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Mechanism of Action of a 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitor on Apolipoprotein B-100 Kinetics in Visceral Obesity

Departments of Medicine (D.C.C., G.F.W., P.H.R.B., T.A.M., L.J.B.) and Physiology (T.G.R.), University of Western Australia, Western Australian Institute for Medical Research, Royal Perth Hospital, Perth, Western Australia 6847

Address all correspondence and requests for reprints to: Associate Professor G. F. Watts, University Department of Medicine, University of Western Australia, Royal Perth Hospital, G.P.O. Box X2213, Perth, Western Australia, WA 6847. E-mail: . gfwatts{at}cyllene.uwa.edu.au

Abstract

We examined the effect of atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the kinetics of apolipoprotein B-100 (apoB) metabolism in 25 viscerally obese men in a placebo-controlled study. Very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB kinetics were measured using an iv bolus injection of [²H₃]leucine. ApoB isotopic enrichment was measured using gas chromatography-mass spectrometry. Kinetic parameters were derived by using a multicompartmental model (SAAM-II). Compared with the placebo group, atorvastatin treatment resulted in significant (P < 0.001) decreases in total cholesterol (-34%), triglyceride (-19%), LDL cholesterol (-42%), total apoB (-39%), and lathosterol (-86%); VLDL-apoB, IDL-apoB, and LDL-apoB pool sizes also fell significantly (P < 0.002) by -27%, -22%, and -41%, respectively. This was associated with an increase in the fractional catabolic rates of VLDL-apoB (+58%, P = 0.019), IDL-apoB (+40%, P = 0.049), and LDL-apoB (+111%, P = 0.001). However, atorvastatin did not significantly alter the production and conversion rates of apoB in all lipoproteins. We conclude that in obese subjects, atorvastatin decreases the plasma concentration of all apoB-containing lipoproteins chiefly by increasing their catabolism and not by decreasing their production or secretion. This may be owing to up-regulation of hepatic receptors as a consequence of inhibition of cholesterogenesis.

OBESITY IS A major risk factor for cardiovascular disease (CVD), type 2 diabetes mellitus, and hypertension. Visceral obesity, the most clinical important topographical form, is typically seen in overweight men. It is associated with dyslipidemia and may account for the increased risk of CVD (1). Although the precise mechanism whereby visceral obesity results in dyslipidemia has not been established, it may involve increased hepatic secretion of very-low-density lipoprotein (VLDL) apolipoprotein B-100 (apoB) (2) as well as impaired catabolism of VLDL, intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB (3). These abnormalities may be consequent on insulin resistance (4).

Although hypocaloric diets and other lifestyle measures may be effective in achieving significant weight loss and correcting dyslipidemia in obesity (3), adherence to such programs is universally disappointing (5). Clinical management of dyslipidemia in obesity may therefore require the use of lipid-regulating pharmacotherapy (6). Subgroup analyses of recent clinical trials support the use of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) inhibitors (statins) to treat dyslipidemia associated with insulin resistance and obesity (7). Statins inhibit cholesterol synthesis and consequently up-regulate hepatic LDL receptors (8) and possibly other receptors that mediate the disposal of triglyceride-rich lipoproteins (9). The precise mechanisms of action of statins on apoB kinetics in obesity still remain to be elucidated.

Although statins are known to up-regulate hepatic LDL receptors, their effect on hepatic secretion of apoB and the forward transport of apoB through the endogenous pathway of lipoprotein metabolism has been less well recognized. Experimental and human evidence supports the notion that hepatic output of apoB is controlled by cholesterol substrate availability (10, 11, 12, 13, 14). In cross-sectional and interventional studies, we have previously reported a direct correlation between cholesterol synthesis and hepatic secretion of VLDL-apoB in normolipidemic subjects (15, 16). In visceral obesity we have also shown increased rates of both de novo cholesterol synthesis and hepatic secretion of apoB (2). These observations provide a rationale for the use of HMG CoA reductase inhibitors in correcting abnormal lipoprotein transport in obesity, even in the presence of insulin resistance. Insulin resistance, because of its effect in regulating apoB metabolism in vivo (17, 18, 19), may modulate the effect of a statin on apoB metabolism, but this remains to be elucidated.

In this present study, we employed a stable isotopic technique to test the hypothesis that inhibition of cholesterogenesis by atorvastatin, an HMG CoA reductase inhibitor, has a dual mechanism of action not only to decrease the hepatic secretion but also to increase catabolic rates of VLDL, IDL, and LDL apoB in viscerally obese men. We were specifically interested in testing this hypothesis in patients who were insulin resistant.

Subjects and Methods

Subjects

Twenty-five obese men were recruited from the community by newspaper advertisement. Obesity was defined as a waist circumference of more than 100 cm, waist to hip ratio of more than 0.97, and body mass index of more than 29 kg/m². All subjects had plasma triglyceride of more than 1.2 mmol/liter and cholesterol of more than 5.2 mmol/liter while consuming ad libitum, weight-maintaining diets. None of the subjects had diabetes mellitus (excluded by oral glucose tolerance test), apolipoprotein E2/E2 genotype, macroproteinuria, creatinemia (>120 µmol/liter), hypothyroidism, or abnormal liver enzymes; or consumed more than 30 g alcohol/d. None reported a history of CVD or was taking medication or other agents known to affect lipid metabolism. All subjects provided written consent, and the study was approved by the Ethics Committee of the Royal Perth Hospital.

Study design and clinical protocols

The study reported here represents a substudy of a larger randomized, doubled-blind, placebo-controlled intervention trial examining the effect of atorvastatin and other agents on aspects of lipoprotein metabolism in obesity, which will be reported separately. The present report hence refers to a two-arm, parallel group study design. Eligible patients entered a 3-wk run-in diet-stabilizing period, at the end of which they were randomized to a 6-wk treatment period of either atorvastatin (40 mg orally at night) or matching placebo. Advice was given to patients to continue on isocaloric diets and maintain physical activity constant during the study. Compliance with atorvastatin and placebo was checked by tablet count at wk 3 and 6 of the treatment period.

All subjects were admitted to the metabolic ward in the morning after a 14-h fast. They were studied in a semirecumbent position and allowed to drink only water. Venous blood was collected for measurements of biochemical analytes. Plasma volume was determined by multiplying body weight by 0.045 (20) and by a correction factor to adjust for the decrease in relative plasma volume associated with an increase in body weight as described by Riches et al. (2). Arterial blood pressure was recorded after 3 min in the supine position using a Dinamap1846 SX/P monitor (Critikon Inc., Tampa, FL). Dietary intake was assessed for energy and major nutrients using at least two 24-h dietary diaries at the beginning and end of the study. Diets were subsequently analyzed using DIET 4 nutrient calculation software (Xyris Software, Queensland, Australia).

A single bolus of [²H₃]-leucine (5 mg/kg of body weight) was administered iv within a 2-min period into an antecubital vein via a 21G butterfly needle. Blood samples were taken at baseline and after injection of the isotope at 5, 10, 20, 30, and 40 min and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, and 10 h. Subjects were then given a snack and allowed to go home. Additional fasting blood samples were collected in the morning on the following 4 d of the same week (24, 48, 72, and 96 h). All the procedures were repeated after 6 wk of treatment with either atorvastatin or placebo.

Isolation and measurement of isotopic enrichment of VLDL, IDL, and LDL apoB-100

VLDL, IDL, and LDL were isolated from 3 ml plasma by sequential ultracentrifugation in a Centrikon T-1190 centrifuge (Kontron Instruments Ltd., Milan, Italy) at densities of 1.006, 1.019, and 1.063 g/ml, respectively. The apoB-100 fraction was precipitated by addition of an equal volume of 100% isopropanol as described by Egusa et al. (21). The precipitate was then delipidated with isopropanol, dried, and hydrolyzed by the addition of 2 ml of 6 M hydrochloric acid. After the hydrolysis at 105 C for 24 h, samples were dried and reconstituted with 1 ml 50% acetic acid. The free amino acids were separated and purified by cation-exchange chromatography using AG 50 W-x8 resin (Bio-Rad Laboratories, Inc., Richmond, CA). Samples were derivatized using acetonitrile and N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide and reconstituted in toluene for gas chromatography-mass spectrometric analysis (HP 5890 Series II Plus gas chromatograph coupled to an HP 5989B mass spectrometer, Hewlett-Packard Co., Palo Alto, CA). Plasma amino acids were also separated by cation-exchange chromatography, derivatized, and analyzed as described above for the determination of plasma leucine isotopic enrichment. Isotopic enrichment was determined by selected ion monitoring of derivatized samples at a mass to charge ratio of 305 and 302 and using positive-electron-impact ionization. Tracer/tracee ratios were derived from isotopic ratios for each sample.

Quantification of apoB and other analytes

In each individual study from the onset of [²H₃] leucine infusion, five pooled plasma samples were collected from various time points during the study week. The apoB in VLDL, IDL, and LDL fractions from the pooled plasma samples was isolated as described in the previous paragraph using isopropanol method (21). After delipidation, 1 ml of 1 M NaOH was added and the precipitate was heated for 3 h at 60 C. A modified Lowry method was used to determine the apoB-100 concentration in each lipoprotein fraction (22). Briefly, 200 µl sample was reacted with 1 ml Lowery reagent for 10 min at room temperature. Then 100 µl diluted Folin-Ciocalteu phenol reagent was added to the mixture for 30 min. Absorbance of the samples was read using a DU650 spectrophotometer (Beckman Coulter, Inc., Palo Alto, CA) at 740 nm. ApoB concentrations were calculated from the standard curve. The interassay coefficient of variation was less than 5%.

Plasma cholesterol and triglyceride concentrations were determined by standard enzymatic methods using a 917 biochemical analyzer (Hitachi Ltd., Tokyo, Japan). High-density lipoprotein (HDL) cholesterol was measured by an enzymatic colorimetric method using a commercial kit (Roche Molecular Biochemicals, Mannheim, Germany). LDL cholesterol was calculated using the Friedewald equation. Non-HDL cholesterol was derived as total cholesterol minus HDL cholesterol. Apolipoproteins A-I and B were determined by immunonephelometry. Plasma nonesterified fatty acids (NEFAs) were measured by an enzymatic, colorimetric method using a commercial kit (Randox Co., Antrim, UK). Plasma insulin was measured by a RIA (DiaSorini s.r.l., Saluggia, Italy). Plasma glucose concentration was measured by a hexokinase method on a 917 biochemical analyser (Hitachi). Insulin resistance was estimated using the HOMA formula: fasting insulin (mU/liter) x fasting plasma glucose (mmol/liter)/22.5 as described by Matthews et al. (23). Plasma lathosterol concentration was assayed by a modification of the method of Mori et al. (24) using gas chromatography-mass spectrometry. Genomic DNA was extracted by the standard Triton X-100 procedure and the genotype for apoE determined as described by Hixson and Vernier (25). Plasma liver (alanine transferase, asparate transferase, alkaline phosphatase) and muscle (creatinine kinase) enzymes were analyzed on a standard autoanalyzer (917 biochemical analyzer, Hitachi).

Model of apoB metabolism and calculation of kinetic parameters

A multicompartmental model (Fig. 1), similar to that of Phair et al. (26), was used to describe VLDL-, IDL-, and LDL-apoB leucine tracer/tracee ratios. In multicompartmental modeling, each compartment or pool represents a group of kinetically homogenous particles. In this study, the SAAM II program (SAAM Institute, Seattle, WA) was used to fit the model to the observed tracer data. Metabolic parameters were subsequently derived from the model parameters giving the best fit. Part of the model consists of a four-compartment subsystem (compartments 1–4) that describe plasma leucine kinetics. This subsystem is connected to an intrahepatic delay compartment (compartment 5) that accounts for the time required for the synthesis and secretion of apoB into plasma. This model provides for the direct secretion of apoB into the VLDL, IDL, and LDL fractions. Compartments 6–10 are used to describe the kinetics of apoB-100 in the VLDL fraction. Compartments 6–9 represent a delipidation cascade. It is assumed that the residence time of particles in each compartment of the cascade is the same. In addition, the fraction of each compartment in the cascade converted to the slowly turning-over VLDL compartment (compartment 10) is the same. VLDL particles in compartment 9 can be converted to IDL or removed directly from plasma. Plasma IDL kinetics is described by two compartments, compartments 11 and 12. Compartment 12 represents a slowly turning-over pool of IDL particles. IDL in compartment 11 can be converted to LDL or compartment 13 or be removed directly from plasma. The LDL section of the model consists of two compartments. Compartment 13 describes plasma LDL, and compartment 14 is an extravascular LDL exchange compartment. It is assumed that all LDL is cleared via compartment 13. In some patients, the rate constants linking compartments 13 and 14 tended to zero. In such cases, the extravascular compartment was removed from the model. VLDL, IDL, and LDL apoB metabolic parameters, including fractional catabolic rate (FCR), production rate (PR), conversion, and direct synthesis (by %) were derived following a fit of the compartment model to the apoB tracer/tracee ratio data.

View larger version (19K): [in this window] [in a new window]   Figure 1. Multicompartmental model for apoB metabolism.

All analyses were carried out using SPSS, Inc. 10.1 (SPSS, Inc., Chicago, IL). Group characteristics were compared by t tests after logarithmic transformation of skewed variables where appropriate. Adjustment for differences in baseline covariates and changes in variables during the study were performed by analysis of covariance using general linear models. Associations were examined by linear regression methods. Statistical significance was defined at the 5% level using a two-tailed test.

Results

Table 1 shows the clinical and biochemical characteristics of the viscerally obese subjects at baseline. On average, they were middle aged, centrally obese, normotensive, and insulin resistant. There were no significant group differences in any of the variables in Table 1. Eighteen of the subjects were E3/E3 homozygotes, one was E2/E3 heterozygote, five were E3/E4 heterozygotes, and one was E4/E4. The frequency distributions of the apoE genotype were not significantly different between the groups. Average daily energy and nutrient intake of the 25 obese subjects studied (mean ± SD) was: 10031 ± 1390 kJ, 32% ± 6% energy from fat, 42% ± 9% energy from carbohydrates, 19% ± 4% energy from protein, and 7% ± 5% energy from alcohol. Nutrient intake did not differ between patients randomized to atorvastatin or placebo.

Figure 2 shows the isotopic enrichment of VLDL, IDL, and LDL apoB after the infusion of [²H₃]-leucine in the subjects before and after atorvastatin treatment. Tracer curves typically demonstrate a precursor-product relationship among VLDL, IDL, and LDL apoB. In the atorvastatin group, the peak enrichment values were higher and the maximum of the enrichment curve was attained earlier. Enrichment curves in the placebo group did not alter between wk 0 and week 6 (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 2. Isotopic enrichment for VLDL, IDL, and LDL apoB with [2H3]-leucine in the obese subjects before (a) and after (b) in subjects randomized with atorvastatin (placebo data are not shown).

In this double-blind, randomized, placebo-controlled trial, we provide new information about the kinetic effects of HMG CoA reductase inhibition on apoB metabolism in insulin-resistant subjects with visceral obesity. Our results demonstrate that inhibition of cholesterogenesis with atorvastatin decreases the plasma concentrations of all major apoB-containing lipoproteins by a mechanism related to an increase in their fractional catabolism. This effect of atorvastatin was demonstrated in the absence of changes in apoB production, body weight, and insulin resistance.

This is the first study to determine the kinetic effect of a statin on apoB metabolism in obese subjects with insulin resistance. Previous kinetic studies using radiolabeling and stable isotopic techniques to examine the effect of HMG CoA reductase inhibitions on apoB metabolism in nonobese patients have recently been reviewed (27, 28, 29, 30, 31). The findings of these kinetic studies have shown inconsistent effects on the PR and FCR of apoB. Discrepancies among the various studies referred to might have been owing to different subject characteristics, experimental protocols, method of data analysis, and the type and dose of statin employed. Moreover, some studies have been uncontrolled, restricted to a single lipoprotein compartment, and generally of small sample size. We have extended the previous reports by examining a larger number of subjects in a placebo-controlled study design investigating the effect of atorvastatin on apoB metabolism in VLDL, IDL, and LDL compartments in insulin-resistant obese subjects.

It is well recognized that visceral obesity and insulin resistance alter the metabolism of apoB-containing lipoproteins (1, 32). Insulin has diverse physiological effects on lipoprotein metabolism. It reduces hepatic apoB secretion by suppressing the delivery of NEFAs to the liver from adipose tissue (17) and inhibiting hepatic de novo cholesterol synthesis (33, 34). Insulin also enhances the lipolysis and hepatic uptake of triglyceride-rich apoB-containing lipoproteins, including chylomicron remnants by up-regulation of lipoprotein lipase activity (35) and stimulating LDL receptor activity (36), respectively. In subjects with insulin resistance, this normal insulin-mediated regulation of apoB metabolism is diminished. The kinetic effects of insulin resistance on apoB metabolism have been reported in both obese and type 2 diabetic subjects (2, 37). These abnormalities include both oversecretion and delayed clearance of apoB-containing lipoproteins. Consistent with these findings, we have also found that our obese subjects had increased VLDL-apoB production (14.44 ± 1.34 vs. 9.66 ± 1.77 mg/kg per day, P < 0.05) and lower LDL-apoB FCR (0.27 ± 0.02 vs. 0.43 ± 0.07 pools/d, P < 0.01) compared with 10 normolipidemic lean subjects (38). These kinetic defects might have accounted for the principal abnormalities in plasma lipid levels seen in our subjects including elevation in plasma triglyceride-rich lipoproteins and to a lesser extent LDL cholesterol, with a reciprocal decrease in HDL cholesterol. Cholesterol synthesis, as reflected by plasma lathosterol concentration, was also increased in our obese subjects, compared with lean controls (10.11 ± 0.78 vs. 6.66 ± 1.10 µmol/liter, P < 0.01) and might have contributed to the kinetic defects in apoB metabolism.

Inhibition of de novo cholesterol synthesis by HMG CoA reductase inhibitors is well recognized to up-regulate LDL receptor activity in vitro (8), and this is supported by studies in familial hypercholesterolemic patients (39). Given the putative central role of the LDL receptor in the removal of all apoB-containing lipoproteins by the liver (40, 41), one might expect that the clearance of all apoB-containing lipoproteins would be enhanced with statin therapy. Such a concept is consistent with our observation that the FCR of apoB in the VLDL, IDL, and LDL fractions increased with atorvastatin treatment. Despite this, there was no change in the fractions of particles cleared directly from the plasma in the VLDL and IDL fractions. This suggests that all pathways involved in the catabolism of these lipoproteins were enhanced by atorvastatin. Our finding of increased VLDL-apoB and IDL-apoB FCRs with treatment are consistent with effects of statins that up-regulate lipoprotein lipase activities (42) and LDL receptor activities (8), respectively. Statins have recently been reported to enhance the effects of peroxisome proliferator-activated receptor- (42), thereby stimulating lipoprotein lipase activity and inhibiting apoC-III expression (43). However, this mechanism of action of statins on increasing LPL activity has only been demonstrated in vitro and requires further investigation.

Increased de novo cholesterol synthesis in obese subjects is associated with increased hepatic VLDL apoB secretion (2, 3). We have previously reported that, in normolipidemic subjects, inhibition of de novo cholesterol synthesis by HMG CoA reductase inhibitors decreased hepatic secretion of VLDL apoB (16). The reduction of hepatic VLDL apoB secretion by atorvastatin has also been demonstrated in animal and cell culture studies (44, 45, 46). However, our findings did not show a significant effect of atorvastatin on apoB production or secretion. The difference in the effects of statin may be a function of insulin resistance in our study population. In cell culture studies, insulin has direct effect on apoB metabolism (47). We have previously reported in both diabetic and nondiabetic subjects that VLDL-apoB secretion is significantly reduced during a hyperinsulinemic, euglycemic insulin clamp (19, 34). This reduction of hepatic VLDL-apoB secretion by acute hyperinsulinemia may be owing to suppression of the delivery of NEFAs to the liver and direct inhibition on apoB synthesis (17, 48).

In viscerally obese men, we also reported that an improvement in insulin sensitivity following weight loss resulted in a decrease in hepatic VLDL-apoB secretion coupled with an increase in LDL-apoB FCR (3). Because atorvastatin treatment did not improve insulin sensitivity in the present study, one could argue that insulin resistance was still driving the oversecretion of apoB by the liver. Although the reduced availability of cholesterol esters because of statins has been shown to be associated with a decrease in hepatic secretion of VLDL-apoB and LDL-apoB (16), this mechanism may not be sufficient to limit the effect of persistent insulin resistance. This is also supported by our findings that there was no direct association between hepatic VLDL-apoB secretion and de novo cholesterol synthesis, as reflected by plasma lathosterol. In subjects with visceral obesity, it is possible that the availability of triglyceride substrate (49) and other regulatory mechanisms may also account for the variance in VLDL-apoB production. In addition to an inhibitory effect of de novo cholesterol synthesis, statins may also decrease cholesterol esterification rates (50), triglyceride substrate availability (51), the expression of apoB mRNA (52), and possibly other related gene products such as microsomal triglyceride transfer protein. However, these contributions to a reduction of hepatic apoB secretion may be diminished in the presence of insulin resistance. Recent evidence also suggests that inhibition of HMG CoA reductase with statins may promote intestinal absorption of dietary cholesterol (53). This mechanism that potentially increases lipid substrate availability to the liver may attenuate apoB secretion and requires further investigation.

The study has limitations. Subtle changes in insulin sensitivity should preferably have been measured with a hyperinsulinemic, euglycemic clamp (54). However, HOMA scores are well correlated with the clamp technique (23). We did not study the kinetics of VLDL₁, VLDL₂, LDL₂, and LDL₃ subspecies. However, we anticipate that the fractional catabolism of these lipoproteins would be enhanced by statin in the presence of insulin resistance but cannot strictly rule out effects on production (18). Examination of the effect of statins on hepatic triglyceride synthesis and secretion may help to clarify the contribution of triglyceride and cholesterol substrates to apoB metabolism. We have previously shown that certain genetic polymorphisms play a role in regulating apoB metabolism in obesity (55, 56). The role of genetics in determining the mechanism of action of statins requires further investigation.

In conclusion, our data support the hypothesis that inhibition of de novo cholesterol synthesis by an HMG CoA reductase inhibitor, atorvastatin, decreases the concentration of all major apoB-containing lipoproteins in viscerally obese insulin-resistant subjects who on average had mixed hyperlipidemia. This improvement is chiefly owing to increasing their catabolism and not to decreasing their hepatic secretion. Several clinical trials have demonstrated that the reduction of plasma apoB-containing lipoproteins with HMG CoA reductase inhibition decreases cardiovascular events in dyslipidemic patients (57). Our results provide a metabolic basis for these effects in men subjects with visceral obesity. However, given that the production of apoB remains unaltered in the present study, further investigations should explore the incremental effect of weight reduction, fish oil supplementation, or insulin sensitizers added to a statin on the kinetics of apoB in these subjects.

Acknowledgments

We are grateful to Associate Prof. F. van Bockxmeer for the apoE genotyping and the nursing staff of the Clinical Research Studies Unit of the University Department of Medicine (Royal Perth Hospital, University of Western Australia) for providing expert clinical assistance.

Footnotes

This study was supported by grants from the National Heart Foundation of Australia, the National Health Medical Research Council of Australia, the Medical Research Fund of the Royal Perth Hospital, Pfizer, Inc. Australia, and the National Institutes of Health (NCRR 12609).

P.H.R.B. is a Career Development Fellow of the National Heart Foundation.

Abbreviations: apoB, Apolipoprotein B; CVD, cardiovascular disease; FCR, fractional catabolic rate; HDL, high-density lipoprotein; HMG CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; NEFA, nonesterified fatty acid; PR, production rate; VLDL, very-low-density lipoprotein.

Received August 2, 2001.

Accepted January 17, 2002.

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