© 1999 American Heart Association, Inc.
Original Contributions |
Niacin Accelerates Intracellular ApoB Degradation by Inhibiting Triacylglycerol Synthesis in Human Hepatoblastoma (HepG2) Cells
From the Cholesterol Center, Medical Service, Department of Veterans Affairs Medical Center, Long Beach, Calif (F.-Y.J., V.S.K., M.L.K.), University of California, Irvine, Calif (F.-Y.J., V.S.K., M.L.K.) and National University of Singapore (M.L.K.).
Correspondence to Moti L. Kashyap, MD, Director, Cholesterol Center, Department of Veterans Affairs Medical Center, 5901 E Seventh Street (111GE), Long Beach, CA 90822.
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Abstract |
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Abstract—The mechanism by which the potent drug niacin decreases apoB-containing atherogenic lipoproteins and prevents coronary disease is unclear. Utilizing human hepatoblastoma (HepG2) cells as an in vitro model, we have examined the effect of niacin on intracellular degradation of apoB and the regulatory mechanisms involved in apoB processing. Niacin significantly increased apoB degradation in a dose- and time-dependent manner. Treatment of HepG2 cells with calpain inhibitor I [N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of certain protease-mediated apoB degradation], did not alter niacin-induced apoB degradation. Niacin decreased inhibition of oleate-mediated apoB degradation. Niacin dose-dependently inhibited the synthesis of both fatty acids and triacylglycerol (TG) by 20% to 40% as determined by the incorporation of 14C-acetate and 3H-glycerol into fatty acids and TG, respectively. Incubation of HepG2 cells with niacin significantly inhibited (by 12% to 15%) fatty acid esterification to produce TG as assessed by the incorporation of 3H-oleic acid into TG. 14C-acetate incorporation into cholesterol and phospholipids was unchanged. The activity of microsomal triglyceride transfer protein (MTP), a carrier protein for lipids, was not altered by pretreatment of cells with niacin. ApoB mRNA expression and 125I-LDL protein uptake were also unchanged. These data indicate that niacin accelerates hepatic intracellular post-translational degradation of apoB by selectively reducing triglyceride synthesis (through inhibiting both fatty acid synthesis and fatty acid esterification to produce TG) without affecting ALLN-inhibitable protease- or MTP-mediated intracellular apoB processing, resulting in decreased apoB secretion and hence lower circulating levels of the atherogenic lipoproteins.
Key Words: atherosclerosis • coronary disease • hepatic apoB degradation • niacin • fatty acid • triacylglycerol
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Introduction |
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ApoB is the major protein of VLDL, IDL, LDL, and Lp(a). Elevated levels of these apoB-containing lipoproteins, especially LDL, are associated with accelerated atherosclerosis, and its reduction results in a reduction in these complications. A growing body of evidence indicates that a large amount of de novo-synthesized apoB is not secreted but rather degraded intracellularly by cultured hepatocytes.1 2 3 4 5 Furthermore, recent studies have shown that translocation of apoB across the endoplasmic reticulum (ER) regulates the degradative pathway and the subsequent secretion in the plasma.6
In addition to the intracellular processes involved in translocation and degradative mechanisms, the cotranslational association of core lipids with apoB to form nascent lipoproteins appears to play an important role in regulating hepatic secretion of apoB-containing lipoproteins.7 For example, preincubation of HepG2 cells with oleate was shown to markedly reduce apoB degradation, suggesting that the newly synthesized lipids may facilitate the translocation of apoB into the ER lumen, and thus reduce the availability of apoB for degradation by proteases localized either in the cytosol or on the cytosolic side of the ER membrane.8 9 Furthermore, microsomal triglyceride transfer protein (MTP), a lipid carrier protein within the membrane, has been suggested to play an important role in facilitating the secretion of apoB-containing lipoproteins. Mutations in the gene encoding for the 97-kDa subunit of MTP have been implicated in a complete lack of the secretion of apoB-containing lipoproteins in rare autosomal recessive abetalipoproteinemia.10 11 Using transfection of COS-1 cells with truncated forms of apoB and MTP, recent studies indicated that the expression of MTP in heterologous cell systems may be sufficient to direct the assembly and secretion of apoB-containing lipoproteins.12
Although these complex cellular mechanisms regulating apoB secretion are fairly well known, the intracellular processes by which lipid-lowering drugs exert their action to decrease atherogenic plasma apoB-containing lipoproteins are not clearly understood. For example, despite the wide usage of nicotinic acid (niacin) to decrease plasma LDL levels and to treat atherosclerotic cardiovascular disease,13 14 15 16 17 the mechanism of action of niacin to lower LDL levels has not been fully defined. Earlier studies suggested that niacin, by decreasing the production of VLDL, may lower plasma levels of VLDL and LDL.13 18 19 Additional reports indicated that the ability of niacin to decrease the transport (or production) of VLDL-triglycerides may be a predominant mechanism to lower triglyceride levels.13 Nevertheless, these earlier studies did not address the role of niacin in modulating the synthesis and post-translational events involved in hepatic secretion of apoB-containing lipoproteins.
Thus, using a human hepatoblastoma cell line (HepG2) as an in vitro model, we examined the effects of niacin on intracellular apoB degradation and regulatory factors (such as protease-mediated degradation, microsomal triglyceride transfer protein and synthesis of fatty acids and lipids) involved in hepatic apoB secretion. This in vitro model has been shown to be useful in studies related to the hepatic secretion of apoB-containing lipoproteins.3 The data presented indicate that niacin increased intracellular apoB degradation by selectively decreasing hepatic synthesis of triglycerides without affecting certain protease- or MTP-mediated intracellular apoB processing.
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Methods |
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Materials
Tissue culture materials, media, BSA (essentially fatty acid-free), oleic acid (sodium salt), ALLN, and niacin were obtained from Sigma Chemical Company. Fetal bovine serum (FBS) was obtained from Hyclone Laboratories. L-[4,5-3H]leucine and 32P-nucleotide were purchased from Amersham Corporation. Human hepatoblastoma cell line HepG2 and human apoB DNA probe were obtained from American Type Culture Collection (Rockville, MD). Polyclonal antibody for human apoB was obtained from Boehringer Mannheim Biochemicals. Protein A-Sepharose CL-4B was purchased from Pharmacia LKB Biotechnology Inc. SDS-PAGE reagents were obtained from BIO-RAD Laboratories. Niacin stock solution was prepared in the culture medium (DMEM). The pH was adjusted to 7.4 with 1N NaOH, and appropriate concentrations of niacin were added to the culture dishes. All other chemicals used were of analytical grade.
HepG2 Cell Culture and Pulse-Chase Studies for ApoB
Degradation
HepG2 cells were grown in Costar T-75 tissue culture flasks with
15 mL of high glucose Dulbecco's modified Eagle's medium (DMEM)
containing 10% FBS, 2 mmol/L glutamine, 100 U/mL penicillin, 100
µg/mL streptomycin, and 0.25 µg/mL amphotericin B. HepG2 cells were
preincubated for 48 hours with varying concentrations of niacin (0 to
3.0 mmol/L) in DMEM containing 10% FBS media. Cells were washed
twice with PBS and preincubated at 37°C in leucine-free medium
containing 1.5% BSA (essentially fatty acid-free) and the same
concentrations of niacin for 2 hours, then pulsed with
L-[4,5-3H]leucine (100 µCi/mL)
for 15 minutes at 37°C. After the pulsing period, cells were washed
with PBS and chased for 0 to 180 minutes as described in individual
figure legends. At each time point, media were collected and cells
washed with PBS and digested in an ice-cold lysis buffer (150
mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L Tris, 0.5% Triton
X-100, 0.5% sodium deoxycholate, 0.06 mol/L sucrose, and protease
inhibitors, pH 7.4). Aliquots of cell lysate and chase
medium were used for apoB immunoprecipitation or SDS-PAGE
analysis. During the course of the studies we monitored the
potential toxic effects of niacin on HepG2 cells by measuring cell
viability by trypan blue exclusion and DNA or protein content. Niacin,
at various doses (up to 3 mmol/L) had no effect on cell viability.
During the incubation period of 48 hours, 
1% to 2% of the cells
were stained with trypan blue in both control and 0.25 to 3 mmol/L
niacin-treated cells. Additionally, no significant differences in DNA
or protein content were observed between control and niacin-treated
cells.
Immunoprecipitation of ApoB and SDS-PAGE Analysis
Aliquots (500 µL) of medium or cell lysate were mixed with 500
µL of NET buffer (150 mmol/L NaCl, 5 mmol/L EDTA, 50
mmol/L Tris, pH 7.4, 0.5% Triton X-100, and 0.1% SDS)
containing excess monospecific sheep anti-human apoB antiserum and
incubated overnight at 4°C on a rocking platform shaker. Protein
A-Sepharose CL-4B beads (100 µL of a 5% solution in NET buffer) was
added to each tube and the incubation was continued for an additional 3
hours. The antigen/antibody complex bound to protein A-Sepharose CL-4B
beads was collected by brief centrifugation at
12 000g, washed 5 times with 1 mL of NET buffer and
reconstituted in 100 µL of electrophoresis buffer (0.125 mol/L
Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol).
ApoB was removed from the protein A-Sepharose beads by boiling for 4
minutes and the released apoB in the supernatant was collected by
centrifugation and quantified by liquid scintillation
counting. An aliquot of apoB released was subjected to electrophoresis
through a 5% to 15% gradient polyacrylamide gel containing
SDS. The gel was incubated in fluorographic solution (Amplify) for 1
hour, dried, and exposed to x-ray film to detect apoB protein bands.
Cell protein was determined by Lowry's method using BSA as a
standard.
Intracellular ApoB Degradation in the Presence of Oleate or ALLN
and Niacin
HepG2 cells were grown in 6 well plates (35 mm) for 3 days
to attain 70% to 80% confluence in DMEM+10% FBS media. Cells were
preincubated in the absence or presence of varying concentrations (0 to
3.0 mmol/L) of niacin for 48 hours. After the incubation, cells
were washed twice with PBS and incubated in 1 mL of serum-free medium
containing the same concentrations of niacin, 1.5% fetal bovine
albumin (FBA) and either 0.4 mmol/L oleate for 2 hours or
40 µg/mL calpain inhibitor I (ALLN) for 1 hour. Cells
were washed twice with PBS and the medium was replaced with
leucine-free DMEM containing 100 µCi of
3H-leucine. After 15 minutes of pulse labeling,
the medium was removed, then cells were washed twice with PBS and
chased for 1 hour in serum-free DMEM. During the pulse-chasing period,
the same concentrations of niacin and oleate or ALLN were present
in the media. ApoB degradation was assessed as described above using
cell lysate and chase medium.
Uptake of LDL by HepG2 Cells Treated With Niacin
Studies examining the uptake of LDL by HepG2 cells were
performed by utilizing radiolabeled LDL. Radioiodination of LDL total
protein was carried out by incubating freshly isolated LDL
(d=1.03 to 1.05 g/mL) with carrier-free
125I as described earlier by
McFarlane.20 After the iodination, unreacted
125I was removed by gel filtration followed by
exhaustive dialysis against PBS. The specific activity of
125I-LDL protein was 186 cpm/ng protein. Uptake
studies were initiated by preincubating HepG2 cells with varying
concentrations of niacin (0 to 3.0 mmol/L) for 48 hours at 37°C.
The media were replaced with fresh DMEM containing fetal bovine
albumin (5 mg/mL) and 125I-LDL (50 µg
protein) was added. After 6 hours of incubation at 37°C, cell
monolayers were washed thoroughly (4 to 5 times with PBS) and digested
with 1N sodium hydroxide solution. An aliquot was used for
radioactivity measurement. The uptake of radiolabeled LDL particles by
HepG2 cells was expressed in terms of cellular protein.
Northern Blot Analysis of ApoB mRNA
HepG2 cells were preincubated with varying concentrations of
niacin (0 to 3.0 mmol/L) for 48 hours. Total RNA was isolated from
HepG2 cells using the protocol of Chomcynski and Sacchi.21
Twenty micrograms of total RNA was loaded into individual wells of a
1.2% agarose gel containing formaldehyde and
electrophoresed,22 the RNA transferred onto MSI nylon
membranes, UV-cross-linked and hybridized overnight with the
[
-32P]dCTP-labeled cDNA probe for human
apoB. After washing the membrane, mRNA detection was performed by
autoradiography. Blots were then rehybridized with
human GAPDH cDNA probe as an internal control to assess RNA quantity
and integrity. mRNA signals were quantified by densitometric
scanning of autoradiographic bands (LKB laser densitometer,
Pharmacia, LKB Biotech) and normalized with GAPDH signals.
Microsomal Triglyceride Transfer Protein (MTP)
Activity Assay
MTP activity was measured by using an MTP Assay Kit according to
manufacturer's instructions (Diagnescent Technologies, Inc). Briefly,
HepG2 cells were treated with niacin at varying concentrations (0 to
3.0 mmol/L) for 48 hours. After incubation, cells were washed with
PBS and collected in homogenization buffer (10
mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5
mmol/L phenylmethylsulfonyl flouride and 20 µg/mL leupeptin).
The cell suspension was sonicated and 50 µg of homogenate
protein was used in the assay. MTP assay was performed by incubating 10
µL of donor and 10 µL of acceptor solutions with 5 µL HepG2 cell
homogenate (50 µg protein, MTP source) in a total volume
of 0.5 mL buffer (10 mmol/L Tris pH 7.4, 150 mmol/L NaCl,
2 mmol/L EDTA) and incubated for 6 to 12 hours at 37°C. The MTP
activity was calculated by measuring the fluorescence at
excitation wavelength of 465 nm and emission wavelength of 535 nm.
Fatty Acid, Cholesterol, Cholesteryl
Ester, Phospholipid and Triacylglycerol (TG)
Synthesis Assay
HepG2 cells were preincubated for 48 hours with varying
concentrations of niacin (0 to 3.0 mmol/L). Cells were washed
twice with serum-free DMEM and incubated at 37°C in the serum-free
DMEM medium containing 5 µCi/mL 3H-glycerol
(for TG synthesis) or 1 µCi/mL 14C-acetate (for
fatty acid, TG, cholesterol, cholesteryl
ester or phospholipid synthesis) or 1 µCi/mL of
3H-oleic acid (for assessing fatty acid
esterification to produce TG) and equal concentrations of niacin for 4
hours. After incubation, the medium was removed and cells were washed
thoroughly with PBS. Total cellular lipids were extracted by
chloroform/methanol (2:1, vol/vol), and the lipid extract was dried
under nitrogen and reconstituted in 100 µL hexane. Radiolabeled
lipids were separated by thin-layer chromatography
(TLC) on silica-coated plates using hexane/diethylether/acetic acid
(80:20:1, vol/vol) as a developing solvent. Lipids were visualized by
exposure to iodine vapors, the bands corresponding to authentic lipid
standards (eg, fatty acids, TG, cholesterol,
cholesteryl ester, or phospholipids) were scraped
and the radioactivity was measured by liquid scintillation counting and
expressed as CPM/mg cell protein.
Statistical Analysis
The mean data presented are average values for each set
of experiments done in triplicate. Each experiment was repeated 3
times. Statistical significance was calculated by using the Student's
t test and a P value <0.05 was considered
significant.
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Results |
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ApoB degradation studies performed in HepG2 cells (pulsed with 3H-leucine for 15 minutes, chased for 1 hour) indicated that the preincubation of cells with niacin (0 to 3 mmol/L) for 48 hours dose-dependently decreased apoB accumulation in the media by 9% to 23% during a 1-hour chase period (Table
). To understand intracellular
mechanisms of hepatic apoB secretion in response to niacin treatment,
we have carried out a series of studies using HepG2 cells as an in
vitro model that examined various intracellular regulatory events
involved in apoB degradation and secretion. Pulse-chase experiments
(pulsed for 15 minutes) performed for 1 hour indicated that the
preincubation of HepG2 cells with varying concentrations of niacin (0
to 3 mmol/L) for 48 hours dose-dependently increased apoB
intracellular degradation and decreased subsequent secretion of apoB
into the culture media (Table) compared with controls. Niacin,
as low as 0.5 mmol/L, significantly increased total (intracellular
plus media) apoB degradation by 26.8% when compared with controls and
the maximum degradation of 35.6% was noted at 3 mmol/L of niacin
(Table). Time-course studies examining the effect of niacin on
apoB degradation at varying incubation times (4 to 48 hours) indicated
that niacin, up to 8 hours incubation, had no significant effect, but
niacin, at 24 hours incubation, resulted in a marginally significant
effect on apoB degradation (data not shown). The maximal effect on apoB
degradation was observed with 48 hours of incubation with niacin, hence
a 48-hour incubation period with niacin was used for all the
experiments.
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Incorporation of 3H-leucine into intracellular
apoB at 0 minutes chase was measured as an index of apoB synthesis.
During the 15-minute pulse period, niacin produced no significant
change in the de novo synthesis of apoB. The apoB synthesis
(3H-leucine incorporation, cpm/mg cell protein)
in control and niacin-treated cells are 20 593±1054 and
18 510±1067, respectively (data shown in Figure 1 legend). The apoB synthesis data (cpm)
in niacin-treated cells were15% lower than control. Although
this was not significant statistically, the lower value could
represent some degradation of apoB during the 15-minute leucine
pulse. Additional pulse-chase experiments (pulse, 15 minutes; chase, 0
to 180 minutes) were performed to determine the effect of niacin on
total apoB degradation (intracellular plus secreted into media) at
various chase periods (0 to 180 minutes). The results from these
studies indicated that the pretreatment of HepG2 cells with niacin
(1.5 mmol/L) for 48 hours significantly increased the total
degradation of apoB during chase periods of 20 to 180 minutes when
compared with the respective controls (Figure 1). Intracellular
apoB degradation, as measured by percent of total initial cellular apoB
decayed (intracellular plus secreted into the media), at as early as 20
minutes chase period, was markedly higher in niacin-treated cells as
compared with controls (apoB degradation: control=36.2± 0.58%,
niacin=50.0±1.15%, P=0.004); apoB degradation at 3 hours
chase period in control and niacin-treated cells was 56.8±1.85% and
72.2±1.84%, respectively (Figure 1, P=0.004). Further confirmatory tests for apoB degradation
using immunoprecipitation techniques were performed by separating apoB
bands via SDS-PAGE and semi-quantifying the degradation by fluorography
of apoB bands. Results indicated that, at each chase period (10 to 180
minutes), niacin-treated cells had markedly reduced full-length apoB
bands when compared with the respective chase-time point of control
cells, indicating increased intracellular apoB degradation by niacin
(Figure 2). However, no degraded apoB
bands (ie, smaller molecular weight bands) were seen on the fluorograms
of either control or niacin-treated cells. The percent of apoB
remaining at various chase periods (10 to 180 minutes) in control and
niacin-treated cells was measured by densitometric scanning of
fluorograms. The results indicated that the percentage values of apoB
remaining in control cells were: 80.7%, 40.1%, 21.2%, 10.7%, and
7.2% during chase periods of 10, 20, 60, 120, and 180 minutes,
respectively; whereas the percentage values of apoB remaining in
niacin-treated cells were: 56.0%, 33.4%, 16.2%, 7.5%, and 4.8%
during chase periods of 10, 20, 60, 120, and 180 minutes,
respectively.
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Because the results presented in the Table
and Figure 1 show increased apoB degradation in niacin-treated cells,
additional studies were designed to delineate regulatory intracellular
mechanisms associated with apoB degradation. Initially, pulse-chase
apoB degradation experiments were done in control and niacin-treated
cells (48 hours) in the presence of ALLN, a protease
inhibitor, to determine whether niacin-induced apoB
degradation is mediated through ALLN-inhibitable protease-dependent
pathways. In these experiments, HepG2 cells were preincubated with
niacin for 48 hours, treated with ALLN for 1 hour and pulse-chase
experiments were performed for 0 to 60 minutes chase period. Congruent
with earlier studies,23 treatment of HepG2 cells with ALLN
significantly decreased apoB degradation when compared with control
cells without ALLN treatment, suggesting the feasibility of our
experimental conditions used to measure apoB degradation. The apoB
degradation (including cell and medium), as measured by percentage
decay of initial during 10 to 60 minutes chase period, was 15.1% to
46.8% and 1.0% to 25.6% in control and ALLN-treated cells,
respectively. To confirm the role of ALLN-mediated inhibition of apoB
degradation in niacin-treated cells, intracellular apoB degradation in
HepG2 cells treated with ALLN alone or in the presence of ALLN and
niacin was compared at various chase periods. As shown in Figure 3, the apoB degradation (as measured by
percentage decay of initial apoB in cell plus medium) in ALLN and ALLN
plus niacin-treated cells was not significantly different. These
results suggest that ALLN-induced effects on apoB degradation were not
altered by niacin treatment of cells.
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Because oleate has been shown to inhibit early apoB degradation,
further experiments were designed to examine whether niacin could alter
oleate-mediated effects on apoB degradation to determine the
involvement of lipids in niacin-induced intracellular apoB processing.
The incubation of niacin-pretreated cells with oleate significantly
increased cellular apoB degradation when compared with oleate treatment
alone (Figure 4). The increased
intracellular apoB degradation induced by varying concentrations of
niacin (0.5 to 3 mmol/L) was significantly inhibited by
coincubation with oleate (percentage apoB degradation at niacin doses
of 0.5 to 3 mmol/L: niacin alone=26.8% to 35.6%, niacin plus
oleate=9.8% to 13.4%). These data indicate that niacin-induced apoB
degradation may be dependent on the pathways involving the synthesis
and association of lipids before apoB processing.
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To determine the direct effects of niacin on cellular lipid synthesis,
experiments were carried out by examining the incorporation of
3H-glycerol and 14C-acetic
acid into triglycerides, cholesterol and
cholesteryl esters. As shown in Figure 5A and 5B, the incubation of HepG2 cells with varying concentrations of
niacin (0 to 3 mmol/L) dose-dependently inhibited fatty acid and
TG synthesis by 20% to 40% as determined by the incorporation of
14C-acetate into newly synthesized fatty acids or
TG. Similarly, the incubation of HepG2 cells with niacin significantly
(20% to 40%) inhibited the incorporation of
3H-glycerol into TG (Figure 6A). Parallel to the results of apoB
degradation in the presence of niacin plus oleate (Figure 3),
niacin, in as low a dose as 0.5 mmol/L, significantly decreased TG
synthesis; the maximal effect was noted at the 3-mmol/L dose (Figure 6A: incorporation, cpm/mg cell
proteinx103: using
3H-glycerol, control=149.7±2.1, 0.5 mmol/L
niacin=134.9±4.5, 3 mmol/L niacin=102.5±5.4; Figure 5A:
using 14C-acetate, control=208.8±7.1, 0.5
mmol/L niacin=194.2±2.7, 3 mmol/L niacin=149.5±15.2). Because
niacin inhibited the synthesis of both fatty acids and TG, we further
examined whether niacin has a significant role in the esterification of
fatty acids to form TG. These studies were performed by examining the
incorporation of radiolabeled oleic acid into TG. The results indicated
that the incubation of HepG2 cells with niacin significantly inhibited
(10% to 15%) oleic acid esterification to form TG (Figure 6B).
The incubation of niacin at various concentrations (0 to 3 mmol/L)
did not significantly alter the synthesis of cholesterol
and cholesteryl esters as assessed by
14C-acetate incorporation into newly synthesized
cholesterol and cholesteryl ester
(14C-acetate incorporation, cpm/mg cell
proteinx103: in cholesterol,
control=105.6±7.8, 1 mmol/L niacin=102.8±7.2, 3 mmol/L
niacin=99.7±5.2; in cholesteryl ester, control=8.0±0.13, 1
mmol/L niacin=7.7±0.25, 3 mmol/L niacin=7.8±0.48). Similarly,
niacin had no effect on the incorporation of
14C-acetate into phospholipids (data not
shown).
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Because MTP, a carrier protein of lipids from their site of synthesis to nascent lipoproteins within the ER, has been implicated in intracellular apoB processing, additional studies were carried out to examine whether niacin-induced apoB degradation is mediated through alterations in MTP activity in HepG2 cells. The incubation of HepG2 cells with varying concentrations of niacin (0 to 3 mmol/L) did not significantly alter MTP activity when compared with controls (MTP activity, nanomoles triacylglycerol transferred per hour per milligram protein: control=371±15, niacin at 1-, 2-, and 3-mmol/L doses=371±11, 367±2.4, and 374±6.9, respectively).
In addition to the role of niacin in intracellular apoB degradation, further experiments were planned to examine the effect of niacin on the steady-state mRNA expression of apoB and the uptake of radiolabeled apoB by HepG2 cells to determine the role of niacin on apoB synthesis and uptake by cells. The incubation of HepG2 cells with niacin (0 to 3 mmol/L) for 48 hours did not significantly alter the steady-state mRNA expression of apoB when compared with controls. Quantitative analysis by densitometric scanning of apoB mRNA blots and normalization with GAPDH message were performed. The densitometric arbitrary values of apoB mRNA expression for control and 0.5- to 3-mmol/L niacin-treated cells were 4.22 and 4.23 to 4.34, respectively. Additional studies were carried out to examine the effect of niacin on the uptake of radiolabeled LDL by HepG2 cells. Uptake studies were performed by incubating control or niacin-pretreated HepG2 cells (48 hours) with 125I-LDL for 6 hours. The results from these studies showed that the preincubation of HepG2 cells with niacin (0 to 2 mmol/L) for 48 hours did not significantly alter the uptake of 125I-LDL as compared with the respective controls (125I-LDL uptake, cpm/mg cell protein: control=40 571±322, niacin-treated cells at doses of 0.5, 1.0, and 2.0 mmol/L=40 559±1926, 37 947±1502, and 41 319±3254, respectively).
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Discussion |
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Niacin is widely used to regulate abnormalities in plasma lipid metabolism and in the treatment of atherosclerotic cardiovascular disease. In pharmacologic doses, niacin reduces total plasma cholesterol, triglycerides, VLDL and LDL and increases HDL.13 24 25 26 27 Furthermore, various clinical trials, including the Coronary Drug Project, the Cholesterol-Lowering Atherosclerosis Study and the Familial Atherosclerosis Treatment Study, indicated that treatment with niacin significantly reduced total mortality and coronary events and retarded the progression of coronary atherosclerosis.15 16 17 Although these studies clearly indicated the beneficial effects of niacin in the treatment of atherosclerotic cardiovascular disease, the cellular and molecular mechanisms by which niacin exerts its effect on lipoprotein metabolism is not clearly defined. Using HepG2 cells as an in vitro model, we have recently reported that niacin selectively decreases hepatic removal of HDL apoAI but not cholesteryl esters, suggesting a new concept with regard to the mechanism by which niacin raises HDL.28 However, the hepatocellular mechanisms of niacin in reducing apoB-containing lipoproteins are largely obscure. To address this question, we have performed a series of studies examining the role of niacin on intracellular apoB degradation and key regulatory mechanisms that govern hepatic post-translational processing of apoB and its secretion in HepG2 cells.
The results presented in this report indicate that niacin
significantly increased intracellular apoB degradation as assessed by
pulse-chase experiments. During a 1-hour chase period, niacin at 0.5 to
3 mmol/L concentration retained less intracellular
radiolabeled apoB by 31.5% to 42% as compared with control cells.
Similarly, niacin treatment resulted in a lowered amount of
radiolabeled apoB secreted into the culture media by 18% to 24% when
compared with controls (Table), strongly indicating enhanced
apoB degradation as 1 possible mechanism. This increase in apoB
degradation by niacin was further demonstrated by varying the chase
period from 10 to 180 minutes and showed cellular apoB degradation of
25.5% to 72.2% when compared with an apoB degradative rate of 19.4%
to 56.8% in control cells. Because cellular radiolabeled apoB measured
by immunoprecipitation techniques may give rise to a nonspecific
artifact, we further analyzed the amount of cellular
radiolabeled full-length or degraded apoB by SDS-PAGE and fluorography
in control and niacin-treated cells at various chase-time points.
Similar to the data obtained by immunoprecipitation techniques,
SDS-PAGE analysis also showed comparable increases in apoB
degradation in cells treated with niacin when compared with controls,
and thus validated the use of immunoprecipitation techniques to measure
apoB degradation in all of the remaining studies presented in
this investigation. Although niacin treatment showed lower signals for
full-length apoB bands as compared with controls, we could not detect
any degraded apoB bands (ie, smaller molecular weight bands) in either
control or niacin-treated cells. This may be due to various factors;
the degraded apoB fragments may not be recognized by anti-apoB used for
immunoprecipitation or the signals for the degraded products may be
too weak to be detected on the fluorogram.
Although the detailed intracellular processes that lead to the assembly
of apoB-containing lipoproteins in or within the ER and its subsequent
secretion are not fully described, available data suggest that
protease(s) localized within the cytosol of the ER may play a crucial
role in apoB degradation before its secretion.23
Furthermore, the synthesis and association of core lipids (eg,
triglycerides and cholesteryl esters) has been proposed to
protect newly synthesized apoB from intracellular
degradation,8 9 suggesting that newly synthesized lipids
play an important role in targeting apoB for secretion. Earlier studies
used ALLN (calpain inhibitor I), a potent
inhibitor of the degradation of both
3-hydroxy-3-methylglutaryl-coenzyme A and the T-cell receptor
subunit in the ER, to determine the role of certain protease-mediated
pathways in intracellular apoB degradation.23 29 30
It has been shown that the treatment of HepG2 cells with ALLN markedly
decreased intracellular apoB degradation and thus may serve as an
important tool to understand ALLN-inhibitable protease-dependent apoB
degradation in response to various agonists.
In this study, using ALLN, we examined whether niacin-induced apoB degradation is dependent or independent of ALLN-inhibitable protease-mediated pathways. The incubation of HepG2 cells with ALLN markedly inhibited intracellular apoB degradation when compared with control cells, an observation similar to those reported in earlier studies using HepG2 cells.6 23 Addition of ALLN to either control cells or niacin-treated cells showed comparable degrees of apoB degradation as compared with their respective controls. Because of the similar percent reduction of apoB degradation by ALLN in control and niacin-treated cells, these results suggest that the effect of niacin to induce apoB degradation is independent of ALLN-inhibitable protease-mediated pathways. These observations also rule out the possibility of other putative proteases that may be sensitive to niacin stimulation because decreased ALLN-induced apoB degradation was unaffected by niacin.
Additional studies were designed to determine whether alterations in the association of newly synthesized triglycerides with apoB are involved in niacin-induced cellular apoB degradation. To explore this possibility, we have used oleic acid, a known agonist that has been employed to decrease intracellular apoB degradation. Earlier studies have indicated that the treatment of HepG2 cells with oleic acid markedly inhibited intracellular apoB degradation with a parallel increase in apoB secretion into culture media without altering mRNA expression of apoB.7 8 9 Furthermore, Boren et al7 suggested that the cotranslational addition of lipids (eg, triglyceride) was important for the rapid transfer of apoB to the ER lumen and subsequent secretion. Using acetyl coenzyme A carboxylase inhibitor and HMG-CoA reductase inhibitor, agents to block fatty acid and cholesterol synthesis, respectively, recent studies have indicated that the rate of triglyceride but not cholesterol synthesis regulates apoB degradation in HepG2 cells.31 Furthermore, these authors also showed that the rate of triglyceride synthesis rather than the total intracellular mass of triglycerides determines the secretion of apoB. Because the mobilization of stored lipid droplets for VLDL assembly is impaired in HepG2 cells,32 Benoist and Grand-Perret suggested that neutral lipid synthesis is compartmentalized and that the triglycerides available for lipidation of apoB and its subsequent secretion are synthesized in the proximity of lipoprotein assembly sites, whereas stored triglycerides are not directly available for the assembly of apoB-containing lipoproteins and subsequent secretion.31 Sakata et al,23 using a combination of the ALLN and oleate approaches, proposed that oleate treatment, by increasing triglyceride synthesis, reduces degradation of apoB by accelerating its translocation from the protease compartment of the ER membrane.
In this investigation, cellular apoB degradation in HepG2 cells treated with niacin plus oleic acid was significantly higher than in cells treated with oleic acid alone. This difference in apoB degradation was significant at various chase-time points from 10 to 60 minutes. Because oleate-mediated increased TG synthesis did not protect against niacin-induced apoB degradation, we hypothesized that niacin, through alterations in TG synthesis, may increase apoB degradation. To confirm this possibility, we examined the direct effects of niacin on the synthesis of TG, cholesterol, cholesteryl ester, and phospholipids by measuring the incorporation of 3H-glycerol or 14C-acetate into corresponding lipids. Results showed that the incubation of HepG2 cells with niacin significantly inhibited TG synthesis by 20% to 40% as determined by radiolabeled acetate or glycerol incorporation, respectively. Because TG synthesis is regulated by cellular processes involved in fatty acid synthesis and their esterification to form TG, we examined the effect of niacin on fatty acid synthesis and on the esterification of fatty acids to produce TG. Niacin significantly inhibited the synthesis of fatty acids and the esterification of fatty acids to synthesize TG. Thus, the data indicate that niacin inhibits TG production at 2 synthetic sites: (a) fatty acid synthesis from acetate, and (b) esterification of fatty acids to form TG. Because these 2 cellular processes are regulated by various enzyme systems and esterification reactions, niacin may have yet unknown roles in modulating these processes, which are outside the scope of this investigation. In contrast to triglyceride synthesis, niacin had no effect on the synthesis of cholesterol or cholesteryl esters in HepG2 cells. Our in vitro findings on triglyceride synthesis are in line with earlier in vivo studies in humans by Grundy et al13 demonstrating that the treatment with niacin reduced VLDL-triglyceride synthetic rates (transport) as determined by multi-compartmental kinetic analysis after the injection of radiolabeled glycerol as a precursor. It has been suggested that the observed reduced influx of VLDL-triglycerides in niacin-treated patients may be associated with the ability of niacin to inhibit the release of free fatty acids by adipose tissue.33 34 35 The quantitative roles of the effect of niacin on decreased fatty acid mobilization versus decreased hepatic triglyceride synthesis is unclear. Because niacin decreases triglyceride content in VLDL, it is possible that such triglyceride-poor particles may result in smaller and denser VLDL particles as suggested by kinetic studies.13 Increased hepatocyte apoB degradation by niacin would also decrease the number of VLDL (and their catabolic product, LDL) particles secreted, and explain the lower apoB concentrations observed clinically after niacin treatment.17
Thus, we propose that niacin, by inhibiting triglyceride synthesis through multiple mechanisms, may hinder the lipidation and translocation of apoB across the ER membrane and may create a favorable environment for protease-mediated intracellular apoB degradation. The inability of niacin to alter cholesterol or cholesteryl ester synthesis is congruent with previous studies indicating that apoB degradation is not dependent on cholesteryl ester synthesis.31 36 In addition to the synthesis of triglyceride, MTP, a protein that catalyzes the transfer of triglycerides, cholesteryl esters and phosphatidylcholine between membranes, has been recently shown to influence apoB degradation and secretion.10 11 12 The treatment of HepG2 cells with niacin did not alter MTP activity, suggesting that the effect of niacin on apoB degradation is independent of MTP-mediated pathways.
In addition to the effects of niacin on apoB degradation, decreased apoB secretion in niacin-treated cells may be the result of alterations in apoB mRNA expression and synthesis, and the uptake of apoB-containing lipoproteins by HepG2 cells. Because niacin did not alter either apoB mRNA expression or radiolabeled LDL uptake, we rule out the possibility that changes in apoB gene expression or protein uptake (via LDL receptor) contributed to the decreased apoB. Data presented in this communication indicate that niacin is directly responsible for the effects observed. Although it would be important to make some correlation of in vitro niacin concentration used in our studies to those of plasma concentrations of niacin in patients, we feel that it would be difficult to make such correlations based on the following points. The pharmacokinetics of niacin have not been clearly established in humans. However, it has been noted that at pharmacologic doses (>1 g/day) niacin is almost completely absorbed and reaches peak plasma concentrations of 0.12 to 0.24 mmol/L within 30 to 60 minutes. It has also been noted that nicotinuric acid is the major metabolite of niacin. After absorption, most of the niacin is taken up in the liver and very little enters the systemic circulation. Thus, plasma levels are not reflective of biological activity. The exact concentration of niacin in the hepatic interstitial compartment has not been established. However, our in vitro concentrations of a 10-fold higher order of concentration than plasma concentrations achieved clinically are not out of line. The possibility also exists that niacin and/or its metabolites (eg, nicotinuric acid) may participate in these effects. Additional research is needed to elucidate this question.
In summary, these results suggest that niacin, by increasing hepatic post-translational intracellular degradation of apoB, but not altering mRNA expression or uptake, exerts its action to lower the secretion of apoB-containing lipoproteins. Furthermore, the selective inhibition of triglyceride synthesis by niacin may limit the lipidation of apoB required for its translocation across the ER membrane and thus facilitate intracellular apoB degradation, suggesting an explanation for reductions in apoB-containing lipoproteins observed clinically after niacin treatment.
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Acknowledgments |
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This investigation was supported in part by a grant from Kos Pharmaceuticals and by the Long Beach Research Foundation. We thank Noah Wagner for his expert assistance in typing this manuscript and Mark Hubbard for preparation of figures.
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Footnotes |
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Part of this work was presented in abstract form at the Annual Meeting of the American Heart Association, in New Orleans in November 1996, and done during a visiting professorship at the National University of Singapore by Dr Kashyap.
Received February 23, 1998; accepted October 6, 1998.
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