This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sakai, T. Articles by Kashyap, M. L. Search for Related Content PubMed PubMed Citation Articles by Sakai, T. Articles by Kashyap, M. L. Related Collections Lipids Cardiovascular Pharmacology Lipid and lipoprotein metabolism

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1783.)
© 2001 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Niacin, but Not Gemfibrozil, Selectively Increases LP-AI, a Cardioprotective Subfraction of HDL, in Patients With Low HDL Cholesterol

Takaaki Sakai; Vaijinath S. Kamanna; Moti L. Kashyap

From the Cholesterol Research Center, Department of Veterans Affairs Healthcare System, Long Beach, Calif, and the Department of Medicine, University of California, Irvine.

Correspondence to Moti L. Kashyap, MD, Director, Cholesterol Research Center, Department of Veterans Affairs Healthcare System, Professor of Medicine, University of California, Irvine, 5901 E Seventh St (11/111-I), Long Beach, CA 90822. E-mail moti.kashyap{at}med.va.gov

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—— Evidence indicates that the high density lipoprotein (HDL) subfraction containing apolipoprotein A-I without apolipoprotein AII (LP-AI) is more antiatherogenic than HDL particles containing apolipoprotein A-I and apolipoprotein A-II (LP-AI+AII). This study examined the effect of extended-release niacin (niacin-ER) and gemfibrozil on LP-AI and LP-AI+AII particles in patients with low levels of HDL cholesterol (HDL-C). Mechanisms by which these agents modulate HDL particles were investigated by in vitro studies using human hepatoblastoma (Hep G2) cells. A total of 139 patients with low HDL-C (40 mg/dL) were randomized to niacin-ER or gemfibrozil in a multicenter double-blind trial. Patients were dose-escalated with once-nightly niacin-ER (1 to 2 g) or gemfibrozil (1.2 g) for 19 weeks. Niacin-ER had a greater effect in raising HDL-C and apolipoprotein A-I levels than did gemfibrozil. Niacin-ER at 1- and 2-g doses increased LP-AI levels by 8.7±4.0% (P=0.033) and 24.0±4.4% (P<0.001), respectively. Gemfibrozil had no consistent effect on LP-AI levels. LP-AI+AII levels increased 5% to 8% by both agents. In vitro studies showed that niacin, but not gemfibrozil, selectively decreased the uptake of ¹²⁵I-labeled LP-AI holoparticles by Hep G2 cells. The uptake of [³H]cholesterol ester was 75% greater from LP-AI versus LP-AI+AII particles, but neither niacin nor gemfibrozil affected cholesterol ester uptake. These data indicate that unlike gemfibrozil, niacin selectively increases LP-AI compared with LP-AI+AII particle concentration in patients with low HDL-C levels. The mechanism of action of increased LP-AI concentration appears to be mediated by decreased hepatic removal of LP-AI particles, which are more efficient in reverse cholesterol transport, thus suggesting an additional mechanism by which niacin mediates its antiatherogenic properties.

Key Words: lipoproteins • drugs • atherosclerosis • apolipoproteins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Niacin is a commonly used antidyslipidemic agent that effectively decreases total plasma levels of cholesterol, triglycerides (TGs), and LDL cholesterol (LDL-C) and increases HDL cholesterol (HDL-C).^1,2 Niacin also lowers plasma levels of Lp(a).³ Treatment of patients with niacin has been shown to decrease myocardial infarction and stroke and, in combination with bile acid-binding regimen, to prevent progression and cause regression of coronary atherosclerosis.^4,5 Although the use of niacin has been associated with adverse effects (eg, flushing and hepatic toxicity^6–9), recent studies have indicated that a once-nightly extended-release niacin (niacin-ER) formulation (Niaspan, Kos Pharmaceuticals) has shown reduced flushing with minimal to no hepatic toxicity with comparable effects on plasma lipid profile.^10–12 Gemfibrozil is another widely used lipid-regulating agent and has been shown to reduce plasma cholesterol, TGs, VLDL, and LDL and to elevate HDL; in a primary prevention study, it significantly reduced coronary events.¹³

See page 1707

The recently reported HDL Intervention Trial (HIT) showed for the first time that increasing HDL-C without altering LDL-C in patients treated with gemfibrozil results in significant reductions in the risk of major cardiovascular events.^14,15 This clinical trial offers stronger rationale and significance in treatment of the low HDL syndrome to prevent atherosclerotic cardiovascular disease. The cardioprotective effects of HDL have been largely attributed to the ability of apo A-I-containing HDL particles to initiate cholesterol efflux and thereby facilitate the removal of excess cholesterol from peripheral tissues and its delivery to the liver for elimination through the reverse cholesterol transport pathway.¹⁶ General consensus is that LP-AI particles (a subfraction of HDL particles containing apoA-I without apoA-II) are more potent in effluxing cellular cholesterol than are LP-AI+AII particles (HDL particles containing apoA-I and apoA-II¹⁷). Furthermore, studies have shown that LP-AI particles are more efficient donors of cholesterol esters (CEs) than are LP-AI+AII particles.¹⁸ Clinical studies have indicated that the increased levels of LP-AI particles are associated negatively with the degree of arteriographically defined coronary disease.^19,20 Premenopausal women have higher levels of LP-AI, suggesting that this may have a beneficial effect in reducing cardiovascular risk in these subjects.²¹ Oral estrogen replacement therapy in postmenopausal women was shown to increase LP-AI levels.²² Using human hepatoblastoma cells, we have shown that estradiol selectively stimulates the synthesis of LP-AI particles.²³

Although niacin is the most potent clinically used agent for elevating HDL-C and apoA-I levels in dyslipidemic patients (see review²⁴), the effect of niacin on plasma levels of LP-AI and LP-AI+AII is not established. In the present study, using a multicenter double-blind randomized design, we have examined the effect of niacin-ER versus gemfibrozil on plasma levels of LP-AI and LP-AI+AII particles in patients with low HDL-C. Previously, we have shown that niacin, by inhibiting the uptake/removal of HDL-apoA-I (without influencing apoA-I synthesis), increased the accumulation of apoA-I in the culture media.²⁵ In the present study, we hypothesized that the increase in LP-AI particles in niacin-ER-treated patients may be due to decreased hepatic removal/uptake of LP-AI particles. To address this issue and to understand the mechanism by which niacin or gemfibrozil modulates these HDL subfractions, in vitro studies examining the uptake of these particles by human hepatoblastoma (Hep G2) cells were carried out. The data in the present study indicate that niacin, but not gemfibrozil, selectively increases LP-AI particles in these patients. The in vitro experiments show that niacin selectively decreases the uptake of LP-AI by cultured Hep G2 cells, whereas gemfibrozil does not, indicating the cellular site of action of niacin on these particles. Uptake of CEs was greater from LP-AI than LP-AI+AII particles, but neither agent affected CE uptake, thus defining the mechanism by which niacin increases LP-AI and reverse cholesterol transport.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Study Design and Patient Selection
A total of 139 patients were randomized to receive either niacin-ER or gemfibrozil. These patients were a subset of the original study from 11 participating institutions detailed previously.²⁶ The protocols were approved by the institutional review boards of the participating institutions, and written informed consent was obtained from each patient. All lipid-lowering medications were withdrawn, and patients were counseled on a National Cholesterol Education Program Step 1 diet for a 4-week lead-in period. Baseline mean lipid profile was obtained from consecutive blood samples taken 7 to 10 days apart from the dietary lead-in period. The eligibility criteria for patients to be included in the study were as follows: HDL-C 40 mg/dL, TGs 400 mg/dL, and LDL-C 160 mg/dL or <130 mg/dL with documented coronary disease. Patients with gallbladder or peptic ulcer disease, active gout, clinically significant hyperuricemia, renal or hepatic disease, cardiac arrhythmias, or other serious cardiac abnormalities were excluded. Diabetic patients were excluded, but non-insulin-requiring diabetic patients with hemoglobin A_Ic within the normal range and fasting glucose 120 mg/dL were included in the study.

In the double-blind, placebo-controlled treatment phase, patients were randomly assigned to receive either niacin-ER (n=72) or gemfibrozil (n=67). Niacin-ER was administered as a once-a-night dose at bedtime after a low-fat snack. During an initial 3-week titration period, niacin-ER was given initially at 375 mg and was increased at weekly intervals to 500 mg and then to 750 mg. The dose of niacin-ER was subsequently escalated to 1000 mg for the next 4 weeks, 1500 mg for 4 weeks, and then 2000 mg for 8 weeks. Gemfibrozil was given a dose of 600 mg twice daily, 30 minutes before the morning and evening meals for the entire 19-week period.

Measurement of Plasma Lipid Profile, ApoA-I, LP-AI, and LP-AI+AII Particles
Blood samples were collected after a 12-hour fast. Plasma cholesterol, TGs, HDL-C, and LDL-C concentrations were analyzed at the Lipid Research Laboratory, Washington University, St. Louis, Mo, which is certified by the Lipid Standardization Program of the Centers for Disease Control. Plasma concentrations of apoA-I, apoB, and Lp(a) were measured by previously described procedures²⁷ at the Northwest Lipid Research Laboratory.

Plasma concentrations of LP-AI particles were measured by the Hydragel LPAI kit (No. 4055, Sebia, Inc) according to the manufacturer‘s instructions. In brief, LP-AI particles were quantified in serum by the electroimmunodiffusion method on agarose gels containing monospecific anti-apoA-I and excess anti-apoA-II antibodies.^21,22,28 After electrophoretic migration, the resulting rockets were stained with acid violet solution, and excess stain was removed with an acid-alcohol mixture. The Sebia gel kit uses excess anti-apoA-II incorporated into the agarose gels to block the migration of LP-AI+AII particles. The LP-AI particles continue to migrate and react with anti-apoA-I antibodies, and the height of the resulting immunoprecipitation rocket is proportional to the LP-AI concentration. Serum LP-AI concentrations were calculated by measuring the rocket heights and were compared with the calibration curve generated with the LP-AI standard serum pool. Plasma concentrations of LP-AI+AII particles were derived by subtracting LP-AI values from the total serum apoA-I concentration (as measured by radioimmunoassay²⁷). The within-day and between-day coefficient of variation for LP-AI assay was 4.2% and 5.4%, respectively.

In Vitro Studies on the Uptake of LP-AI and LP-AI+AII Particles by Hep G2 Cells
In these studies, the isolation of LP-AI and LP-AI+AII particles from normal serum, radiolabeling, and their uptake by Hep G2 cells were performed by the following procedures.

LP-AI and LP-AI+AII particles from normal human serum were isolated by immunoaffinity column chromatography with the use of anti-apoA-I and anti-apoA-II columns according to modified previously described methods.^29,30 In brief, affinity columns specific for apoA-I or apoA-II were prepared by coupling polyclonal antibodies for human apoA-I or apoA-II to CNBr-activated Sepharose 4B (Pharmacia) according to the procedures described in the instruction manual. Serum samples were loaded onto the apoA-II affinity column and incubated at 4°C for 16 to 18 hours to allow binding of apolipoprotein particles to the specific antibody. The unretained fraction was collected. The retained fraction on the anti-apoA-II column was eluted with 3 mol/L NaSCN, pH 6.0, to obtain apoA-II-containing particles (ie, LP-AI+AII). The unretained fraction from the anti-apoA-II column was passed through the anti-apoA-I column, and the retained particles were eluted with 3 mol/L NaSCN, pH 6.0, to obtain LP-AI particles. After elution, the contact of HDL subfractions with NaSCN was minimized by immediate passage through the Sephadex G-25 columns and dialysis against PBS.¹⁸ During initial standardization, we have established that the unretained washing fractions from anti-apoA-I and anti-apoA-II affinity columns were devoid of apo-A-I and apoA-II (as measured by ELISA), respectively, suggesting that the amount of plasma used for immunoaffinity separation did not exceed the column capacity. We have previously validated the specificity of these immunoaffinity procedures for the isolation of LP-AI and LP-AI+AII particles.²⁹ We have established that the LP-AI subfraction did not contain apoA-II.

Radioiodination of LP-AI and LP-AI+AII particles was carried out by incubating freshly isolated particles with carrier-free ¹²⁵I, as described earlier by McFarlane.³¹ After radioiodination, unreacted ¹²⁵I was removed by gel filtration, followed by exhaustive dialysis against PBS, pH 7.4.^25,29 The specific radioactivities in LP-AI and LP-AI+AII were 109 and 105 cpm/ng protein, respectively. We have previously established that radioiodination of HDL-apoA-I with this procedure yielded radioiodinated HDL-apoA-I containing 93% to 97% of radioactivity in trichloroacetic acid-precipitable material, suggesting that the majority of the radioactivity is associated with the protein component of HDL. The iodination methodology and observations are in line with previous reports.^32–34 For CE-labeled LP-AI and LP-AI+AII particle isolation, initially normal human serum was incubated with [1,2(n)-³H]cholesterol for 18 hours at 37°C to allow CE formation via the lecithin:cholesterol acyltransferase enzyme reaction.³⁵ The [³H]CE-labeled LP-AI and LP-AI+AII particles were isolated by immunoaffinity column chromatography as described above. We have used identical radiolabeling methodology for LP-AI and LP-A+AII particles (in terms of concentration of protein, incubation time, and tracer amount), and the specific radioactivities in these 2 particles were similar (specific radioactivities in ³H-LP-AI and ³H-LP-AI+AII were 67 and 64 cpm/ng protein, respectively).

Hep G2 cells were obtained from American Type Culture Collection. Cells were grown in high-glucose DMEM containing 10% FBS, 1% glutamine-penicillin-streptomycin, and 1% amphotericin B in a humidified incubator at 37°C. The uptake of radiolabeled LP-AI and LP-AI+AII by Hep G2 cells was measured as described earlier.²⁵ Hep G2 cells were preincubated with either niacin (0 to 3 mmol/L) or gemfibrozil (0 to 400 µmol/L) for 48 hours at 37°C. The doses of niacin and gemfibrozil used in this investigation are based on our previous studies that established the optimal concentrations and duration of incubation with these agents necessary to affect HDL/apoA-I uptake or the production in Hep G2 cells.^25,29 After the appropriate incubation, the medium was replaced with fresh DMEM containing fetal bovine albumin (FBA, 5 mg/mL), and ¹²⁵I-LP-AI and ¹²⁵I-LP-AI+AII were added. After 16 hours of incubation at 37°C, cell monolayers were washed thoroughly (4 or 5 times with PBS) and digested with 1N sodium hydroxide solution. An aliquot was used for radioactivity measurement. The uptake of [³H]CE-labeled LP-AI and AI+A-II was performed by incubating Hep G2 cells with niacin (0 to 3.0 mmol/L) or gemfibrozil (0 to 400 mmol/L) for 48 hours at 37°C. The medium was replaced with DMEM containing FBA (5 mg/mL), and [³H]CE-labeled LP-AI or AI+A-II was added. Cells were harvested 6 hours later, washed thoroughly, and digested with 1 mL of 1N NaOH. Radioactivity was measured and expressed as counts per minute per milligram cellular protein.

Statistical Analysis
Lipid profile data presented are mean±SEM. Multiple comparison analysis was used to determine statistical significance at each postbaseline visit. ANOVA was used to compare baseline levels and changes from baseline between the niacin-ER and gemfibrozil groups. For studies examining the uptake of LP-AI and LP-AI+AII particles by Hep G2 cells, the values are mean±SD of 3 experiments performed in triplicate. Statistical significance was calculated by using the Student t test, and a value of P<0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
The data presented in this report on LP-AI and LP-AI+AII levels in patients are a substudy of a multicenter clinical trial assessing the efficacy of niacin-ER versus gemfibrozil for treatment of low levels of HDL-C. The detailed study design and clinical data, including lipid profile, clinical chemistry, and hematologic parameters, and adverse effects or events during treatment are reported elsewhere.²⁶ Baseline characteristics of patients in the study groups indicated that the patients were predominantly white men (93%) with a mean age and body mass index of 53 years and 29 kg/m², respectively.²⁶ The in vitro studies were specifically conducted to assess the mechanism of action of niacin and gemfibrozil on LP-AI±AII particle concentrations.

Effect of Niacin-ER and Gemfibrozil on Lipid Profile
Lipid and apolipoprotein profile analyses of patients in this substudy showed that niacin-ER significantly decreased total cholesterol (TC, at 19 weeks of treatment), TGs, TC/HDL-C ratio, and apoB compared with baseline values (at 7, 11, and 19 weeks of treatment; Table 1). Niacin-ER significantly increased HDL-C and apoA-I during all treatment periods (Table 1). Patients treated with gemfibrozil showed a significant decrease in TC (at 7 weeks of treatment), in TGs and the TC/HDL-C ratio (at all treatment periods), and in apoB levels (at 7 and 11 weeks, Table 1). Gemfibrozil increased plasma concentrations of HDL-C at all treatment periods and apoA-I at 11 weeks of treatment (Table 1). Comparative analysis between values after niacin-ER versus gemfibrozil treatment compared with the respective baseline values (at the 19-week treatment period) had the following effects on lipid profile: reduction in TGs by 30% versus 45% and TC/HDL-C by 22% versus 13% and increase in HDL-C by 25% versus 13% and apoA-I by 10% versus 2.5%, respectively (P<0.01 for all parameters).

View this table: [in this window] [in a new window]   Table 1. Plasma Lipid and Apolipoprotein Values at Baseline and During Treatment With Niacin-ER and Gemfibrozil

Effect of Niacin-ER and Gemfibrozil on LP-AI and LP-AI+AII Levels
Measurement of HDL particles without apoA-II (LP-AI) and with apoA-II (LP-AI+AII) showed that niacin-ER significantly and dose-dependently increased LP-AI particles by 8.7% to 24.0%, respectively, during all the treatment periods (between 7 to 19 weeks) compared with the baseline values (Table 2). Niacin-ER also significantly increased LP-AI+AII levels by 5.2% to 9.5% compared with the baseline values; however, the effect was less when LP-AI+AII levels were compared with LP-AI levels (Table 2). Treatment of patients with gemfibrozil did not consistently increase LP-AI levels compared with the baseline values (Table 2). However, gemfibrozil significantly increased LP-AI+AII concentrations by 4.2% to 8.0% compared with their baseline values (Table 2).

View this table: [in this window] [in a new window]   Table 2. Plasma Lp-AI and Lp-AI+All Values at Baseline and During Treatment With Niacin-ER and Gemfibrozil

Effect of Niacin and Gemfibrozil on the Uptake of LP-AI and LP-AI+AII Particles by Hep G2 Cells
The next series of in vitro experiments were performed on the uptake of radiolabeled-LP-AI and LP-AI+AII particles by Hep G2 cells to understand the mechanisms by which niacin and gemfibrozil affect LP-AI and LP-AI+AII levels. In these in vitro studies, we used optimal concentrations of niacin or gemfibrozil and incubation period based on initial studies and our previous reports.^25,29 For example, niacin during 6 to 16 hours had a minimal effect on HDL particle uptake by Hep G2 cells. At 24 hours of incubation, there were some marginal effects, and the effect was maximal at 48 hours of incubation with niacin. Similar experiments were performed for incubation periods with gemfibrozil. The incubation of Hep G2 cells with niacin (1 to 3 mmol/L) for 48 hours significantly inhibited the uptake of ¹²⁵I-LP-AI particles by 15% to 17% (Figure, panel A), whereas the preincubation of Hep G2 cells with gemfibrozil (50 to 400 µmol/L) for 48 hours had no significant effect on the uptake of ¹²⁵I-LP-AI particles (Figure, panel A). Neither niacin nor gemfibrozil affected the uptake of ¹²⁵I-LP-AI+AII particles by Hep G2 cells (Figure, panel B). Further experiments were performed to examine the effect of niacin and gemfibrozil on the uptake of [³H]CE from LP-AI and LP-AI+AII particles by Hep G2 cells. Incubation of Hep G2 cells with either niacin (0.5 to 3 mmol/L) or gemfibrozil (50 to 400 µmol/L) for 48 hours did not significantly affect the uptake of [³H]CE from LP-AI and LP-AI+AII particles by Hep G2 cells (Table 3). However, the CE uptake from LP-AI particles was 75% greater than that from LP-AI+AII particles, indicating that LP-AI particles were more efficient donors of CE per unit of protein.

View larger version (17K): [in this window] [in a new window]   Effect of niacin and gemfibrozil on the uptake of 125I-LP-AI (A) and 125I-LP-AI+AII (B) particles by Hep G2 cells. Cells were preincubated with varying concentrations of niacin (0 to 3 mmol/L) or gemfibrozil (0 to 400 µmol/L) for 48 hours. Fresh DMEM containing 5 mg/mL FBA, niacin, or gemfibrozil and 125I-labeled particles was added. Cells were harvested 16 hours later, washed thoroughly with PBS, and digested with 1N NaOH. Radioactivity was measured and expressed in terms of total cellular protein. Data are mean±SD of 3 experiments performed in triplicate. The degree of variation between experiments was in the range of 1.5% to 4.0%.

View this table: [in this window] [in a new window]   Table 3. Effect of Niacin and Gemfibrozil on the Uptake of [3H]CE-Labeled Lp-AI and Lp-AI+All Particles by Hep G2 Cells

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although the measurement of HDL-C and apoA-I are important lipid profile parameters for cardiovascular risk assessment, recent studies have emphasized that the levels of the LP-AI HDL subfraction may offer greater utility, as indicated in the introduction to the present study. The mechanisms of action of niacin and gemfibrozil to raise HDL are incompletely understood. Using human Hep G2 cells, we have shown that niacin, through inhibiting the uptake/removal of HDL-apoA-I, increases the accumulation of apoA-I in the culture media without affecting apoA-I synthesis.²⁵ The mechanism by which gemfibrozil increases apoA-I was shown at the level of stabilization of apoA-I mRNA transcription but not at the level of uptake/removal of HDL-apoA-I, leading to the increased production of apoA-I by hepatocytes.²⁹ This is consistent with earlier turnover studies in dyslipidemic patients in which gemfibrozil increased the transport (synthetic) rate of apoA-I and apoA-II.³⁶

The major focus of the present study was to assess the effect of niacin-ER and gemfibrozil on LP-AI and LP-AI+AII HDL subfractions in patients with low HDL-C. The data indicated that niacin-ER (2 g daily) raised HDL-C by 25%, an effect twice as much as the HDL-C increase afforded by gemfibrozil (1200 mg daily). The HDL-C-raising effect of niacin-ER is consistent with a previous report showing a comparable HDL-C increase provided by niacin-ER and immediate-release niacin.¹² These observations clearly indicated that the specific extended-release characteristic of niacin-ER formulation and its once-nightly schedule of administration can retain an HDL-C-raising effect that is comparable to that of immediate-release niacin, with significantly reduced episodes of adverse events, such as flushing.¹² The data on the effect of gemfibrozil to raise HDL-C presented in the present study is in accord with previous studies.¹³

The data on HDL subfraction measurements showed that niacin-ER (2 g daily) during the 19-week treatment period significantly increased LP-AI levels by 24.0% above baseline. Treatment of patients with niacin-ER also significantly increased LP-AI+AII particles by 9.5% above baseline; however, the effect was less when LP-AI+AII levels were compared with LP-AI levels. Contrary to niacin-ER, gemfibrozil had no consistent effect on LP-AI levels. However, gemfibrozil increased LP-AI+AII levels by 4.2%, to 8.0% of their baseline values, an effect comparable to that of niacin-ER. These data suggest that niacin-ER, but not gemfibrozil, significantly increased LP-AI particles, a cardioprotective subfraction of HDL in patients with a low HDL state.

To define the potential mechanisms of action of niacin and gemfibrozil to modulate HDL subfractions, we performed additional experiments with the use of Hep G2 cells as an in vitro model system. Previously, we have shown that niacin had no effect on apoA-I synthesis but selectively inhibited the uptake/removal of HDL-apoA-I, resulting in increased accumulation of apoA-I in Hep G2 cell culture media.²⁵ As an extension of our previous study,²⁵ in the present study, we proposed that the increase in LP-AI particles in niacin-ER-treated patients may be due to decreased hepatic removal/uptake of LP-AI particles. In support of this possibility, our data indicated that niacin significantly inhibited the uptake of radiolabeled LP-AI particles by Hep G2 cells. However, niacin had no significant effect on the uptake of LP-AI+AII particles by Hep G2 cells. These data suggest that niacin, by selectively inhibiting the hepatic removal/uptake of LP-AI particles, may lead to the increased retention of LP-AI particles in the circulation. Our observation of greater CE uptake from LP-AI particles compared with LP-AI+AII particles is in line with an earlier report¹⁸ and confirms that LP-AI is a better donor for CEs. Thus, by selectively decreasing hepatic LP-AI uptake/removal, niacin may have a greater effect not only on LP-AI mass but also in reverse cholesterol transport function. The inability of niacin to affect LP-AI+AII particle uptake by Hep G2 cells does not fully explain the moderate increased plasma levels of LP-AI+AII particles observed in niacin-ER-treated patients and may reflect limitations of in vitro studies to in vivo observations. Because LP-AI±AII particles, used in uptake studies in Hep G2 cells, were isolated from normal human plasma (but not from individual patients treated with niacin-ER or gemfibrozil), the in vitro data reported do not provide information on the potential in vivo modification of LP-AI±AII particles with respect to their uptake properties by hepatocytes in patients treated with niacin-ER or gemfibrozil. Because of inadequate availability of plasma samples from patients treated with niacin-ER and gemfibrozil to isolate large amounts of LP-AI and LP-AI+AII particles required for uptake studies, we were unable to measure uptake of these HDL subfractions isolated from patients treated with either drug. This was beyond the scope of the present study protocol. Also, the in vivo catabolism of HDL is complex, and extrahepatic tissues (especially kidney) are significantly involved. However, their role in the catabolism of HDL subfractions is unknown and may also account for lack of a more complete correlation between in vitro and clinical measurements. Although these in vitro studies reported in the present study provide a novel approach for the mechanism of action of niacin to raise LP-AI particles, caution should be taken in direct extrapolation of these data to the in vivo observations in humans. Additional direct plasma kinetic studies with LP-AI and LP-AI+AII particles in humans would be very much needed to address the synthetic or catabolic aspects of these particles in control and niacin-treated patients.

Although various regulatory processes involved in the catabolism of HDL-apoA-I or HDL-C are not clearly established, recent studies have indicated that the components of HDL (apoA-I and cholesterol) are taken up or transported by mainly 2 cellular proteins, including (1) cubilin and (2) scavenger receptor type B class I (SR-BI). Using yolk-sac endoderm-like cells, Hammad et al³⁷ and Kozyraki et al³⁸ identified cubilin (the recently described endocytic receptor for intrinsic factor-vitamin B₁₂) as the receptor that mediates HDL holoparticle endocytosis. However, cubilin has not been reported to be present on liver cells. The SR-BI receptor was shown to bind HDL and to mediate the selective uptake or removal of HDL-CEs without the uptake/degradation of HDL-apoA-I.³⁹ It has been proposed that the SR-BI receptor acts as a "docking" site so that CEs can be removed after which the HDL particle (depleted of CEs) "undocks" and recirculates to pick up more CEs from peripheral tissues. However, there is no evidence that cubilin or SR-B1 is involved in plasma apoA-I metabolism. Hepatic lipase has been shown to play a major role in HDL catabolism by hydrolyzing TGs and phospholipids within HDL particles (see reviews^40,41). Recently, Dugi et al⁴² examined the in vivo role of hepatic lipase in HDL metabolism by injecting recombinant adenovirus expressing hepatic lipase in hepatic lipase-deficient mice. These authors showed that the mice expressing hepatic lipase exhibited significant reductions in cholesterol, HDL-C, apoA-I, and apoA-II and showed enhanced plasma clearance of HDL-apoA-I, HDL-apoA-II, and HDL-CEs. It is conceivable that modulation of these HDL catabolic pathways may play an important role in regulating plasma levels of HDL particles.

Because hepatic lipase is associated with enhanced plasma clearance of HDL-apoA-I/A-II and reduction in HDL,⁴² it may be possible that the ability of niacin to decrease hepatic lipase⁴³ may, at least in part, play a role in reduced hepatic removal/uptake of LP-AI particles. Because niacin had no effect on HDL-CE uptake²⁵ and on LP-AI-CE uptake, we suggest that niacin may not modulate SR-B1-mediated uptake/removal of HDL-CEs. Unlike niacin, gemfibrozil did not inhibit LP-AI uptake by Hep G2 cells. Extrapolation of these in vitro data may, at least in part, explain the inability of gemfibrozil to affect LP-AI particle concentration in patients.

In summary, we have shown that niacin but not gemfibrozil selectively increased LP-AI particles in patients with low HDL. In vitro studies have indicated that niacin but not gemfibrozil significantly decreases the uptake of LP-AI particles by Hep G2 cells. These data suggest that unlike gemfibrozil, niacin by selective inhibition of hepatic LP-AI removal significantly elevates the HDL subfraction LP-AI concentration in patients with the isolated low HDL state. Elevated levels of LP-AI, in turn, may facilitate more efficient reverse CE transport via cholesterol efflux and its efficient removal via the SR-B1 receptor pathway. These data suggest an additional mechanism by which niacin exerts its beneficial effect on atherosclerotic cardiovascular disease.

	Acknowledgments

 
The present study was supported, in part, by Kos Pharmaceuticals and the Long Beach Research Foundation. We thank Phillip D. Simmons for statistical analyses and Shobha H. Ganji for assistance in manuscript preparation.

	Footnotes

 
Presented in part at the Annual Meeting of the American College of Cardiology, 2000, in abstract form (J Am Coll Cardiol. 2000;35:300A).

Received June 8, 2001; accepted July 20, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Altschul R, Hoffer A, Stephen JD. Influence of nicotinic acid on serum cholesterol in man. Arch Biochem Biophys. 1955; 54: 558–559.
2. Grundy SM, Mok HY, Zech L, Berman M. Influence of nicotinic acid on metabolism of cholesterol and triglycerides in man. J Lipid Res. 1981; 22: 24–36.[Abstract]
3. Carlson LA, Hamsten A, Asplund A. Pronounced lowering of serum levels of lipoprotein Lp(a) in hyperlipidemic subjects treated with nicotinic acid. J Intern Med. 1989; 226: 271–276.[Medline] [Order article via Infotrieve]
4. Canner PL, Berge KG, Wenger NK, Stamler J, Friedman L, Prineas RJ, Friedewald W. Fifteen year mortality in coronary drug project patients: long-term benefit with niacin. J Am Coll Cardiol. 1986; 8: 1245–1255.[Abstract]
5. Blankenhorn DH, Nessim SA, Johnson RL, Sanmarco ME, Azen SP, Cashin-Hemphill L. Beneficial effects of combined colestipol-niacin therapy on coronary atherosclerosis and coronary venous bypass grafts. JAMA. 1987; 257: 3233–3240.[Abstract]
6. Knopp RH, Ginsberg J, Albers JJ, Hoff C, Ogilvie JT, Warnick GR, Burrows E, Retzlaff B, Poole M. Contrasting effects of unmodified and time-release forms of niacin on lipoproteins in hyperlipidemic subjects. Metabolism. 1985; 34: 642–650.[Medline] [Order article via Infotrieve]
7. Stern RH, Spence JD, Freeman DJ, Parbtani A. Tolerance of nicotinic acid flushing. Clin Pharmacol Ther. 1991; 50: 66–70.[Medline] [Order article via Infotrieve]
8. McKenney JM, Proctor JD, Harris S, Chinchili VM. A comparison of the efficacy and toxic effects of sustained-vs immediate-release niacin in hypercholesterolemic patients. JAMA. 1994; 271: 672–677.[Abstract]
9. Gray DR, Morgan T, Chretien SD. Efficacy and safety of controlled-release niacin in dyslipoproteinemic veterans. Ann Intern Med. 1994; 121: 252–258.[Abstract/Free Full Text]
10. Morgan JM, Capuzzi DM, Guyton JR, Centor RM, Goldberg R, Robbins DC, DiPette D, Jenkins S, Marcovina S. Treatment effect of Niaspan, a controlled-release niacin, in patients with hypercholesterolemia: a placebo-controlled trial. J Cardiovasc Pharmacol Ther. 1996; 1: 195–202.[Medline] [Order article via Infotrieve]
11. Guyton JR, Goldberg AC, Kreisberg RA, Sprecher DL, Superko HR, O‘Connor CM. Effectiveness of once nightly dosing of extended-release niacin alone and in combination for hypercholesterolemia. Am J Cardiol. 1998; 82: 737–743.[Medline] [Order article via Infotrieve]
12. Knopp RH, Alagona P, Davidson M, Goldberg AC, Kafonek SD, Kashyap M, Sprecher D, Superko HR, Jenkins S, Marcovina S. Equivalent efficacy of a time-release form of niacin (Niaspan) given once-a-night versus plain niacin in the management of hyperlipidemia. Metabolism. 1998; 47: 1097–1104.[Medline] [Order article via Infotrieve]
13. Frick MH, Elo O, Haapa K, Heinonen OP, Heinsalmi P, Helo P, Huttunen JK, Kaitaniemi P, Koskinen P, Manninen V, et al. Helsinki Heart Study: primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia; safety of treatment, changes in risk factors, and incidence of coronary heart disease. N Engl J Med. 1987; 317: 1237–1245.[Abstract]
14. Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, Faas FH, Linares E, Schaefer EJ, Schectman G, et al. Gemfibrozil for the secondary prevention of coronary heart disease in men with low levels of high-density lipoprotein cholesterol: Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study Group. N Engl J Med. 1999; 341: 410–418.[Abstract/Free Full Text]
15. Robins SJ, Collins D, Wittes JT, Papademetriou V, Deedwania PC, Schaefer EJ, McNamara JR, Kashyap ML, Hershman JM, Wexler LF, et al. Relation of gemfibrozil treatment and lipid levels with major coronary events: VA-HIT: a randomized controlled trial. JAMA. 2001; 285: 1585–1591.[Abstract/Free Full Text]
16. Kashyap ML. Basic considerations in the reversal of atherosclerosis: significance of high density lipoproteins in stimulating reverse cholesterol transport. Am J Cardiol. 1989; 63: 56H–59H.[Medline] [Order article via Infotrieve]
17. Barbaras R, Puchois P, Fruchart J-C, Ailhaud G. Cholesterol efflux from cultured adipose cells is mediated by LP A-I particles but not by LP A-I:A-II particles. Biochem Biophys Res Commun. 1987; 142: 63–69.[Medline] [Order article via Infotrieve]
18. Rinninger F, Kaiser T, Windler E, Greten H, Fruchart J-C, Castro G. Selective uptake of cholesteryl esters from high-density lipoprotein-derived LPA-I and LPA-I:A-II particles by hepatic cells in culture. Biochim Biophys Acta. 1998; 1393: 277–291.[Medline] [Order article via Infotrieve]
19. Puchois P, Kandoussi A, Fievet P, Fourrier JL, Bertrand M, Koren E, Fruchart JC. Apolipoprotein A-I-containing lipoproteins in coronary artery disease. Atherosclerosis. 1987; 68: 35–40.[Medline] [Order article via Infotrieve]
20. Amouyel P, Isorez D, Bard JM, Goldman M, Lebel P, Zylberberg G, Fruchart JC. Parental history of early myocardial infraction is associated with decreased levels of lipoparticle A-I in adolescents. Arterioscler Thromb. 1993; 13: 1640–1644.[Abstract/Free Full Text]
21. Parra HJ, Mezdour H, Ghalim N, Bard JM, Fruchart JC. Differential electroimmunoassay of human LP-AI lipoprotein particles on ready-to-use plates. Clin Chem. 1990; 36: 1431–1435.[Abstract/Free Full Text]
22. Brinton EA. Oral estrogen replacement therapy in postmenopausal women selectively raises levels and production rates of lipoprotein A-I and lowers hepatic lipase activity without lowering the fractional catabolic rate. Arterioscler Thromb Vasc Biol. 1996; 16: 431–440.[Abstract/Free Full Text]
23. Jin FY, Kamanna VS, Kashyap ML. Estradiol stimulates apolipoprotein A-I- but not A-II-containing particle synthesis and secretion by stimulating mRNA transcription rate in Hep G2 cells. Arterioscler Thromb Vasc Biol. 1998; 18: 999–1006.[Abstract/Free Full Text]
24. Kashyap ML. Mechanistic studies of high-density lipoproteins. Am J Cardiol. 1998; 82: 42U–48U.[Medline] [Order article via Infotrieve]
25. Jin F-Y, Kamanna VS, Kashyap ML. Niacin decreases removal of high-density lipoprotein apolipoprotein A-I but not cholesterol ester by Hep G2 cells. Arterioscler Thromb Vasc Biol. 1997; 17: 2020–2028.[Abstract/Free Full Text]
26. Guyton JR, Blazing MA, Hagar J, Kashyap ML, Knopp RH, McKenney JM, Nash DT, Nash SD. Extended-release niacin versus gemfibrozil for treatment of low levels of high density lipoprotein cholesterol. Arch Intern Med. 2000; 160: 1177–1184.[Abstract/Free Full Text]
27. Marcovina SM, Albers JJ, Henderson LO, Hannon WH. International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B, III: comparability of apolipoprotein A-I values by use of international reference material. Clin Chem. 1993; 39: 773–781.[Abstract/Free Full Text]
28. Steinmetz J, Choukaife A, Visvikis S, Henny J, Siest G. Biological factors affecting concentrations of serum LP AI lipoprotein particles in serum and determination of reference limits. Clin Chem. 1990; 36: 677–680.[Abstract/Free Full Text]
29. Jin FY, Kamanna VS, Chuang MY, Morgan K, Kashyap ML. Gemfibrozil stimulates apolipoprotein A-I synthesis and secretion by stabilization of mRNA transcripts in human hepatoblastoma cell line (Hep G2). Arterioscler Thromb Vasc Biol. 1996; 16: 1052–1062.[Abstract/Free Full Text]
30. Malmendier CL, Lontie JF, Lagrost L, Delcroix C, Dubois DY, Gambert P. In vivo metabolism of apolipoprotein A-IV and A-I associated with high density lipoprotein in normolipidemic subjects. J Lipid Res. 1991; 32: 801–808.[Abstract]
31. McFarlane AS. Efficient trace-labeling of proteins with iodine. Nature. 1958; 182: 53.[Medline] [Order article via Infotrieve]
32. Barnard GF, Erickson SK, Cooper AD. Regulation of lipoprotein receptors on rat hepatoma in vivo. Biochim Biophys Acta. 1986; 879: 301–312.[Medline] [Order article via Infotrieve]
33. Chao Y, Windler EE, Chen GC, Havel RJ. Hepatic catabolism of rat and human lipoproteins in rats treated with 17--ethinyl estradiol. J Biol Chem. 1979; 254: 11360–11366.[Free Full Text]
34. Roheim PS, Rachmilewitz D, Stein O, Stein Y. Metabolism of iodinated high density lipoproteins in the rat. Biochim Biophys Acta. 1971; 248: 315–329.[Medline] [Order article via Infotrieve]
35. Fielding CJ, Fielding PE. Evidence for a lipoprotein carrier in human plasma catalyzing sterol efflux from cultured fibroblasts and its relationship to lecithin: cholesterol acyltransferase. Proc Natl Acad Sci U S A. 1981; 78: 3911–3914.[Abstract/Free Full Text]
36. Saku K, Gortside PS, Hynd BA, Kashyap ML. Mechanism of action of gemfibrozil on lipoprotein metabolism. J Clin Invest. 1985; 75: 1702–1712.[Medline] [Order article via Infotrieve]
37. Hammad SM, Steingrimur S, Twal WO, Drake CJ, Fleming P, Remaley A, Brewer B, Argraves WS. Cubilin, the endocytic receptor for intrinsic factor-vitamin B12 complex, mediates high-density lipoprotein holoparticle endocytosis. Proc Natl Acad Sci U S A. 1999; 96: 10158–10163.[Abstract/Free Full Text]
38. Kozyraki R, Fyfe J, Kristiansen M, Gerdes C, Jacobsen C, Cui S, Christensen EI, Aminoff M, de la Chapelle A, Krahe R, et al. The intrinsic factor-vitamin B12, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein. Nat Med. 1999; 5: 656–661.[Medline] [Order article via Infotrieve]
39. Acton S, Riggoti A, Landschutz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996; 271: 518–520.[Abstract]
40. Rader DJ, Ikewaki K. Unravelling high density lipoprotein-apolipoprotein metabolism in human mutants and animal models. Curr Opin Lipidol. 1996; 7: 117–123.[Medline] [Order article via Infotrieve]
41. Santamarina-Fojo S, Haudenschild C, Amar M. The role of hepatic lipase in lipoprotein metabolism and atherosclerosis. Curr Opin Lipidol. 1998; 9: 211–219.[Medline] [Order article via Infotrieve]
42. Dugi KA, Amar MJ, Haudenschild CC, Shamburek RD, Bensadoun A, Hoyt RFJr, Fruchart NJ, Madj Z, Brewer HBJr, Santamarina-Fojo S. In vivo evidence for both lipolytic and nonlipolytic function of hepatic lipase in the metabolism of HDL. Arterioscler Thromb Vasc Biol. 2000; 20: 793–800.[Abstract/Free Full Text]
43. Zambon A, Hokanson J, Brown BG, Brunzell JD. Evidence for a new pathophysiological mechanism for coronary artery disease regression, hepatic lipase-mediated changes in LDL density. Circulation. 1999; 99: 1959–1964.[Abstract/Free Full Text]

This article has been cited by other articles:

				L.-H. Zhang, V. S. Kamanna, M. C. Zhang, and M. L. Kashyap Niacin inhibits surface expression of ATP synthase {beta} chain in HepG2 cells: implications for raising HDL J. Lipid Res., June 1, 2008; 49(6): 1195 - 1201. [Abstract] [Full Text] [PDF]

				J. G. Richman, M. Kanemitsu-Parks, I. Gaidarov, J. S. Cameron, P. Griffin, H. Zheng, N. C. Guerra, L. Cham, D. Maciejewski-Lenoir, D. P. Behan, et al. Nicotinic Acid Receptor Agonists Differentially Activate Downstream Effectors J. Biol. Chem., June 22, 2007; 282(25): 18028 - 18036. [Abstract] [Full Text] [PDF]

				P. Barter Options for therapeutic intervention: how effective are the different agents? Eur. Heart J. Suppl., October 1, 2006; 8(suppl_F): F47 - F53. [Abstract] [Full Text] [PDF]

				A. Kontush and M. J. Chapman Functionally Defective High-Density Lipoprotein: A New Therapeutic Target at the Crossroads of Dyslipidemia, Inflammation, and Atherosclerosis Pharmacol. Rev., September 1, 2006; 58(3): 342 - 374. [Abstract] [Full Text] [PDF]

				A. D. Mooradian, M. J. Haas, and N. C. W. Wong The Effect of Select Nutrients on Serum High-Density Lipoprotein Cholesterol and Apolipoprotein A-I Levels Endocr. Rev., February 1, 2006; 27(1): 2 - 16. [Abstract] [Full Text] [PDF]

				H. Duez, B. Lefebvre, P. Poulain, I. P. Torra, F. Percevault, G. Luc, J. M. Peters, F. J. Gonzalez, R. Gineste, S. Helleboid, et al. Regulation of Human ApoA-I by Gemfibrozil and Fenofibrate Through Selective Peroxisome Proliferator-Activated Receptor {alpha} Modulation Arterioscler. Thromb. Vasc. Biol., March 1, 2005; 25(3): 585 - 591. [Abstract] [Full Text] [PDF]

				R. S. Birjmohun, B. A. Hutten, J. J.P. Kastelein, and E. S.G. Stroes Efficacy and safety of high-density lipoprotein cholesterol-increasing compounds: A meta-analysis of randomized controlled trials J. Am. Coll. Cardiol., January 18, 2005; 45(2): 185 - 197. [Abstract] [Full Text] [PDF]

				A. M. Gotto Jr and E. A. Brinton Assessing low levels of high-density lipoprotein cholesterol as a risk factor in coronary heart disease: A working group report and update J. Am. Coll. Cardiol., March 3, 2004; 43(5): 717 - 724. [Abstract] [Full Text] [PDF]

				B. G. Brown, M. C. Cheung, A. C. Lee, X.-Q. Zhao, and A. Chait Antioxidant Vitamins and Lipid Therapy: End of a Long Romance? Arterioscler. Thromb. Vasc. Biol., October 1, 2002; 22(10): 1535 - 1546. [Abstract] [Full Text] [PDF]

				L. Saucan and E. A. Brinton Lp A-I and Niacin: New Views of an Antiatherogenic Duo Arterioscler. Thromb. Vasc. Biol., November 1, 2001; 21(11): 1707 - 1709. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sakai, T. Articles by Kashyap, M. L. Search for Related Content PubMed PubMed Citation Articles by Sakai, T. Articles by Kashyap, M. L. Related Collections Lipids Cardiovascular Pharmacology Lipid and lipoprotein metabolism