 h after the last dose of atorvastatin or
simvastatin was administered, the pigs were killed and sections of
liver and small intestine were removed and stored at -80 C before
analyses. Hepatic free and esterified cholesterol concentrations
were significantly less in atorvastatin- treated pigs when compared
with simvastatin-treated animals (Table 4). Furthermore, a trend toward a lower
hepatic microsomal free cholesterol and triglyceride concentrations was
observed with atorvastatin compared with simvastatin treatment. Hepatic
HMG-CoA reductase activity increased (35–46%) in microsomes, and
hepatic ACAT actvity decreased (36–56%) in crude liver homogenates
with statin treatment when compared with control animals. However, no
significant differences were observed between treatments. It is likely
that the induction of hepatic reductase activity is underestimated in
microsomes from atorvastatin-treated animals. It has been shown in rats
that atorvastatin is more tightly bound to hepatic microsomal HMG-CoA
reductase than lovastatin and hence more difficult to remove with
washing (37). Although hepatic HMG-CoA reductase activities were
similar between the two statins, abundance of HMG-CoA reductase mRNA
was 105% higher (P = 0.038) and abundance of LDL
receptor mRNA was 40% higher (P = 0.047) in
atorvastatin-treated pigs, when compared with simvastatin-treated
animals (Table 5). Abundances of hepatic
and intestinal apoB mRNA was unchanged.
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summarizes the major apoB
kinetic parameters observed in this study for atorvastatin, 10
mg/kg/day, and simvastatin, 10 mg/kg/day. For comparison purposes, apoB
kinetic parameters obtained previously in a similar study (26) using
atorvastatin, 3 mg/kg/day, are included. In general, the magnitude of
effect of HMG-CoA reductase inhibition in this pig model on VLDL apoB
and LDL apoB kinetic parameters was atorvastatin, 10 mg/kg/day >
simvastatin 10 mg/kg/day = atorvastatin 3 mg/kg/day.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The primary effect of atorvastatin and simvastatin on apoB metabolism in miniature pigs fed normal amounts of fat and cholesterol was a reduction in the secretion of hepatic apoB containing lipoproteins into plasma. However, at the 10 mg/kg/day doses used, the decrease with atorvastatin was of greater magnitude than simvastatin. In previous in vivo apoB kinetic studies from this laboratory, using atorvastatin, 3 mg/kg/day (26), a decrease in VLDL apoB secretion into plasma was also a primary mechanism of action, although the effect was less than that observed for atorvastatin, 10 mg/kg/day, and similar to that observed for simvastatin, 10 mg/kg/day. This suggests that the magnitude of reduction in VLDL apoB secretion is related to the extent of HMG-CoA reductase inhibition. Furthermore, these observations are consistent with the concept that the extent of reductase inhibition determines the magnitude of the reduction in the ER regulatory cholesterol pool involved in VLDL assembly and secretion.
Perturbations of free cholesterol concentrations elicit rapid responses in the rates of cholesterol biosynthesis, LDL receptor expression, and cholesterol esterification in cultured cells (38). HMG-CoA reductase inhibition should decrease the hepatic free cholesterol concentration of the ER. In whole liver, when compared with simvastatin-treated animals, significant reductions in both free and esterified cholesterol concentrations were observed with atorvastatin treatment. In contrast, hepatic free or esterified cholesterol concentrations were unaffected by low-dose atorvastatin treatment (26). Trends toward lower hepatic microsomal free cholesterol and triglyceride concentrations were observed with atorvastatin compared with simvastatin treatment. Despite significant decreases in apoB secretion, Conde et al. (39), in guinea pigs receiving doses of atorvastatin similar to those used in the present study (10 mg/kg/day), were unable to demonstrate significant reductions in either hepatic free or esterified cholesterol concentrations, whereas, microsomal free cholesterol was significantly decreased by 30%. Taken together, these results would suggest that a putative regulatory pool of microsomal cholesterol (40, 41) is decreased by HMG-CoA reductase treatment, resulting in reduced availability for either lipoprotein surface, or as a substrate for ACAT, thereby, reducing cholesteryl ester availability for lipoprotein core formation. Only when HMG-CoA reductase is inhibited to a large extent are decreases in free and esterified cholesterol concentrations in whole liver apparent.
Inhibition of cholesterol synthesis with an HMG-CoA reductase inhibitor should cause an overexpression of HMG-CoA reductase as a result of a positive feedback-regulatory mechanism (1, 2). As anticipated, hepatic microsomal HMG-CoA reductase activity was greater for both statins compared with controls with no difference between atorvastatin- and simvastatin-treated pigs. However, when compared with simvastatin-treated animals, atorvastatin resulted in a significant 105% induction of hepatic HMG-CoA reductase gene expression. The changes described for hepatic HMG-CoA reductase mirrored those of LDL receptor gene expression with the induction of LDL receptor expression occurring to a greater extent with atorvastatin treatment. These results are consistent with the lower hepatic free cholesterol content in atorvastatin-treated animals. Collectively, these results suggest that HMG-CoA reductase was inhibited to a greater extent with atorvastatin compared with simvastatin when given at equal doses.
LDL apoB production was significantly decreased by both HMG-CoA reductase inhibitors. The magnitude of the reduction was greater with atorvastatin than simvastatin treatment. Atorvastatin decreased both LDL apoB direct synthesis and VLDL apoB to LDL apoB conversion. The reduced conversion was due to the marked reduction of VLDL apoB production by atorvastatin. Simvastatin treatment also decreased LDL direct synthesis, but had a lesser effect on reduction of VLDL apoB production and no significant effect on the conversion of VLDL to LDL. When compared with pigs treated with low-dose atorvastatin (3 mg/kg/day) (26), the animals receiving high-dose atorvastatin demonstrated a more marked reduction in LDL apoB production (38 vs. 22%), less effect on the conversion of VLDL apoB to LDL apoB (22 vs. 34%), but a greater decrease in LDL apoB direct synthesis (28 vs. 12%). Major alterations in VLDL composition were not responsible for the decreased conversion observed with atorvastatin. Furthermore, the percentage of total VLDL apoB production converted to LDL apoB, or removed directly, did not change with either statin.
According to current dogma, inhibition of hepatic cholesterol synthesis by HMG-CoA reductase inhibitors decreases plasma LDL cholesterol concentrations by increasing LDL receptor expression, resulting in increased LDL clearance. Consistent with our previous findings with low-dose atorvastatin (26), no significant effect was seen on LDL apoB FCR in simvastatin-treated animals. The lack of change in LDL FCR was not due to changes in LDL composition or on changes in LDL subfraction distribution. These results are consistent with our observation that abundance of hepatic LDL receptor mRNA was not significantly changed. Thus, these findings support the concept that simvastatin, 10 mg/kg/day, and atorvastatin, 3 mg/kg/day, decrease a newly synthesized pool of cholesterol that is required for lipoprotein assembly, a pool not in complete equilibrium with the LDL receptor-regulatory pool (42). In contrast, we were able to demonstrate a significant 15% increase in LDL apoB FCR with high-dose atorvastatin. Increases in hepatic microsomal LDL receptor number have been reported in guinea pigs treated with 20 mg/kg/day of atorvastatin (39), raising the possibility that increased LDL clearance may become evident at higher atorvastatin doses. Consistent with this finding, significant increases in LDL receptor gene expression were observed in atorvastatin-treated pigs when compared with simvastatin-treated or control animals. No significant effects were noted on percent composition of plasma LDL for any of the parameters measured in animals treated with either inhibitor. Collectively, our results suggest that in this animal model, hepatic apoB secretion is most sensitive to HMG-CoA reductase inhibition and that LDL receptor expression is increased only at higher levels of reductase inhibition.
Clinical studies have demonstrated an increased dose efficacy of atorvastatin over simvastatin (15) and other statins (16) in hypercholesterolemic subjects. Naoumova et al. (19) in FH heterozygotes have recently shown a prolonged inhibition in cholesterol synthesis with high-dose atorvastatin treatment when compared with high-dose simvastatin. Although the initial degrees of suppression were similar, they postulated that the greater efficacy of atorvastatin related to its hepatic selectivity and longer duration of action in the liver. These findings are entirely consistent with the present study.
The mechanisms involved in the regulation of the assembly and secretion of apoB-containing lipoproteins within the hepatocyte by HMG-CoA reductase inhibitors remain incompletely understood (11). We hypothesize that reduced cholesterol synthesis and/or cholesteryl esterification may compromise the ability of apoB to fold appropriately and/or reduce full lipidation and subsequent secretion. In turn, the rates of apoB degradation and/or translocation of apoB across the ER membrane may be affected. Moreover, the apoB translocation and degradation rates can determine the fate of newly synthesized apoB (11). In cultured HepG2 cells, atorvastatin has been shown to decrease apoB secretion (43, 44, 45). Wilcox et al. (45) and Mohammadi et al. (43) demonstrated that atorvastatin increases intracellular apoB degradation rates. In addition, Mohammadi et al. (43) observed that atorvastatin also decreases apoB translocation. Whether increased apoB degradation occurs in the livers of pigs treated with atorvastatin or simvastatin is unknown.
Inhibition of triglyceride (46) and phospholipid synthesis (47) in cultured hepatocytes has been shown to decrease apoB secretion. However, studies from our laboratory in HepG2 cells demonstrated that neither atorvastatin nor simvastatin at concentrations up to 10 µM had any significant effect on triglyceride or phospholipid synthesis (45). Taken together with our observation in the present study that hepatic triglyceride concentrations did not change, we conclude that the decrease in total apoB secretion by both atorvastatin- and simvastatin-treated pigs was not secondary to altered hepatic triglyceride or phospholipid metabolism.
Inhibition of newly synthesized cholesteryl ester catalyzed by ACAT has been shown to decrease apoB secretion in cell culture models and perfused monkey livers as well as in several small animal models (11). ApoB kinetic studies in miniature pigs demonstrated that the ACAT inhibitors, DuP 128 and avasimibe (previously known as CI-1011), significantly decreased hepatic VLDL apoB secretion (31, 48, 49). HMG-CoA reductase inhibition may decrease the ACAT free cholesterol substrate pool, thereby reducing the expression and/or activity of ACAT and hence apoB secretion. The reductions in hepatic ACAT activities observed with both statins in the present study are consistent with this concept and with the findings in atorvastatin-treated guinea pigs (39). Microsomal triglyceride transfer protein (MTP) is capable of facilitating apoB translocation (50, 51), in addition to its role in mediating delivery of core lipid to nascent apoB (52). Although not measured in the present study, the HMG-CoA reductase inhibitor-induced reduction in apoB secretion may be mediated, in part, to a decreased MTP expression and/or activity, secondary to reduced cholesterol synthesis.
This study adds further support to the concept that a reduction in the assembly and secretion of apoB synthesis and secretion is an important mechanism whereby HMG-CoA reductase inhibitors lower the plasma concentration of apoB-containing lipoproteins (11). Atorvastatin and simvastatin decrease both VLDL and LDL apoB PRs in miniature pigs. The reduction in LDL apoB production relates to a marked reduction in VLDL apoB synthesis and a reduction primarily in direct LDL apoB synthesis, i.e. mechanistically the two statins appear similar. However, in this large-animal model, LDL apoB FCRs were increased with atorvastatin, but not with simvastatin treatment. Consistent with this finding, hepatic LDL receptor and HMG-CoA reductase gene expression were significantly increased and hepatic free and esterified cholesterol concentrations decreased in atorvastatin-treated pigs. We conclude that both atorvastatin and simvastatin decrease hepatic VLDL apoB secretion in vivo, but that magnitude is determined by the extent of HMG-CoA reductase inhibition.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Core Clinical Pathology and
Biochemistry, Royal Perth Hospital, Perth, West Australia, 6001,
Australia.
3 Current address: Esperion Therapeutics Inc., 3621 South State
Street, 695 KMS Place, Ann Arbor, Michigan 48108.
Received May 26, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D. Sahoo, T. C. Trischuk, T. Chan, V. A. B. Drover, S. Ho, G. Chimini, L. B. Agellon, R. Agnihotri, G. A. Francis, and R. Lehner ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes J. Lipid Res., June 1, 2004; 45(6): 1122 - 1131. [Abstract] [Full Text] [PDF]
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B. H. C. M. T. Prinsen, J. A. Romijn, P. H. Bisschop, M. M. J. de Barse, P. H. R. Barrett, M. Ackermans, R. Berger, T. J. Rabelink, and M. G. M. de Sain-van der Velden Endogenous cholesterol synthesis is associated with VLDL-2 apoB-100 production in healthy humans J. Lipid Res., July 1, 2003; 44(7): 1341 - 1348. [Abstract] [Full Text] [PDF]
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 K. Ouguerram, T. Magot, Y. Zair, J. S. Marchini, B. Charbonnel, H. Laouenan, and M. Krempf Effect of Atorvastatin on Apolipoprotein B100 Containing Lipoprotein Metabolism in Type-2 Diabetes J. Pharmacol. Exp. Ther., July 1, 2003; 306(1): 332 - 337. [Abstract] [Full Text] [PDF]
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D. E. Telford, J. Y. Edwards, S. M. Lipson, B. Sutherland, P. H. R. Barrett, J. R. Burnett, E. S. Krul, B. T. Keller, and M. W. Huff Inhibition of both the apical sodium-dependent bile acid transporter and HMG-CoA reductase markedly enhances the clearance of LDL apoB J. Lipid Res., May 1, 2003; 44(5): 943 - 952. [Abstract] [Full Text] [PDF]
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M. W. Huff, D. E. Telford, J. Y. Edwards, J. R. Burnett, P. H. R. Barrett, S. R. Rapp, N. Napawan, and B. T. Keller Inhibition of the Apical Sodium-Dependent Bile Acid Transporter Reduces LDL Cholesterol and ApoB by Enhanced Plasma Clearance of LDL ApoB Arterioscler. Thromb. Vasc. Biol., November 1, 2002; 22(11): 1884 - 1891. [Abstract] [Full Text] [PDF]
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J. L. Dixon, S. Shen, J. P. Vuchetich, E. Wysocka, G. Y. Sun, and M. Sturek Increased atherosclerosis in diabetic dyslipidemic swine: protection by atorvastatin involves decreased VLDL triglycerides but minimal effects on the lipoprotein profile J. Lipid Res., October 1, 2002; 43(10): 1618 - 1629. [Abstract] [Full Text] [PDF]
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N. Roglans, E. Sanguino, C. Peris, M. Alegret, M. Vazquez, T. Adzet, C. Diaz, G. Hernandez, J. C. Laguna, and R. M. Sanchez Atorvastatin Treatment Induced Peroxisome Proliferator-Activated Receptor alpha Expression and Decreased Plasma Nonesterified Fatty Acids and Liver Triglyceride in Fructose-Fed Rats J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 232 - 239. [Abstract] [Full Text] [PDF]
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K. G. Parhofer, P. H. R. Barrett, and P. Schwandt Atorvastatin Improves Postprandial Lipoprotein Metabolism in Normolipidemic Subjects J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4224 - 4230. [Abstract] [Full Text]
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), IDL (•), and LDL (
) after injection
of [131I]VLDL. Data points
represent the observed data, and the lines are the best
fit generated by the kinetic model. Panel A represents a control pig;
panel B, an atorvastatin-treated pig.