271
Pathogenesis of Carbohydrate-Induced
Hypertriglyceridemia Using HepG2 Cells
as a Model System
Katherine Cianflone, Smadar Dahan, Juan Carlos Monge, and Allan D. Sniderman
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This study compares the effects of glucose and fatty acid on hepatic lipid synthesis and apolipoproteln
(apo) B secretion. To do so, varying concentrations of either glucose or oleic acid were added to the
medium in which HepG2 cells were being incubated. Intracellular triacylglycerol and cholesteryl ester
synthesis and secretion were measured by addition of radioisotopic tracers and by determination of mass,
whereas apo B concentration in the medium was measured by a specific enzyme-linked immunosorbent
assay. The data indicate that increasing concentrations of glucose in the medium resulted in increased
synthesis of triacylglycerol within the cell and increased secretion of triacylglycerol into the medium. Apo
B secretion into the medium, however, did not change, and intracellular synthesis and secretion of
cholesteryl ester did not change as well. By contrast, addition of oleic acid to the medium resulted in
increased synthesis and secretion of both cholesteryl ester and triacylglycerol, and this was associated with
increased secretion of apo B into the medium. Thus, a carbohydrate load resulted in secretion of normal
numbers of triacylgtycerol-enriched apo B particles by this hepatocyte cell line, whereas a fatty acid load
led to the secretion of increased numbers of apo B particles, which were essentially normal in composition.
(Arteriosclerosis and Thrombosis 1992;12:271-277)
KEY WORDS • hypertriglyceridemia • apolipoprotein B • HepG2 cells
H
ypertriglyceridemia can result from disorders of
clearance or from disorders of overproduction.1-2 It is obviously, therefore, not a unitary
entity. Two different clearance defects have been recognized: the first involves the rate of triacylglycerol
hydrolysis (lipoprotein lipase and apolipoprotein [apo]
CH deficiency fall in this category1), whereas the second
involves impaired removal of chylomicron and very low
density lipoprotein (VLDL) remnants (with type III
hyperlipoproteinemia being the prototype of this class
of disorders).3 Two different genetic forms of hepatic
overproduction of VLDL have also been recognized:
familial hypertriglyceridemia and familial combined hyperlipidemia. The former is characterized by the secretion of normal numbers of triacylglycerol-enriched
VLDL particles, whereas the latter is characterized by
secretion of increased numbers of VLDL particles of
normal composition.4-6
However, little is yet known of the processes that
regulate VLDL secretion. Almost all available in vitro
evidence supports the view that apo B production rates
are modulated by posttranslational mechanisms,7-8 and
we have advanced the hypothesis that cholesteryl ester
synthesis is critical in this regard, at least so far as the
response to increased delivery of fatty acids to the liver
From the Royal Victoria Hospital, McGill Unit for the Prevention of Cardiovascular Disease, McGill University, Montreal,
Canada.
Supported by a Baxter Intramural Grant.
Address for correspondence: Dr. Allan D. Sniderman, Royal
Victoria Hospital, 687 Pine Avenue West, Room M4.76, Montreal,
Canada H3A 1A1.
Received June 7, 1991; revision accepted October 29, 1991.
is concerned.9 Nevertheless, it has been well documented that carbohydrate feeding also can lead to
increased plasma triacylglycerols, 1011 presumably
through increased delivery of glucose to the liver.
Accordingly, the present studies were undertaken to
compare the response of HepG2 cells on the one hand
to an exogenous glucose load and, on the other, to an
exogenous fatty acid load. Our expectation was that the
latter would result in secretion of increased numbers of
apo B particles, whereas the former would result in an
increased content of triacylglycerol per apo B particle.
Our hypothesis was that these differences would relate
to differences in cholesteryl ester synthesis induced by
the two different metabolic loads.
Methods
Tissue Culture
HepG2 cells obtained from the American Tissue
Culture Collection (Rockville, Md.) were routinely
grown in minimum essential medium supplemented
with 10% fetal calf serum and 100 IU penicillin/streptomycin in 75-cm2 flasks with 15 ml medium in a 37°C
incubator with 5% CO2. Flasks were subcultured every
7 days, with a split ratio of 1:3. The cells were dislodged
from the culture flask with 0.25% trypsin in Ca2+- and
Mg2+-free phosphate-buffered saline (PBS) for 5 minutes at 37°C. For experiments, cells were plated out at a
density of 1.3xlO4 cells/cm2 in 17-mm dishes (24-well
plates) or 35-mm dishes. For the density gradient
experiments, cells were used in T75 flasks.
Experimental Conditions
[2-3H]glycerol (final specific activity, 200 mCi/mmol);
[l-l4C]acetic acid, sodium salt (final specific activity,
272
Arteriosclerosis and Thrombosis
Vol 12, No 3 March 1992
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86.4 mCi/mmol); fHJglucose (stock specific activity,
15.5 Ci/mmol); and [9,10-3H(A9]sodium oleate (stock
specific activity, 7.4 Ci/mmol) were purchased from
DuPont-New England Nuclear (Montreal, Canada).
Oleic acid (sodium salt) and fatty acid-free bovine
serum albumin (BSA), fraction V, were obtained from
Sigma Chemical Co. (St. Louis, Mo.). For experiments
with varying glucose concentrations, the amount of
radioactive [3H]glucose was adjusted to give a final
average specific activity of 10 dpm/pmol. Similarly for
experiments with varying oleate concentration, the
amount of radioactive oleate was adjusted to yield a
final average specific activity of 5.9 dpm/pmol to avoid
isotope dilution. Cholesteryl ester was determined from
the incorporation of [MC]acetate (average specific activity, 105 dpm/pmol). Fatty acid was added to the medium at the indicated concentrations complexed to BSA
at a ratio of 5:1 according to the method of Van Harken
et al.12 Tissue-culture medium was purchased from Flow
Laboratories (MacLean, Va.).
Before the experiments were performed, cells were
preincubated for 24 hours at 37°C in 5% CO2 in
serum-free medium supplemented with 1% BSA. Cells
were then changed to 1% BSA in serum-free medium
with the indicated supplements and incubated for the
indicated times at 37°C. At the end of the incubation
period, the cells were placed on ice, and the medium
was removed and set aside for analysis. The cells were
washed three times with 1 ml ice-cold PBS, and the cell
lipids were extracted with 2 ml heptane/isopropanol
(3:2, vol/vol). After 30 minutes, the extracts were
removed and the cells were washed once with an
additional 1 ml heptane/isopropanol (3:2, vol/vol) and
added to the previous extract. The soluble cell protein
was dissolved in 1 ml 0.1N NaOH and collected.
Lipid Quantification
Cell lipid extracts were evaporated under nitrogen
and redissolved in a known volume of chloroform/
methanol (2:1, vol/vol). An aliquot was applied to a
thin-layer chromatography plate prewashed in chlorofonn/methanol (2:1, vol/vol). The lipids were separated
by running in hexane/ether/acetic acid (75:25:1, vol/vol/
vol); the lipid spots were visualized by exposure to
iodine vapor and identified by comparison to reference
lipids (cholesteryl ester, triacylglycerol, and phospholipids). The phospholipids were taken as the nonmigTating
polar lipids in this solvent system and include phosphatidylcholine and sphingomyelin. The silica gel was
scraped into vials containing scintillation cocktail, and
the radioactivity was counted in a scintillation counter
(Beckman Instruments, Palo Alto, Calif.). For mass
assays, the triacylglycerol band was scraped from the
plates, eluted with isopropanol, and measured by the
method of Neri and Frings.13 Cholesteryl ester mass was
quantified by gas-liquid chromatography on a HewlettPackard Model 5830A gas chromatograph as described
by Huff et al.14
Aliquots of the medium were also extracted with 5
volumes of heptane/isopropanol (1:1, vol/vol), and the
organic phase was washed twice with 1 ml isopropanol/
heptane (4:1, vol/vol) and 1 ml 0.05% KOH to remove
the remaining radioactive free oleate. The sample was
then processed in the same way as the cell extracts to
measure medium triacylglycerol and cholesteryl ester.
For density gradient experiments, cells in T75 flasks
were incubated as described previously, and the media
from two to three flasks were pooled. The medium was
concentrated at least 10-fold by dialysis against 50%
polyethylene glycol in 10,000 molecular-weight-cutoff
dialysis tubing. A 1-ml sample was then adjusted to a
density of 1.10 g/ml and layered under a discontinuous
gradient of 1 ml of d= 1.07 g/ml, 1 ml of d=1.05 g/ml, 1
ml of <2=1.02 g/ml, and 1 ml of d= 1.0063 g/ml using
potassium bromide salt solutions (total volume, 5 ml).
The sample was centrifuged for 24 hours at lOO.OOOg in
a Beckman L8-80 ultracentrifuge to form a linear
gradient. The bottom of the tube was then pierced, and
0.5-ml aliquots were collected. Each fraction, including
the starting fractions, was analyzed for apo B by enzyme-linked immunosorbent assay, for [3H]glycerol incorporation into triacylglycerol, and for [14C]acetate
incorporation into cholesteryl ester. The density of the
solution was measured by refractive index and compared against a set of calibrated potassium bromide
standard solutions.
Protein and Apolipoprotein Quantification
Cell protein was measured by the method of Bradford,15 using BSA as a standard. Apo B and apo A-I
concentrations in the medium were measured by sandwich enzyme-linked immunoassay16 as modified by Ortho Diagnostics (La Jolla, Calif.), using a standard curve
of 0.025-0.400 /xg/ml apo B. Each point in each experiment is the average of triplicate determinations and is
expressed per milligram cell protein ±SEM. Significance
was measured by paired Student's t test between the test
value and the control value. An individual control was
run for each experiment.
Results
The effects of increasing concentration of oleate in
the medium on intracellular lipid synthesis and secretion of apo B and apo A-I into the medium are shown in
Figure 1. Consistent with our previous results,9 triacylglycerol and cholesteryl ester synthesis increased in
parallel. Apo B secretion into the medium also increased substantially, while apo A-I release was unaltered. These results contrast with those obtained by
increasing the medium concentration of glucose (Figure
2). In this instance, intracellular triacylglycerol synthesis
increased to the same extent as in the oleate experiments (373±103% glucose versus 344±33% oleate
nmol/mg cell protein), but neither cholesteryl ester
synthesis nor phospholipid synthesis changed significantly. Once again, apo A-I secretion was unaffected,
but this time apo B secretion was unaffected as well.
Cells exposed to 50 mM glucose or 1 mM oleate
manifested cytoplasmic lipid droplets as determined by
light microscopy.
The changes in cellular lipids as determined by
radioactive tracer were confirmed by mass analysis of
the triacylglycerol and cholesteryl ester as shown in
Table 1. There was a three to fourfold increase in
cellular triacylglycerol mass on incubation with either
oleate or glucose for 24 hours. In contrast, there was no
Cianflone et al Carbohydrate-Induced Hypertriglyceridemia in HepG2 Cells
TG(nmol/mg cell P)
0.2
0.4
C£(x 10)
0.0
0.8
1
273
ug/ml/mg cell protein
0
0-2
a*
0.8
0.8
1
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[OLEATE] mM
[OLEATE] mM
FIGURE 1. Line plots showing effect ofoleate on intraceUidar lipid synthesis and apoUpoprotein (apo) secretion. HepG2 cells were
preincubated (see "Methods") and then incubated in 1% bovine serum albumin medium supplemented with 0-1 mM oleate for
24 hours. Left panel: IntraceUular triacylgfycerol (TG; +) and cholesteryl ester (CE; a) were measured as nanomoles per
milligram soluble cell protein (P)±SEM. Right panel: Apo B (o) and apo A-I (•) secretion into the medium was measured as
micrograms per milUUterper milligram sohible cellprotein±SEM. *p<0.025, **p<0.01, ***p<0.0025.
change in cellular cholesteryl ester mass with glucose,
but there was an increase with the oleate incubation.
Not only did the number of secreted apo B particles
differ in the two circumstances, but also their composition differed as well. These results are shown in Figure
3. With the oleate challenge, there was little difference
TG{nmol/mg cell P)
10
20
CE(x 10)
30
40
[GLUCOSE] mM
50
in either the cholesteryl ester to apo B or the triacylglycerol to apo B ratio over the range of oleate concentrations examined (Figure 3, left panel). On the other
hand, carbohydrate challenge changed the composition
of the secreted apo B particle but not the number of
particles, in that there was a pronounced increase in the
ug/ml/mg cell protein
10
20
30
40
60
[GLUCOSE] mM
FIGURE 2. Line plots showing effect of glucose on intraceUular Upid synthesis and apoUpoprotein (apo) secretion. HepG2 cells
were preincubated (see "Methods") and then incubated in 1% bovine serum albumin medium supplemented with 5.5-50 mM
ghicosefor 24 hours. Left panel: IntraceUular triacylgfycerol (TG; +) and cholesteryl ester (CE; n) were measured as nanomoles
per milligram sohible ceU protein (P)±SEM. Right panel: Apo B (o) and apo A-I (•) secretion into the medium was measured
as micrograms per milliliter per milligram sohible cell protein±SEM. *p<0.05.
274
Arteriosclerosis and Thrombosis
Vol 12, No 3 March 1992
TABLE 1. Determination of HepG2 Cellular Lipid Mass: Triacylglycerol and Cholesteryl Ester
Triacylglycerol
Cholesteryl ester
(nmol/mg cell protein)
Addition
1% BSA
246+42
4.2+0.4
1% B S A + 5 0 mM glucose
791±191
4.1+0.4
P
1% B S A + 0 . 8 mM oleate
P
0.05
NS
l,170±355
6.1+0.4
0.005
0.OOO5
Cells were incubated in 35-mm dishes as described in "Methods"
in the indicated media for 24 hours. Cellular mass was determined
and expressed as nanomoles per milligram cell protein±SD (n=S).p
values were calculated by t test for two means against basal ( 1 %
bovine serum albumin [BSA]). NS, not significant
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triacylglycerol to apo B ratio but no change in the
cholesteryl ester to apo B ratio over the range of glucose
concentrations tested (Figure 3, right panel). When
both glucose and oleate were added to the medium,
more apo B particles were secreted and they were
triglyceride enriched (results not shown).
The effect of addition of glucose or oleate on the
distribution of the secreted apo B particles within a
density gradient was also examined. In essence, the
increased triacylglycerol to apo B ratio induced by
glucose was associated with a shift in the secreted apo B
particles toward the more buoyant densities. This can be
seen in the density gradient profile of the medium
lipoproteins shown in Figure 4. The upper panel shows
the distribution of secreted lipoproteins in basal medium ( 1 % BSA) as measured by apo B mass and
[3H]glycerol incorporation into triacylglycerol. After
oleate incubation, there was no change in size distribution (middle panel). However, in the presence of glucose, the density shifted toward a less dense fraction
100
(lower panel). The amount of apo B associated with the
peaks was 74%, 81%, and 79% of the total. Similarly,
the amount of triacylglycerol associated with the peaks
was 77%, 82%, and 73%.
The importance of the duration of the incubation and
the concentration of glucose in the medium is illustrated in
Figure 5. At the lowest concentration of glucose (5 mM),
there was little increase in triacylglycerol in the medium
even after an 8-day incubation, whereas at 25 mM and
even more markedly at 50 mM, triacylgrycerol secreted
into the medium rose progressively and markedly over the
8-day time course (Figure 5, upper panel). This contrasts
with the results for cholesteryl ester secreted into the
medium, for which there was little change (Figure 5,
middle panel). Of importance, there was no difference in
apo B concentration in the medium for any glucose
concentration at any time point (Figure 5, lower panel).
Discussion
The mechanism of carbohydrate-induced hypertriglyceridemia has long been of interest, and we believe
the present data allow this phenomenon to be viewed
from yet another perspective, a perspective that provides insights into the mechanisms that regulate secretion of hepatic apo B particles. In these experiments,
triacylglycerol synthesis has been augmented either by
increasing delivery of fatty acids to the liver or by
increasing de novo fatty acid synthesis within the liver.
Although triacylglycerol synthesis increased in both
cases, the consequences differed. With a fatty acid
challenge, the number of apo B particles secreted
increased, although their composition did not change.
With a carbohydrate challenge, the number of apo B
particles secreted did not change, but their composition
did. In the first instance, the extra triacylglycerol synthesized was removed from the cells in more particles,
TQ/B
CE/B
60
so
20
- 10
O2
0.4
oe
[OLEATE] mM
as
i
o
io
20
30
40
50
[GLUCOSE] mM
FIGURE 3. Line plots showing effect of oleate and glucose on secreted triacylglycerol (TG) and cholesteryl ester (CE) to
apolipoprotein (apo) B (B) ratio. Cells were incubated for 24 hours as described in the legends to Figures 1 and 2. Triacylgfycerol
(o) and cholesteryl ester (•) secreted into the medium are expressed per secreted apo B (nanomoles lipid per microgram apo
B)±SEM. *p<0.0125, **p<0.025.
Cianflonc et al Carbohydrate-Induced Hypertriglyceridenila in HepG2 Cells
275
nmol/ mo call protein
. medium TG
tOI
102
103
104
105
10«
107
106
109
DENSITY (g/mU
nmol/ mg coll protsln
* DUtrt button
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medium CE
101
102
103
104
106
WO
DENSITY (g/mL)
107
108
109
0
1
2
3
*
8
6
7
8
* Habitation
medium apoB
101
102
103
w
ut
toe
DENSITY (g/mU
tor
tot
too
FIGURE 4. Line plots showing effect of oleate and glucose
on the size distribution of secreted lipoprotein particles.
HepG2 cells in T75 flasks were incubated with basal 1%
bovine serum albumin (BSA) medium (upper panel), 1%
BSA+0.8 mM oleate (middle panel), or 1% BSA+50 mM
glucose (lower panel) for 24 hours. The percentage distributions of apolipoprotein B (o) and triacylgfycerol (*) are
shown in each panel. The medium was then concentrated
and separated on a continuous density gradient as described
in "Methods."
•
5
8
7
8
[DAYS]
FIGURE 5. Line plots showing long-term effect of glucose on
lipoprotein lipid and apoUpoprvtein (apo) B secretion in HepG2
cells. Cells were incubated as described in "Methods" for 24
hours-8 days in 1% bovine serum albumin medium supplemented with 5.5 (o), 25 (*), or 50 (a) mM glucose. Results are
expressed as nanomoles triacylglycerol (TG) per milligram
soluble cell protein (upper panel), nanomoles cholesteryl ester
(CE) per milligram cell protein (middle panel), or mkrograms
apo B per milliliter medium per milligram cell protein (lower
panel)±S£M *p<0.01, **p<0.0025.
276
Arteriosclerosis and Thrombosis
Vol 12, No 3 March 1992
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while in the second, the same end was achieved by
packaging more triacylglycerol per particle, resulting in
a more buoyant particle.
We have previously proposed that the critical event
regulating the apo B response to increased delivery of
fatty acids to the liver is the rate of cholesteryl ester
synthesis in the rough endoplasmic reticulum.9 Our
model posits that the newly synthesized cholesteryl
ester molecule causes a newly formed apo B molecule to
enter the phospholipid bilayer of the rough endoplasmic
reticulum and then to move along to the junction with
the smooth endoplasmic reticulum where triglyceride is
added to the apo B-cholesteryl ester complex, following
which the lipid-apo B complex is extruded into the
lumen of the endoplasmic reticulum. The fact that
cholesteryl ester synthesis is much less in toto than
triacylglycerol synthesis does not argue against the
model, in that only a small number of cholesteryl ester
molecules synthesized in the rough endoplasmic reticulum may be necessary to achieve a critical effect.
The present results are in accord with this model.
With an exogenous fatty acid load, cholesteryl ester
synthesis increases in parallel with triacylglycerol synthesis and apo B secretion increases as well. By contrast,
with an endogenous fatty acid push, that is, fatty acids
generated by de novo synthesis, cholesteryl ester synthesis does not increase and apo B secretion does not
change either. Therefore, in situations where triacylglycerol and cholesteryl ester synthesis can be dissociated, apo B secretion follows cholesteryl ester synthesis,
not triacylglycerol synthesis.
All these studies, however, have been conducted in
HepG2 cells. Notwithstanding that they retain many of
the biologic capacities of normal hepatocytes, HepG2
cells are a transformed cell line and do not primarily
secrete VLDL.17-19 Nevertheless, there is a close correspondence between the present results and much that
has been done before. Ahrens and colleagues10 were the
first to demonstrate that increased dietary intake of
carbohydrate resulted in increased plasma triacylglycerol levels and that this phenomenon occurs in normals
as well as in hypertriglyceridemic patients. Since then,
in both rats and humans, several studies have shown
that carbohydrate loads result in the secretion of triacylgrycerol-enriched VLDL particles without a change
in the number of such particles secreted.20-23
The present results may also provide insight into the
molecular defects responsible for familial combined
hyperlipidemia and familial hypertriglyceridemia. The
former is characterized by the secretion of increased
numbers of VLDL particles. Were fatty acid flux to the
liver to increase for any reason, our data demonstrate
that the consequences would be an increased rate of
intracellular triacylglycerol synthesis and an increased
secretion rate of VLDL particles of normal composition. The metabolic features of familial combined hyperlipidemia and hyperapobetalipoproteinemia are
identical,24 and one defect has now been identified in a
subset of patients: reduced cell membrane receptors for
acylation stimulating protein,23-26 which might reduce
the maximal rate of triacylglycerol synthesis in the
peripheral tissues and which, as a consequence, would
result in increased fatty acid influx to the liver.
On the other hand, the present data also suggest a
hypothesis for the nature of the metabolic defect in
familial hypertriglyceridemia, namely that intracellular
synthesis of fatty acids from glucose is increased and that
this results in the secretion of normal numbers of triacylglycerol-enriched apo B particles. Although this hypothesis remains to be tested, the present results at least amplify
the evidence that it is intrahepatic cholesteryl ester synthesis and not triacylglycerol synthesis that plays the key
role in regulating hepatic apo B secretion.
Acknowledgments
We appreciate the help of Murray Huff and Cindy Sawyez
for the determination of the cholesteryl ester mass in the
HepG2 cells.
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Pathogenesis of carbohydrate-induced hypertriglyceridemia using HepG2 cells as a model
system.
K Cianflone, S Dahan, J C Monge and A D Sniderman
Arterioscler Thromb Vasc Biol. 1992;12:271-277
doi: 10.1161/01.ATV.12.3.271
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville
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Copyright © 1992 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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