Randomized controlled trial of the effect of n–3 fatty acid supplementation on the metabolism of apolipoprotein B-100 and chylomicron remnants in men
1 From the Departments of Medicine (DCC, GFW, TAM, PHRB, and LJB) and Physiology (TGR), University of Western Australia, West Australian Institute for Medical Research, Royal Perth Hospital, Perth.x{)z\2, 百拇医药
2 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 Australia, and the National Institutes of Health (NCRR 12609). PHRB is a Career Development Fellow of the National Heart Foundation. The Omacor capsules and corn oil were a gift from Provona Biocare, Oslo. x{)z\2, 百拇医药
ABSTRACTx{)z\2, 百拇医药
Background: Lipid abnormalities may contribute to the increased risk of atherosclerosis and coronary disease in visceral obesity. Fish oils lower plasma triacylglycerols, but the underlying mechanisms are not fully understood.x{)z\2, 百拇医药
Objective: We studied the effect of fish oils on the metabolism of apolipoprotein B-100 (apo B) and chylomicron remnants in obese men.
Design: Twenty-four dyslipidemic, viscerally obese men were randomly assigned to receive either fish oil capsules (4 g/d, consisting of 45% eicosapentaenoic acid and 39% docosahexaenoic acid as ethyl esters) or matching placebo (corn oil, 4 g/d) for 6 wk. VLDL, intermediate-density lipoprotein (IDL), and LDL apo B kinetics were assessed by following apo B isotopic enrichment with the use of gas chromatography–mass spectrometry after an intravenous bolus injection of trideuterated leucine. Chylomicron remnant catabolism was measured with the use of an intravenous injection of a chylomicron remnant–like emulsion containing cholesteryl [13C]oleate, and isotopic enrichment of 13CO2 in breath was measured with isotope ratio mass spectrometry. Kinetic values were derived with multicompartmental models.c*8o, 百拇医药
Results: Fish oil supplementation significantly (P < 0.05) lowered plasma concentrations of triacylglycerols (-18%) and VLDL apo B (-20%) and the hepatic secretion of VLDL apo B (-29%) compared with placebo. The percentage of conversions of VLDL apo B to IDL apo B, VLDL apo B to LDL apo B, and IDL apo B to LDL apo B also increased significantly (P < 0.05): 71%, 93%, and 11%, respectively. Fish oils did not significantly alter the fractional catabolic rates of apo B in VLDL, IDL, or LDL or alter the catabolism of the chylomicron remnant–like emulsion.
Conclusion: Fish oils effectively lower the plasma concentration of triacylglycerols, chiefly by decreasing VLDL apo B production but not by altering the catabolism of apo B–containing lipoprotein or chylomicron remnants.nr}*%, 百拇医药
Key Words: Fish oils • apolipoprotein B • chylomicron remnants • visceral obesity • mennr}*%, 百拇医药
INTRODUCTIONnr}*%, 百拇医药
Obesity is a major risk factor for type 2 diabetes and cardiovascular disease. Visceral obesity, the most clinically important topographical form, is typically seen in overweight men. It is strongly associated with dyslipidemia and may account for the increased risk of cardiovascular disease in these men (1). Dyslipidemia in visceral obesity is principally due to insulin resistance, which may perturb the metabolism of apolipoprotein (apo) B-100 (apo B) and chylomicron remnants (2). These lipid abnormalities involve increased hepatic secretion of VLDL apo B (3), as well as impaired catabolism of VLDL, intermediate-density lipoprotein (IDL), LDL, and chylomicron remnants (4–6).
Efforts to treat dyslipidemia in obese persons with the use of hypocaloric diets and other lifestyle measures have largely been disappointing, owing to poor adherence to such programs (7). Effective management of dyslipidemia in persons with obesity and other high-risk states often requires the use of lipid-regulating pharmacotherapy (8). Fish oils are a rich source of long-chain n-3 fatty acids, primarily eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Fish oil supplementation has been shown to lower plasma triacylglycerols (9) and may therefore constitute a potentially effective treatment for obesity-related dyslipidemia. Previous evidence suggests that n-3 fatty acids decrease the secretion of hepatic VLDL (10, 11), increase the conversion of VLDL to LDL (12, 13), and increase the clearance of chylomicron (14), but these studies were not specific to obese subjects. In studies of visceral obesity, we have previously reported increased hepatic secretion of apo B and delayed catabolism of chylomicron remnants (3, 5, 6). These observations provide a rationale for the use of n-3 fatty acids in correcting lipoprotein abnormalities in obese subjects. The effects of n-3 fatty acids on apo B and chylomicron remnant metabolism in viscerally obese subjects with insulin resistance have not been examined previously. It may be anticipated that, by reducing the secretion of VLDL apo B, n-3 fatty acids may decrease the competition of lipolysis and high-affinity clearance pathways that are impaired in obesity and insulin resistance. In contrast, n-3 fatty acids may increase the production of IDL and LDL by enhancing the catabolism of VLDL. We therefore investigated these effects of n-3 fatty acids in obese subjects by using stable isotope–labeled techniques that measure the kinetics of apo B and chylomicron remnants. Chylomicron remnant catabolism was assessed with the use of a novel breath test that we previously described and validated for a functional measure of chylomicron remnant metabolism (15, 16). The effect of fish oils on this breath test has not yet been investigated.
SUBJECTS AND METHODSep, 百拇医药
Subjectsep, 百拇医药
Twenty-four obese men were recruited from the community by newspaper advertisement. Obesity was defined as a waist circumference >100 cm, waist-to-hip ratio >0.97, and body mass index (in kg/m2) >29. All obese subjects were dyslipidemic and had concentrations of plasma triacylglycerols >1.2 mmol/L and cholesterol >5.2 mmol/L while they were consuming weight-maintaining diets ad libitum. None of the subjects had diabetes mellitus (excluded by oral-glucose-tolerance test), apolipoprotein E2/E2 genotype, macroproteinuria, creatinemia (>120 µmol/L), hypothyroidism, or abnormal liver enzymes; no subject consumed >30 g alcohol/d. None reported a history of cardiovascular disease or was taking any medication or other agents known to affect lipid metabolism. Ten normolipidemic, lean, insulin-sensitive men (body mass index, 25 ± 3; waist-to-hip ratio, 0.92 ± 0.06; plasma triacylglycerol, 0.77 ± 0.25 mmol/L; total cholesterol, 4.33 ± 0.34 mmol/L; HDL cholesterol, 1.28 ± 0.30 mmol/L; insulin, 24 ± 4 mU/L; and glucose, 5.4 ± 0.3 mmol/L) who were similar in age to our obese subjects (53 ± 9 y) were also recruited by newspaper advertisement from the community as control subjects. These subjects were described in detail elsewhere (6). All subjects provided written informed consent, and the study was approved by the Ethics Committee of the Royal Perth Hospital.
Study designm, 百拇医药
The study reported here represents a substudy of a larger, randomized, double-blind, placebo-controlled intervention trial examining the effect of fish oils and other agents on aspects of lipoprotein metabolism in obesity, which will be reported separately. The present report 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 randomly assigned to a 6-wk treatment period of 4 g/d doses of either fish oil capsules of 45% EPA and 39% DHA as ethyl esters (Omacor; Provona Biocare, Oslo) or matching placebo capsules (corn oil; Pronova Biocare). Patients were advised to continue on isocaloric diets and to maintain their accustomed degree of physical activity during the study. Compliance with the fish oil and placebo protocols was checked by capsule count at weeks 3 and 6 of the treatment period.m, 百拇医药
Clinical protocolsm, 百拇医药
All subjects were admitted to the clinical investigation unit 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 (17). The plasma volume was modified by multiplying a factor that uses the formula [(relative body weight – 310)/-215] to adjust for the decrease in relative plasma volume associated with an increase in body weight as described by Riches et al (3, 4). After 3 min, arterial blood pressure was recorded with the use of a monitor (Dinamap1846 SX/P; Critikon Inc, Tampa, FL) while subjects were in a supine position. Dietary intake was assessed for energy and major nutrients with the use of at least two 24-h dietary diaries at both the beginning and the end of the study. Diets were analyzed with the use of DIET 4 Nutrient Calculation Software (Xyris Software, Highgate Hill, Australia). A single bolus of trideuterated leucine (5 mg/kg body wt) and the sterile, isotopically labeled chylomicron remnant–like emulsion containing cholesteryl [13C]oleate (14 mL) was administered intravenously within 2 min into an antecubital vein via a 21-gauge butterfly needle. Blood samples were taken at baseline and at 5, 10, 20, 30, and 40 min and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, and 10 h after injection of the isotope. End-expiratory breath samples were collected into an evacuated tube at baseline and after injection every 10 min for the first hour, every 20 min for the second hour, every 30 min for the next 5 h, and every hour for another 3 h. Subjects were then given a snack and allowed to go home. They were requested to refrain from vigorous activity and to eat a light supper before retiring. Additional fasting blood samples were collected in the morning of the next 4 d (at 24, 48, 72, and 96 h). Two additional breath samples were also collected on the following day (at 24 h). All the procedures were repeated after 6-wk treatment with either fish oils or placebo.
Preparation of stable isotope–labeled remnant-like emulsionsna+}, 百拇医药
Stable isotope–labeled remnant-like emulsions were prepared as described previously (15). Briefly, pure lipid mixtures containing triolein (135 mg), phosphatidylcholine (75 mg), cholesterol (24 mg) (Nu-Chek Prep, Elysian, MN), and cholesteryl [13C]oleate (70 mg) were emulsified by sonication for 1 h in 2.2% glycerol in water. The mixture was then centrifuged for 10 min to remove titanium fragments and filtered into sterile vessels. All emulsions were confirmed to be sterile and pyrogen-free (Pharmacy Department, Royal Perth Hospital, Perth, Australia). Uniformly labeled [13C]oleate was purchased from Novachem Pty Ltd (Victoria, Australia), and cholesteryl [13C]oleate was synthesized from cholesterol and [13C]oleic acid as described previously (16).na+}, 百拇医药
Isolation and measurement of isotopic enrichment of apo Bna+}, 百拇医药
VLDL, IDL, and LDL were isolated from 3 mL plasma by sequential ultracentrifugation at 177500 x g for 24 h at 4°C in a Centrikon T-1190 centrifuge (Kontron Instruments, Milan) at densities of 1.006, 1.019, and 1.063 g/mL, respectively. The apo B fraction was precipitated by addition of an equal volume of 100% isopropanol as described by Egusa et al (18). The precipitate was then delipidated with isopropanol, dried, and hydrolyzed by the addition of 2 mL of 6 mol hydrochloric acid/L. After undergoing 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 with the use of resin (AG 50 W-X8; Bio-Rad, Richmond, CA). Samples were derivatized with acetonitrile and N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide and reconstituted in toluene for gas chromatography–mass spectrometry analysis (HP 5890 Series II Plus gas chromatograph coupled to an HP 5989B mass spectrometer; Hewlett-Packard, 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 (m/z) of 305 and 302 and with the use of electron-impact ionization. Tracer-to-tracee ratios were derived from isotopic ratios for each sample.
Quantification of apo B and other analytes(, 百拇医药
Plasma samples were combined to yield 5 pooled VLDL, IDL, and LDL samples per patient study (ie, 3 plasma aliquots pooled at regular intervals during the day of isotope infusion and 2 aliquots pooled from day 2 or 3 and day 4 or 5 after infusion). The apo B in VLDL, IDL, and LDL fractions from the pooled plasma was isolated as described in the previous paragraph by the use of the isopropanol method. A modified Lowry method was used to determine the apo B concentration in each lipoprotein fraction (19). The CV of plasma apo B concentration in VLDL, IDL, and LDL at several time points during the study was <10%.(, 百拇医药
Biochemical analyses(, 百拇医药
Plasma triacylglycerol and cholesterol concentrations were determined by standard enzymatic methods with the use of a biochemical analyzer (model 917; Hitachi Ltd, Tokyo). VLDL triacylglycerol was measured by an enzymatic method with the use of a commercial kit (Trace Scientific, Clayton, Australia) after ultracentrifiguation as described above. HDL cholesterol was measured by an enzymatic colorimetric method using a commercial kit (Boehringer Mannheim, Mannheim). Non-HDL cholesterol was derived as total cholesterol minus HDL cholesterol. LDL cholesterol was calculated by the Friedewald equation. Remnant-like particle cholestersol was determined from plasma with an assay kit (JIMRO-II; Japan Immunoresearch Laboratories, Takasaki, Japan) and the use of an immunoseparation technique. Apo A-I and apo B were determined by immunonephelometry (Beckman Instruments Inc, Palo Alto, CA). Apo C-III was measured by an immunoturbidimetric assay (Daichi, Toyko). Plasma nonesterified fatty acids were measured by an enzymatic, colorimetric method using a commercial kit (Randox, Crumlin, United Kingdom). Plasma insulin was measured by radioimmunoassay (DiaSorini, Saluggia, Italy) and glucose concentration by a hexokinase method on a Hitachi 917 analyzer. Insulin resistance was estimated by the homeostasis model assessment that uses the formula [fasting insulin (mU/L) x fasting plasma glucose (mmol/L)/22.5], as described by Matthews et al (20). Plasma lathosterol concentration was assayed by a modification of the method of Mori et al (21) with the use of gas chromatography–mass spectrometry. Apo E genotype was determined by use of methods from Hixson and Vernier (22) after the extraction of genomic DNA. Plasma liver [alanine aminotransferase (EC 2.6.1.2), asparate aminotransferase (EC 2.6.1.1), and alkaline phosphatase (EC 3.1.3.1)] and muscle [creatine kinase (EC 2.7.3.2)] enzymes were measured with a Hitachi 917 analyzer. The interassay CV of all measurements was <6%.
The expired CO2 in the exhaled breath samples was analyzed at each time point by isotope ratio-mass spectroscopy using a Finnigan BreathPlus instrument (Thermoquest Systems Pty Ltd, Sydney). The ratio of 13CO2 to 12CO2 was referenced to Peedeebelimnite standard values, and the delta unit value was calculated with the use of BREATHMAT software (Finigan MAT, Bremen, Germany). The delta units reference a sample of limestone, a standard in the 13-carbon isotope ratio field, and basal (nonenriched) values correspond to <1% 13C (15, 16):zy;, 百拇医药
where R is 13C:12C.zy;, 百拇医药
Kinetic analysiszy;, 百拇医药
Model of apo B metabolismzy;, 百拇医药
A multicompartmental model (Figure 1) was used to describe VLDL, IDL, and LDL apo B leucine tracer-to-tracee ratios (23). In multicompartmental modeling, each compartment or pool represents a group of kinetically homogenous particles. In this study, the SAAM II software program (SAAM Institute, Seattle) was used to fit the model to the observed tracer data. Metabolic values were subsequently derived from the model values giving the best fit. Part of the model consists of a 4-compartment subsystem (compartments 1–4) that describes plasma leucine kinetics. This subsystem is connected to an intrahepatic delay compartment (compartment 5) that accounts for the time required for the synthesis of apo B and its secretion into plasma. This model provides for the direct secretion of apo B into the VLDL, IDL, and LDL fractions. Compartments 6 through 10 are used to describe the kinetics of apo B in the VLDL fraction. Compartments 6 through 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 are described by 2 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 (compartment 13) or removed directly from plasma. The LDL section of the model consists of 2 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. VLDL, IDL, and LDL apo B metabolic values, including fractional catabolic rate (FCR), production rate, percentage conversion, and direct synthesis, were derived by following a fit of the compartment model to the apo B tracer-to-tracee ratio data.
fig.ommittedj@s9/:, http://www.100md.com
FIGURE 1. Multicompartmental model for apolipoprotein B (apo B) metabolism. Compartments 1–4 represent plasma leucine; compartment 5 is an intrahepatic delay compartment for apo B synthesis and secretion; compartments 6–10 represent a delipidation cascade for VLDL apo B; compartment 11 represents plasma intermediate-density lipoprotein (IDL) converting to LDL or being removed directly from plasma; compartment 12 represents a slowly-turning-over pool of IDL apo B; compartment 13 represents plasma LDL; and compartment 14 represents an extravascular LDL-exchange compartment.j@s9/:, http://www.100md.com
Model of chylomicron remnant–like emulsionj@s9/:, http://www.100md.com
A compartmental model describing the appearance of labeled carbon dioxide in breath was developed with SAAM II software (SAAM Institute) as described previously (15, 16). The compartmental model was fitted to the observed 13CO2 breath data, and estimates of the FCR were then determined.j@s9/:, http://www.100md.com
Statistical analysis
All analyses were carried out with SPSS software, version 10.1 (SPSS Inc, Chicago). Group characteristics were compared by t tests, after logarithmic transformation of skewed variables where appropriate. Chi-square analysis was used to compare the distribution of the apo E alleles. Adjustment for differences in baseline covariates and changes in variables during the study were performed by analysis of covariance with the use of general linear models. Associations were examined by Pearson’s correlation analysis. Statistical significance was defined at the 5% level by use of a two-tailed test.9m-1-v2, 百拇医药
RESULTS9m-1-v2, 百拇医药
Subject characteristics9m-1-v2, 百拇医药
The pretreatment clinical characteristics of the viscerally obese subjects randomly assigned to receive fish oils or placebo are shown in Table 1. The subjects were middle-aged and normotensive. There were no significant group differences in any of the variables. Sixteen subjects were E3/E3 homozygotes, 6 were E3/E4 heterozygotes, and 2 were E4/E4 heterozygotes. The frequency distributions of the apo E genotype were not significantly different between the groups. Mean (±SD) daily energy and nutrient intakes of the 24 obese subjects studied were 9700 ± 1479 kJ and 33 ± 5% energy from fat, 41 ± 7% energy from carbohydrate, 20 ± 6% energy from protein, and 6 ± 6% energy from alcohol. Nutrient intake did not differ significantly between the groups. Capsule counts confirmed that compliance with random assignment to receive fish oils or placebo was >95%. No subject reported untoward clinical side effects or showed significant increases in liver or muscle enzymes.
TABLE 1 Baseline clinical characteristics in subjects randomly assigned to receive fish oil or placebo1#}^-g-], 百拇医药
Effect of fish oil on biochemical values#}^-g-], 百拇医药
The plasma lipid, lipoprotein, apolipoprotein, lathosterol, nonesterified fatty acid, glucose, and insulin concentrations and the homeostasis model assessment score before and after 6-wk intervention in the fish oil and placebo groups are shown in Table 2. There were no significant group differences in the variables at baseline. Compared with the placebo, fish oils significantly (P < 0.05) lowered plasma triacylglycerols (-18%) and VLDL triacylglycerols (-33%). Plasma concentrations of total cholesterol, non-HDL cholesterol, LDL cholesterol, remnant-like particle cholesterol, apo B, apo C-III, and lathosterol also dropped, and HDL cholesterol and apo A-I rose by 3% and 5%, respectively. However, none of these changes were statistically significant. There were no significant changes in blood pressure with fish oil supplementation: posttreatment systolic and diastolic blood pressures were 131 ± 15 and 74 ± 9 mm Hg, respectively. Body weight, waist-to-hip ratio, plasma nonesterified fatty acids, glucose, insulin, homeostasis model assessment score, and dietary and alcohol intake (data not shown) did not change significantly during the study in either group.
fig.ommitted!1, 百拇医药
TABLE 2 Changes in plasma lipids, lipoproteins, and apolipoproteins and in biochemical characteristics after 6 wk of intervention in the fish oil and placebo groups1!1, 百拇医药
Effect of fish oil on apo B and chylomicron remnant–like emulsion kinetics!1, 百拇医药
The isotopic enrichment of VLDL, IDL, and LDL apo B after the infusion of trideuterated leucine in a representative subject before and after fish oil supplementation is shown in Figure 2. All tracer curves were of similar contour and showed a precursor-product relation between VLDL, IDL, and LDL apo B. As seen, the peak apo B enrichment in the VLDL fraction was higher with fish oils than without them. However, there were no differences in the apo B enrichment curves for the IDL and LDL apo B fractions before and after fish oil supplementation. The enrichment curves in the VLDL, IDL, and LDL fractions were of similar contour in the placebo group before and after treatment. The enrichment of 13CO2 in the breath after the injection of the emulsion in a representative subject before and during treatment with fish oil supplementation over time is shown in Figure 3. There was a rapid increase in the enrichment of 13CO2 that peaked at <4 h, which reflected the catabolism of remnant-like particles via lipolysis and hepatic removal and which was followed by a slow decline over the subsequent 20 h. The enrichment curve was of similar contour and peak enrichment between before and after fish oil supplementation.
fig.ommittedu/, http://www.100md.com
FIGURE 2. Plasma isotopic tracer curves for VLDL apolipoprotein B , intermediate-density lipoprotein (IDL) apolipoprotein B , and LDL apolipoprotein B after the administration of trideuterated leucine in a representative subject from the fish oil group before (top) and after (bottom) treatment. Observed values (symbols) and model-predicted values (lines) are shown.u/, http://www.100md.com
fig.ommittedu/, http://www.100md.com
FIGURE 3. Enrichment of 13CO2 in breath after the administration of [13C]oleate chylomicron remnant–like particle emulsion in a representative subject from the fish oil group before (top) and after (bottom) treatment. Observed values (symbols) and model-predicted values (lines) are shown.u/, http://www.100md.com
Compared with the lean control subjects (n = 10; see reference 6), the 24 obese subjects had significantly higher VLDL apo B production (9.7 ± 1.8 and 15.5 ± 2.2 mg·kg-1·d-1, respectively; P < 0.05), less conversion of VLDL apo B to LDL apo B (64.0 ± 1.2% and 28.4 ± 7.3%, respectively; P < 0.05), and lower FCRs of LDL apo B (0.43 ± 0.07 and 0.28 ± 0.03 pools/d, respectively; P < 0.05). The FCR of the emulsion also tended to be lower in the obese group than in the lean group (1.90 ± 0.19 and 2.30 ± 0.22 pools/d, respectively; P = 0.076), which is consistent with the significant differences found in comparison with larger sample sizes (6).
Compared with the placebo , fish oils significantly (P < 0.05) lowered VLDL apo B pool size (-20%) and the VLDL apo B production rate (-29%), without any significant change in IDL and LDL apo B production. The percentage conversion of VLDL apo B to IDL apo B, VLDL apo B to LDL apo B, and IDL apo B to LDL apo B also increased significantly (P < 0.05): 71%, 93%, and 11%, respectively. However, the FCRs of apo B and chylomicron remnant–like emulsion did not change significantly with fish oil supplementation.s', 百拇医药
fig.ommitteds', 百拇医药
TABLE 3 Metabolic variables for apolipoprotein (apo) B-100 and the fractional catabolic rate (FCR) of the remnant-like emulsion before (baseline) and after 6 wk of intervention in the fish oil and placebo groups1s', 百拇医药
DISCUSSIONs', 百拇医药
In this double-blind, randomized, placebo-controlled study, we provide new information concerning the kinetic effects of n-3 fatty acids on apo B metabolism in viscerally obese subjects with insulin resistance. Our results show that n-3 fatty acids lower plasma triacylglycerols by means of a reduction in VLDL apo B production. These effects were shown in the absence of changes in body weight, insulin resistance, and FCRs of apo B and chylomicron remnant–like particles.
This is the first study to determine the kinetic effect of n-3 fatty acids on apo B metabolism in obese subjects with insulin resistance. Previous kinetic studies examined the effect of n-3 fatty acids on apo B metabolism in nonobese subjects only. By using radiolabeling techniques, Nestel et al (10) found that fish oil supplementation reduced VLDL apo B synthesis in 5 normolipidemic subjects and 2 hyperlipidemic patients. Illingworth et al (24) found in 7 normolipidemic subjects that fish oil reduced LDL apo B synthesis. Fisher et al (13) reported in 5 patients with type 2 diabetes mellitus that fish oil increased the conversion of VLDL to LDL. In an uncontrolled trial using stable isotopy, Bordin et al (11) found that fish oil decreased VLDL apo B production in 10 normolipidemic subjects. The discrepant findings of those studies, particularly with regard to LDL apo B metabolism, might be accounted for by differences in subject characteristics, experimental protocols, methods of data analysis, and the type and dose of fish oil employed. Moreover, most studies have been uncontrolled, restricted to a single lipoprotein compartment, and generally of small sample size. We extended these reports by examining, in a placebo-controlled study design, insulin-resistant obese subjects with dyslipidemia. In addition, we investigated the effects of n-3 fatty acids on apo B metabolism in VLDL, IDL, and LDL compartments and on chylomicron remnant metabolism.
It is well recognized that visceral obesity and insulin resistance alter the metabolism of apo B–containing lipoproteins (1). Insulin has diverse physiologic effects on lipoprotein metabolism. It reduces hepatic apo B secretion by suppressing the delivery of nonesterified fatty acids to the liver from adipose tissue and by inhibiting new hepatic cholesterol synthesis (25, 26). Insulin also enhances the lipolysis and hepatic uptake of triacylglycerol-rich apo B–containing lipoproteins, including chylomicron remnants, by the up-regulation of lipoprotein lipase activity (27) and the stimulation of LDL receptor activity (28), respectively. In subjects with insulin resistance, this "normal" insulin-mediated regulation of apo B metabolism is diminished. This occurrence is consistent with our observation that our obese subjects had higher VLDL apo B production and less conversion of VLDL apo B to LDL apo B, as well as lower FCRs of LDL apo B and the remnant-like emulsion than lean control subjects had.
Studies in animals and humans have shown that the hypotriacylglycerolemic effect of n-3 fatty acids primarily involves the suppression of hepatic VLDL apo B production (9–11). This suppression is chiefly due to a decrease in triacylglycerol synthesis and an increase in fatty acid mitochondrial ß-oxidation (29). Such a concept is consistent with our observation that the VLDL apo B pool size and production rate decreased with fish oil treatment without any significant change in cholesterol synthesis, as reflected in the plasma lathosterol concentration. We also showed a significant increase in the percentage conversion of VLDL apo B to LDL apo B. Given that there was a reduction in hepatic VLDL apo B secretion with fish oil, the absolute flux of VLDL apo B into the circulation would drop in parallel. The system would accordingly be less saturated with VLDL particles, so that a greater proportion of particles would conceivably be converted into LDL down the delipidation cascade. Although we did not find a statistically significant decrease in the ratio of VLDL triacylglycerol to VLDL apo B after supplementation with fish oils, our study could have been underpowered to detect a true effect of fish oils in decreasing VLDL particle size. This potential effect may be physiologically meaningful in relation to increased conversion to IDL and LDL apo B. Consistent with this, other studies have shown that enrichment of n-3 fatty acids in VLDL particles favor the conversion of VLDL to LDL (30), although the studies have not specifically referred to insulin-resistant obese subjects. Moreover, fish oil itself might have down-regulated hepatic receptor activity and hindered the removal of apo B. The net effect of fish oil in our insulin-resistant subjects may therefore, in percentage terms, increase apo B conversion down the lipolytic pathway. In spite of increased apo B conversion, we did not find a significant increase in IDL or LDL apo B pool sizes, which suggests that fish oils did not result in a significant concomitant change in the individual FCR of these lipoproteins. Hence, we suggest that the reduction in plasma VLDL particle numbers (as opposed to VLDL size or composition) with n-3 fatty acids has a significant effect in modulating apo B trafficking without any alteration in overall apo B clearance. We previously reported that, in viscerally obese men, an improvement in insulin sensitivity after weight loss resulted in an increase in LDL apo B FCR (4). Because fish oil treatment did not alter insulin sensitivity in the present study, one could argue that insulin resistance was still down-regulating the LDL receptors and LDL activities. Given that the primary mechanism of action of n-3 fatty acids is on VLDL production, we would expect the FCRs of apo B to remain suppressed in the setting of persistent insulin resistance. The effect of fish oils on other lipoprotein-related variables, including hepatic lipase, apo C-II, and hepatic receptor activity, may also modulate apo B clearance and thus may merit further investigation.
Our results also show that VLDL particle size, as reflected by the ratio of VLDL triacylglycerols to VLDL apo B, did not change significantly with fish oil supplementation. On the assumption that the FCRs for VLDL apo B and VLDL triacylglycerols are the same when the plasma VLDL triacylglycerol-to-VLDL apo B ratio remains constant in a steady state condition, hepatic triacylglycerol synthesis can be estimated as described by Sigurdsson et al (31). Accordingly, our results showed that n-3 fatty acids significantly decreased hepatic triacylglycerol synthesis by 35% (from 174 ± 27 to 112 ± 22 mg·kg-1·d-1, P < 0.03). This suggests that the inhibition of triacylglycerol synthesis by n-3 fatty acids would be accompanied by a commensurate inhibition of apo B synthesis (32) and could have contributed to a reduction in VLDL apo B production. Further kinetic examinations of VLDL subspecies and hepatic triacylglycerol synthesis may help to clarify the contribution of triacylglycerol and apo B to VLDL production.
Previous kinetic studies examining chylomicron remnant metabolism showed that n-3 fatty acids enhance postprandial chylomicron and chylomicron remnant clearance (14, 33). In this present study, we did not show any significant change in plasma remnant-like particle cholesterol and the FCR of chylomicron remnant–like emulsion. The concentration of apo B-48 in plasma also did not change with fish oil supplementation (DC Chan, JCL Mamo, GF Watts, unpublished observations, 2002). These results suggest that n-3 fatty acids do not have a significant effect on chylomicron remnant metabolism in insulin-resistant obese subjects. Given that there was no change in IDL and LDL apo B pool sizes after supplementation with fish oil in our study, we suggest that chylomicron remnants were still competing with VLDL remnants for the same hepatic LDL receptors. This suggestion is consistent with our findings that n-3 fatty acids do not affect the FCRs of apo B or chylomicron remnant–like emulsion. However, our results might have been different had we used postprandial fat challenges (33, 34). The breath test provides a specific functional assessment of chylomicron remnant metabolism and has been validated in patients with familial dyslipidemias (15). However, it depends not only on the plasma clearance of remnant-like emulsions, but also on the subsequent oxidation of fatty acids hydrolyzed from the emulsion of cholesteryl ester. The n-3 fatty acids have been reported to stimulate mitochondrial ß-oxidation of fatty acid in the liver (29). Nevertheless, this effect would only enhance the FCR of remnant-like emulsion measured by the breath test and therefore would not confound the results reported in this study.
In conclusion, our data support the hypothesis that n-3 fatty acids lower plasma triacylglycerols and reduce VLDL apo B pool size in viscerally obese subjects with dyslipidemia. These improvements are chiefly due to a reduction in VLDL apo B secretion by the liver. Fish oil supplementation does not alter the catabolism of apo B in all lipoproteins or chylomicron remnants. In view of the differential effects of EPA and DHA on serum lipids (35, 36), it may be of interest to examine the effect of pure DHA and EPA, as well as that of certain genetic polymorphisms, on apo B and chylomicron remnant metabolism (37). Further investigations should also explore the incremental effect of weight reduction, statin treatment, or the addition of insulin sensitizers to fish oils on the kinetics of apo B in these subjects.4l/, 百拇医药
ACKNOWLEDGMENTS4l/, 百拇医药
We thank Frank van Bockxmeer for the apolipoprotein E 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.
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Watts GF, Riches FM, Humphries SE, Talmud PJ, van Bockxmeer FM. Genotypic associations of the hepatic secretion of VLDL apolipoprotein B-100 in obesity. J Lipid Res 2000;41:481–8. Received for publication November 27, 2001. Accepted for publication April 30, 2002.(Dick C Chan Gerald F Watts Trevor A Mori P Hugh R Barrett Trevor G Redgrave and Lawrence J Beilin)
2 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 Australia, and the National Institutes of Health (NCRR 12609). PHRB is a Career Development Fellow of the National Heart Foundation. The Omacor capsules and corn oil were a gift from Provona Biocare, Oslo. x{)z\2, 百拇医药
ABSTRACTx{)z\2, 百拇医药
Background: Lipid abnormalities may contribute to the increased risk of atherosclerosis and coronary disease in visceral obesity. Fish oils lower plasma triacylglycerols, but the underlying mechanisms are not fully understood.x{)z\2, 百拇医药
Objective: We studied the effect of fish oils on the metabolism of apolipoprotein B-100 (apo B) and chylomicron remnants in obese men.
Design: Twenty-four dyslipidemic, viscerally obese men were randomly assigned to receive either fish oil capsules (4 g/d, consisting of 45% eicosapentaenoic acid and 39% docosahexaenoic acid as ethyl esters) or matching placebo (corn oil, 4 g/d) for 6 wk. VLDL, intermediate-density lipoprotein (IDL), and LDL apo B kinetics were assessed by following apo B isotopic enrichment with the use of gas chromatography–mass spectrometry after an intravenous bolus injection of trideuterated leucine. Chylomicron remnant catabolism was measured with the use of an intravenous injection of a chylomicron remnant–like emulsion containing cholesteryl [13C]oleate, and isotopic enrichment of 13CO2 in breath was measured with isotope ratio mass spectrometry. Kinetic values were derived with multicompartmental models.c*8o, 百拇医药
Results: Fish oil supplementation significantly (P < 0.05) lowered plasma concentrations of triacylglycerols (-18%) and VLDL apo B (-20%) and the hepatic secretion of VLDL apo B (-29%) compared with placebo. The percentage of conversions of VLDL apo B to IDL apo B, VLDL apo B to LDL apo B, and IDL apo B to LDL apo B also increased significantly (P < 0.05): 71%, 93%, and 11%, respectively. Fish oils did not significantly alter the fractional catabolic rates of apo B in VLDL, IDL, or LDL or alter the catabolism of the chylomicron remnant–like emulsion.
Conclusion: Fish oils effectively lower the plasma concentration of triacylglycerols, chiefly by decreasing VLDL apo B production but not by altering the catabolism of apo B–containing lipoprotein or chylomicron remnants.nr}*%, 百拇医药
Key Words: Fish oils • apolipoprotein B • chylomicron remnants • visceral obesity • mennr}*%, 百拇医药
INTRODUCTIONnr}*%, 百拇医药
Obesity is a major risk factor for type 2 diabetes and cardiovascular disease. Visceral obesity, the most clinically important topographical form, is typically seen in overweight men. It is strongly associated with dyslipidemia and may account for the increased risk of cardiovascular disease in these men (1). Dyslipidemia in visceral obesity is principally due to insulin resistance, which may perturb the metabolism of apolipoprotein (apo) B-100 (apo B) and chylomicron remnants (2). These lipid abnormalities involve increased hepatic secretion of VLDL apo B (3), as well as impaired catabolism of VLDL, intermediate-density lipoprotein (IDL), LDL, and chylomicron remnants (4–6).
Efforts to treat dyslipidemia in obese persons with the use of hypocaloric diets and other lifestyle measures have largely been disappointing, owing to poor adherence to such programs (7). Effective management of dyslipidemia in persons with obesity and other high-risk states often requires the use of lipid-regulating pharmacotherapy (8). Fish oils are a rich source of long-chain n-3 fatty acids, primarily eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Fish oil supplementation has been shown to lower plasma triacylglycerols (9) and may therefore constitute a potentially effective treatment for obesity-related dyslipidemia. Previous evidence suggests that n-3 fatty acids decrease the secretion of hepatic VLDL (10, 11), increase the conversion of VLDL to LDL (12, 13), and increase the clearance of chylomicron (14), but these studies were not specific to obese subjects. In studies of visceral obesity, we have previously reported increased hepatic secretion of apo B and delayed catabolism of chylomicron remnants (3, 5, 6). These observations provide a rationale for the use of n-3 fatty acids in correcting lipoprotein abnormalities in obese subjects. The effects of n-3 fatty acids on apo B and chylomicron remnant metabolism in viscerally obese subjects with insulin resistance have not been examined previously. It may be anticipated that, by reducing the secretion of VLDL apo B, n-3 fatty acids may decrease the competition of lipolysis and high-affinity clearance pathways that are impaired in obesity and insulin resistance. In contrast, n-3 fatty acids may increase the production of IDL and LDL by enhancing the catabolism of VLDL. We therefore investigated these effects of n-3 fatty acids in obese subjects by using stable isotope–labeled techniques that measure the kinetics of apo B and chylomicron remnants. Chylomicron remnant catabolism was assessed with the use of a novel breath test that we previously described and validated for a functional measure of chylomicron remnant metabolism (15, 16). The effect of fish oils on this breath test has not yet been investigated.
SUBJECTS AND METHODSep, 百拇医药
Subjectsep, 百拇医药
Twenty-four obese men were recruited from the community by newspaper advertisement. Obesity was defined as a waist circumference >100 cm, waist-to-hip ratio >0.97, and body mass index (in kg/m2) >29. All obese subjects were dyslipidemic and had concentrations of plasma triacylglycerols >1.2 mmol/L and cholesterol >5.2 mmol/L while they were consuming weight-maintaining diets ad libitum. None of the subjects had diabetes mellitus (excluded by oral-glucose-tolerance test), apolipoprotein E2/E2 genotype, macroproteinuria, creatinemia (>120 µmol/L), hypothyroidism, or abnormal liver enzymes; no subject consumed >30 g alcohol/d. None reported a history of cardiovascular disease or was taking any medication or other agents known to affect lipid metabolism. Ten normolipidemic, lean, insulin-sensitive men (body mass index, 25 ± 3; waist-to-hip ratio, 0.92 ± 0.06; plasma triacylglycerol, 0.77 ± 0.25 mmol/L; total cholesterol, 4.33 ± 0.34 mmol/L; HDL cholesterol, 1.28 ± 0.30 mmol/L; insulin, 24 ± 4 mU/L; and glucose, 5.4 ± 0.3 mmol/L) who were similar in age to our obese subjects (53 ± 9 y) were also recruited by newspaper advertisement from the community as control subjects. These subjects were described in detail elsewhere (6). All subjects provided written informed consent, and the study was approved by the Ethics Committee of the Royal Perth Hospital.
Study designm, 百拇医药
The study reported here represents a substudy of a larger, randomized, double-blind, placebo-controlled intervention trial examining the effect of fish oils and other agents on aspects of lipoprotein metabolism in obesity, which will be reported separately. The present report 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 randomly assigned to a 6-wk treatment period of 4 g/d doses of either fish oil capsules of 45% EPA and 39% DHA as ethyl esters (Omacor; Provona Biocare, Oslo) or matching placebo capsules (corn oil; Pronova Biocare). Patients were advised to continue on isocaloric diets and to maintain their accustomed degree of physical activity during the study. Compliance with the fish oil and placebo protocols was checked by capsule count at weeks 3 and 6 of the treatment period.m, 百拇医药
Clinical protocolsm, 百拇医药
All subjects were admitted to the clinical investigation unit 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 (17). The plasma volume was modified by multiplying a factor that uses the formula [(relative body weight – 310)/-215] to adjust for the decrease in relative plasma volume associated with an increase in body weight as described by Riches et al (3, 4). After 3 min, arterial blood pressure was recorded with the use of a monitor (Dinamap1846 SX/P; Critikon Inc, Tampa, FL) while subjects were in a supine position. Dietary intake was assessed for energy and major nutrients with the use of at least two 24-h dietary diaries at both the beginning and the end of the study. Diets were analyzed with the use of DIET 4 Nutrient Calculation Software (Xyris Software, Highgate Hill, Australia). A single bolus of trideuterated leucine (5 mg/kg body wt) and the sterile, isotopically labeled chylomicron remnant–like emulsion containing cholesteryl [13C]oleate (14 mL) was administered intravenously within 2 min into an antecubital vein via a 21-gauge butterfly needle. Blood samples were taken at baseline and at 5, 10, 20, 30, and 40 min and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, and 10 h after injection of the isotope. End-expiratory breath samples were collected into an evacuated tube at baseline and after injection every 10 min for the first hour, every 20 min for the second hour, every 30 min for the next 5 h, and every hour for another 3 h. Subjects were then given a snack and allowed to go home. They were requested to refrain from vigorous activity and to eat a light supper before retiring. Additional fasting blood samples were collected in the morning of the next 4 d (at 24, 48, 72, and 96 h). Two additional breath samples were also collected on the following day (at 24 h). All the procedures were repeated after 6-wk treatment with either fish oils or placebo.
Preparation of stable isotope–labeled remnant-like emulsionsna+}, 百拇医药
Stable isotope–labeled remnant-like emulsions were prepared as described previously (15). Briefly, pure lipid mixtures containing triolein (135 mg), phosphatidylcholine (75 mg), cholesterol (24 mg) (Nu-Chek Prep, Elysian, MN), and cholesteryl [13C]oleate (70 mg) were emulsified by sonication for 1 h in 2.2% glycerol in water. The mixture was then centrifuged for 10 min to remove titanium fragments and filtered into sterile vessels. All emulsions were confirmed to be sterile and pyrogen-free (Pharmacy Department, Royal Perth Hospital, Perth, Australia). Uniformly labeled [13C]oleate was purchased from Novachem Pty Ltd (Victoria, Australia), and cholesteryl [13C]oleate was synthesized from cholesterol and [13C]oleic acid as described previously (16).na+}, 百拇医药
Isolation and measurement of isotopic enrichment of apo Bna+}, 百拇医药
VLDL, IDL, and LDL were isolated from 3 mL plasma by sequential ultracentrifugation at 177500 x g for 24 h at 4°C in a Centrikon T-1190 centrifuge (Kontron Instruments, Milan) at densities of 1.006, 1.019, and 1.063 g/mL, respectively. The apo B fraction was precipitated by addition of an equal volume of 100% isopropanol as described by Egusa et al (18). The precipitate was then delipidated with isopropanol, dried, and hydrolyzed by the addition of 2 mL of 6 mol hydrochloric acid/L. After undergoing 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 with the use of resin (AG 50 W-X8; Bio-Rad, Richmond, CA). Samples were derivatized with acetonitrile and N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide and reconstituted in toluene for gas chromatography–mass spectrometry analysis (HP 5890 Series II Plus gas chromatograph coupled to an HP 5989B mass spectrometer; Hewlett-Packard, 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 (m/z) of 305 and 302 and with the use of electron-impact ionization. Tracer-to-tracee ratios were derived from isotopic ratios for each sample.
Quantification of apo B and other analytes(, 百拇医药
Plasma samples were combined to yield 5 pooled VLDL, IDL, and LDL samples per patient study (ie, 3 plasma aliquots pooled at regular intervals during the day of isotope infusion and 2 aliquots pooled from day 2 or 3 and day 4 or 5 after infusion). The apo B in VLDL, IDL, and LDL fractions from the pooled plasma was isolated as described in the previous paragraph by the use of the isopropanol method. A modified Lowry method was used to determine the apo B concentration in each lipoprotein fraction (19). The CV of plasma apo B concentration in VLDL, IDL, and LDL at several time points during the study was <10%.(, 百拇医药
Biochemical analyses(, 百拇医药
Plasma triacylglycerol and cholesterol concentrations were determined by standard enzymatic methods with the use of a biochemical analyzer (model 917; Hitachi Ltd, Tokyo). VLDL triacylglycerol was measured by an enzymatic method with the use of a commercial kit (Trace Scientific, Clayton, Australia) after ultracentrifiguation as described above. HDL cholesterol was measured by an enzymatic colorimetric method using a commercial kit (Boehringer Mannheim, Mannheim). Non-HDL cholesterol was derived as total cholesterol minus HDL cholesterol. LDL cholesterol was calculated by the Friedewald equation. Remnant-like particle cholestersol was determined from plasma with an assay kit (JIMRO-II; Japan Immunoresearch Laboratories, Takasaki, Japan) and the use of an immunoseparation technique. Apo A-I and apo B were determined by immunonephelometry (Beckman Instruments Inc, Palo Alto, CA). Apo C-III was measured by an immunoturbidimetric assay (Daichi, Toyko). Plasma nonesterified fatty acids were measured by an enzymatic, colorimetric method using a commercial kit (Randox, Crumlin, United Kingdom). Plasma insulin was measured by radioimmunoassay (DiaSorini, Saluggia, Italy) and glucose concentration by a hexokinase method on a Hitachi 917 analyzer. Insulin resistance was estimated by the homeostasis model assessment that uses the formula [fasting insulin (mU/L) x fasting plasma glucose (mmol/L)/22.5], as described by Matthews et al (20). Plasma lathosterol concentration was assayed by a modification of the method of Mori et al (21) with the use of gas chromatography–mass spectrometry. Apo E genotype was determined by use of methods from Hixson and Vernier (22) after the extraction of genomic DNA. Plasma liver [alanine aminotransferase (EC 2.6.1.2), asparate aminotransferase (EC 2.6.1.1), and alkaline phosphatase (EC 3.1.3.1)] and muscle [creatine kinase (EC 2.7.3.2)] enzymes were measured with a Hitachi 917 analyzer. The interassay CV of all measurements was <6%.
The expired CO2 in the exhaled breath samples was analyzed at each time point by isotope ratio-mass spectroscopy using a Finnigan BreathPlus instrument (Thermoquest Systems Pty Ltd, Sydney). The ratio of 13CO2 to 12CO2 was referenced to Peedeebelimnite standard values, and the delta unit value was calculated with the use of BREATHMAT software (Finigan MAT, Bremen, Germany). The delta units reference a sample of limestone, a standard in the 13-carbon isotope ratio field, and basal (nonenriched) values correspond to <1% 13C (15, 16):zy;, 百拇医药
where R is 13C:12C.zy;, 百拇医药
Kinetic analysiszy;, 百拇医药
Model of apo B metabolismzy;, 百拇医药
A multicompartmental model (Figure 1) was used to describe VLDL, IDL, and LDL apo B leucine tracer-to-tracee ratios (23). In multicompartmental modeling, each compartment or pool represents a group of kinetically homogenous particles. In this study, the SAAM II software program (SAAM Institute, Seattle) was used to fit the model to the observed tracer data. Metabolic values were subsequently derived from the model values giving the best fit. Part of the model consists of a 4-compartment subsystem (compartments 1–4) that describes plasma leucine kinetics. This subsystem is connected to an intrahepatic delay compartment (compartment 5) that accounts for the time required for the synthesis of apo B and its secretion into plasma. This model provides for the direct secretion of apo B into the VLDL, IDL, and LDL fractions. Compartments 6 through 10 are used to describe the kinetics of apo B in the VLDL fraction. Compartments 6 through 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 are described by 2 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 (compartment 13) or removed directly from plasma. The LDL section of the model consists of 2 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. VLDL, IDL, and LDL apo B metabolic values, including fractional catabolic rate (FCR), production rate, percentage conversion, and direct synthesis, were derived by following a fit of the compartment model to the apo B tracer-to-tracee ratio data.
fig.ommittedj@s9/:, http://www.100md.com
FIGURE 1. Multicompartmental model for apolipoprotein B (apo B) metabolism. Compartments 1–4 represent plasma leucine; compartment 5 is an intrahepatic delay compartment for apo B synthesis and secretion; compartments 6–10 represent a delipidation cascade for VLDL apo B; compartment 11 represents plasma intermediate-density lipoprotein (IDL) converting to LDL or being removed directly from plasma; compartment 12 represents a slowly-turning-over pool of IDL apo B; compartment 13 represents plasma LDL; and compartment 14 represents an extravascular LDL-exchange compartment.j@s9/:, http://www.100md.com
Model of chylomicron remnant–like emulsionj@s9/:, http://www.100md.com
A compartmental model describing the appearance of labeled carbon dioxide in breath was developed with SAAM II software (SAAM Institute) as described previously (15, 16). The compartmental model was fitted to the observed 13CO2 breath data, and estimates of the FCR were then determined.j@s9/:, http://www.100md.com
Statistical analysis
All analyses were carried out with SPSS software, version 10.1 (SPSS Inc, Chicago). Group characteristics were compared by t tests, after logarithmic transformation of skewed variables where appropriate. Chi-square analysis was used to compare the distribution of the apo E alleles. Adjustment for differences in baseline covariates and changes in variables during the study were performed by analysis of covariance with the use of general linear models. Associations were examined by Pearson’s correlation analysis. Statistical significance was defined at the 5% level by use of a two-tailed test.9m-1-v2, 百拇医药
RESULTS9m-1-v2, 百拇医药
Subject characteristics9m-1-v2, 百拇医药
The pretreatment clinical characteristics of the viscerally obese subjects randomly assigned to receive fish oils or placebo are shown in Table 1. The subjects were middle-aged and normotensive. There were no significant group differences in any of the variables. Sixteen subjects were E3/E3 homozygotes, 6 were E3/E4 heterozygotes, and 2 were E4/E4 heterozygotes. The frequency distributions of the apo E genotype were not significantly different between the groups. Mean (±SD) daily energy and nutrient intakes of the 24 obese subjects studied were 9700 ± 1479 kJ and 33 ± 5% energy from fat, 41 ± 7% energy from carbohydrate, 20 ± 6% energy from protein, and 6 ± 6% energy from alcohol. Nutrient intake did not differ significantly between the groups. Capsule counts confirmed that compliance with random assignment to receive fish oils or placebo was >95%. No subject reported untoward clinical side effects or showed significant increases in liver or muscle enzymes.
TABLE 1 Baseline clinical characteristics in subjects randomly assigned to receive fish oil or placebo1#}^-g-], 百拇医药
Effect of fish oil on biochemical values#}^-g-], 百拇医药
The plasma lipid, lipoprotein, apolipoprotein, lathosterol, nonesterified fatty acid, glucose, and insulin concentrations and the homeostasis model assessment score before and after 6-wk intervention in the fish oil and placebo groups are shown in Table 2. There were no significant group differences in the variables at baseline. Compared with the placebo, fish oils significantly (P < 0.05) lowered plasma triacylglycerols (-18%) and VLDL triacylglycerols (-33%). Plasma concentrations of total cholesterol, non-HDL cholesterol, LDL cholesterol, remnant-like particle cholesterol, apo B, apo C-III, and lathosterol also dropped, and HDL cholesterol and apo A-I rose by 3% and 5%, respectively. However, none of these changes were statistically significant. There were no significant changes in blood pressure with fish oil supplementation: posttreatment systolic and diastolic blood pressures were 131 ± 15 and 74 ± 9 mm Hg, respectively. Body weight, waist-to-hip ratio, plasma nonesterified fatty acids, glucose, insulin, homeostasis model assessment score, and dietary and alcohol intake (data not shown) did not change significantly during the study in either group.
fig.ommitted!1, 百拇医药
TABLE 2 Changes in plasma lipids, lipoproteins, and apolipoproteins and in biochemical characteristics after 6 wk of intervention in the fish oil and placebo groups1!1, 百拇医药
Effect of fish oil on apo B and chylomicron remnant–like emulsion kinetics!1, 百拇医药
The isotopic enrichment of VLDL, IDL, and LDL apo B after the infusion of trideuterated leucine in a representative subject before and after fish oil supplementation is shown in Figure 2. All tracer curves were of similar contour and showed a precursor-product relation between VLDL, IDL, and LDL apo B. As seen, the peak apo B enrichment in the VLDL fraction was higher with fish oils than without them. However, there were no differences in the apo B enrichment curves for the IDL and LDL apo B fractions before and after fish oil supplementation. The enrichment curves in the VLDL, IDL, and LDL fractions were of similar contour in the placebo group before and after treatment. The enrichment of 13CO2 in the breath after the injection of the emulsion in a representative subject before and during treatment with fish oil supplementation over time is shown in Figure 3. There was a rapid increase in the enrichment of 13CO2 that peaked at <4 h, which reflected the catabolism of remnant-like particles via lipolysis and hepatic removal and which was followed by a slow decline over the subsequent 20 h. The enrichment curve was of similar contour and peak enrichment between before and after fish oil supplementation.
fig.ommittedu/, http://www.100md.com
FIGURE 2. Plasma isotopic tracer curves for VLDL apolipoprotein B , intermediate-density lipoprotein (IDL) apolipoprotein B , and LDL apolipoprotein B after the administration of trideuterated leucine in a representative subject from the fish oil group before (top) and after (bottom) treatment. Observed values (symbols) and model-predicted values (lines) are shown.u/, http://www.100md.com
fig.ommittedu/, http://www.100md.com
FIGURE 3. Enrichment of 13CO2 in breath after the administration of [13C]oleate chylomicron remnant–like particle emulsion in a representative subject from the fish oil group before (top) and after (bottom) treatment. Observed values (symbols) and model-predicted values (lines) are shown.u/, http://www.100md.com
Compared with the lean control subjects (n = 10; see reference 6), the 24 obese subjects had significantly higher VLDL apo B production (9.7 ± 1.8 and 15.5 ± 2.2 mg·kg-1·d-1, respectively; P < 0.05), less conversion of VLDL apo B to LDL apo B (64.0 ± 1.2% and 28.4 ± 7.3%, respectively; P < 0.05), and lower FCRs of LDL apo B (0.43 ± 0.07 and 0.28 ± 0.03 pools/d, respectively; P < 0.05). The FCR of the emulsion also tended to be lower in the obese group than in the lean group (1.90 ± 0.19 and 2.30 ± 0.22 pools/d, respectively; P = 0.076), which is consistent with the significant differences found in comparison with larger sample sizes (6).
Compared with the placebo , fish oils significantly (P < 0.05) lowered VLDL apo B pool size (-20%) and the VLDL apo B production rate (-29%), without any significant change in IDL and LDL apo B production. The percentage conversion of VLDL apo B to IDL apo B, VLDL apo B to LDL apo B, and IDL apo B to LDL apo B also increased significantly (P < 0.05): 71%, 93%, and 11%, respectively. However, the FCRs of apo B and chylomicron remnant–like emulsion did not change significantly with fish oil supplementation.s', 百拇医药
fig.ommitteds', 百拇医药
TABLE 3 Metabolic variables for apolipoprotein (apo) B-100 and the fractional catabolic rate (FCR) of the remnant-like emulsion before (baseline) and after 6 wk of intervention in the fish oil and placebo groups1s', 百拇医药
DISCUSSIONs', 百拇医药
In this double-blind, randomized, placebo-controlled study, we provide new information concerning the kinetic effects of n-3 fatty acids on apo B metabolism in viscerally obese subjects with insulin resistance. Our results show that n-3 fatty acids lower plasma triacylglycerols by means of a reduction in VLDL apo B production. These effects were shown in the absence of changes in body weight, insulin resistance, and FCRs of apo B and chylomicron remnant–like particles.
This is the first study to determine the kinetic effect of n-3 fatty acids on apo B metabolism in obese subjects with insulin resistance. Previous kinetic studies examined the effect of n-3 fatty acids on apo B metabolism in nonobese subjects only. By using radiolabeling techniques, Nestel et al (10) found that fish oil supplementation reduced VLDL apo B synthesis in 5 normolipidemic subjects and 2 hyperlipidemic patients. Illingworth et al (24) found in 7 normolipidemic subjects that fish oil reduced LDL apo B synthesis. Fisher et al (13) reported in 5 patients with type 2 diabetes mellitus that fish oil increased the conversion of VLDL to LDL. In an uncontrolled trial using stable isotopy, Bordin et al (11) found that fish oil decreased VLDL apo B production in 10 normolipidemic subjects. The discrepant findings of those studies, particularly with regard to LDL apo B metabolism, might be accounted for by differences in subject characteristics, experimental protocols, methods of data analysis, and the type and dose of fish oil employed. Moreover, most studies have been uncontrolled, restricted to a single lipoprotein compartment, and generally of small sample size. We extended these reports by examining, in a placebo-controlled study design, insulin-resistant obese subjects with dyslipidemia. In addition, we investigated the effects of n-3 fatty acids on apo B metabolism in VLDL, IDL, and LDL compartments and on chylomicron remnant metabolism.
It is well recognized that visceral obesity and insulin resistance alter the metabolism of apo B–containing lipoproteins (1). Insulin has diverse physiologic effects on lipoprotein metabolism. It reduces hepatic apo B secretion by suppressing the delivery of nonesterified fatty acids to the liver from adipose tissue and by inhibiting new hepatic cholesterol synthesis (25, 26). Insulin also enhances the lipolysis and hepatic uptake of triacylglycerol-rich apo B–containing lipoproteins, including chylomicron remnants, by the up-regulation of lipoprotein lipase activity (27) and the stimulation of LDL receptor activity (28), respectively. In subjects with insulin resistance, this "normal" insulin-mediated regulation of apo B metabolism is diminished. This occurrence is consistent with our observation that our obese subjects had higher VLDL apo B production and less conversion of VLDL apo B to LDL apo B, as well as lower FCRs of LDL apo B and the remnant-like emulsion than lean control subjects had.
Studies in animals and humans have shown that the hypotriacylglycerolemic effect of n-3 fatty acids primarily involves the suppression of hepatic VLDL apo B production (9–11). This suppression is chiefly due to a decrease in triacylglycerol synthesis and an increase in fatty acid mitochondrial ß-oxidation (29). Such a concept is consistent with our observation that the VLDL apo B pool size and production rate decreased with fish oil treatment without any significant change in cholesterol synthesis, as reflected in the plasma lathosterol concentration. We also showed a significant increase in the percentage conversion of VLDL apo B to LDL apo B. Given that there was a reduction in hepatic VLDL apo B secretion with fish oil, the absolute flux of VLDL apo B into the circulation would drop in parallel. The system would accordingly be less saturated with VLDL particles, so that a greater proportion of particles would conceivably be converted into LDL down the delipidation cascade. Although we did not find a statistically significant decrease in the ratio of VLDL triacylglycerol to VLDL apo B after supplementation with fish oils, our study could have been underpowered to detect a true effect of fish oils in decreasing VLDL particle size. This potential effect may be physiologically meaningful in relation to increased conversion to IDL and LDL apo B. Consistent with this, other studies have shown that enrichment of n-3 fatty acids in VLDL particles favor the conversion of VLDL to LDL (30), although the studies have not specifically referred to insulin-resistant obese subjects. Moreover, fish oil itself might have down-regulated hepatic receptor activity and hindered the removal of apo B. The net effect of fish oil in our insulin-resistant subjects may therefore, in percentage terms, increase apo B conversion down the lipolytic pathway. In spite of increased apo B conversion, we did not find a significant increase in IDL or LDL apo B pool sizes, which suggests that fish oils did not result in a significant concomitant change in the individual FCR of these lipoproteins. Hence, we suggest that the reduction in plasma VLDL particle numbers (as opposed to VLDL size or composition) with n-3 fatty acids has a significant effect in modulating apo B trafficking without any alteration in overall apo B clearance. We previously reported that, in viscerally obese men, an improvement in insulin sensitivity after weight loss resulted in an increase in LDL apo B FCR (4). Because fish oil treatment did not alter insulin sensitivity in the present study, one could argue that insulin resistance was still down-regulating the LDL receptors and LDL activities. Given that the primary mechanism of action of n-3 fatty acids is on VLDL production, we would expect the FCRs of apo B to remain suppressed in the setting of persistent insulin resistance. The effect of fish oils on other lipoprotein-related variables, including hepatic lipase, apo C-II, and hepatic receptor activity, may also modulate apo B clearance and thus may merit further investigation.
Our results also show that VLDL particle size, as reflected by the ratio of VLDL triacylglycerols to VLDL apo B, did not change significantly with fish oil supplementation. On the assumption that the FCRs for VLDL apo B and VLDL triacylglycerols are the same when the plasma VLDL triacylglycerol-to-VLDL apo B ratio remains constant in a steady state condition, hepatic triacylglycerol synthesis can be estimated as described by Sigurdsson et al (31). Accordingly, our results showed that n-3 fatty acids significantly decreased hepatic triacylglycerol synthesis by 35% (from 174 ± 27 to 112 ± 22 mg·kg-1·d-1, P < 0.03). This suggests that the inhibition of triacylglycerol synthesis by n-3 fatty acids would be accompanied by a commensurate inhibition of apo B synthesis (32) and could have contributed to a reduction in VLDL apo B production. Further kinetic examinations of VLDL subspecies and hepatic triacylglycerol synthesis may help to clarify the contribution of triacylglycerol and apo B to VLDL production.
Previous kinetic studies examining chylomicron remnant metabolism showed that n-3 fatty acids enhance postprandial chylomicron and chylomicron remnant clearance (14, 33). In this present study, we did not show any significant change in plasma remnant-like particle cholesterol and the FCR of chylomicron remnant–like emulsion. The concentration of apo B-48 in plasma also did not change with fish oil supplementation (DC Chan, JCL Mamo, GF Watts, unpublished observations, 2002). These results suggest that n-3 fatty acids do not have a significant effect on chylomicron remnant metabolism in insulin-resistant obese subjects. Given that there was no change in IDL and LDL apo B pool sizes after supplementation with fish oil in our study, we suggest that chylomicron remnants were still competing with VLDL remnants for the same hepatic LDL receptors. This suggestion is consistent with our findings that n-3 fatty acids do not affect the FCRs of apo B or chylomicron remnant–like emulsion. However, our results might have been different had we used postprandial fat challenges (33, 34). The breath test provides a specific functional assessment of chylomicron remnant metabolism and has been validated in patients with familial dyslipidemias (15). However, it depends not only on the plasma clearance of remnant-like emulsions, but also on the subsequent oxidation of fatty acids hydrolyzed from the emulsion of cholesteryl ester. The n-3 fatty acids have been reported to stimulate mitochondrial ß-oxidation of fatty acid in the liver (29). Nevertheless, this effect would only enhance the FCR of remnant-like emulsion measured by the breath test and therefore would not confound the results reported in this study.
In conclusion, our data support the hypothesis that n-3 fatty acids lower plasma triacylglycerols and reduce VLDL apo B pool size in viscerally obese subjects with dyslipidemia. These improvements are chiefly due to a reduction in VLDL apo B secretion by the liver. Fish oil supplementation does not alter the catabolism of apo B in all lipoproteins or chylomicron remnants. In view of the differential effects of EPA and DHA on serum lipids (35, 36), it may be of interest to examine the effect of pure DHA and EPA, as well as that of certain genetic polymorphisms, on apo B and chylomicron remnant metabolism (37). Further investigations should also explore the incremental effect of weight reduction, statin treatment, or the addition of insulin sensitizers to fish oils on the kinetics of apo B in these subjects.4l/, 百拇医药
ACKNOWLEDGMENTS4l/, 百拇医药
We thank Frank van Bockxmeer for the apolipoprotein E 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.
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