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(Received for publication, March 24, 1995; and in revised form, June 12, 1995) From the
Elevated plasma high density lipoprotein cholesterol (HDL-C)
levels are associated with a decreased risk for coronary heart disease.
Ironically, diets enriched in saturated fat and cholesterol (HF/HC
diets), which tend to accelerate atherosclerotic processes by
increasing LDL cholesterol levels, also raise HDL-C. We have recently
reported, using a human apoA-I (hapoA-1) transgenic mouse model, that
the elevation of HDL-C by a HF/HC diet is attributable, in part, to an
increase in the hepatic production of hapoA-1. To further define the
hepatocellular processes associated with this induction, we have
prepared primary hepatocytes from hapoA-1 transgenic mice. Rates of
hapoA-1 secretion were 40% greater from cells prepared from animals fed
the HF/HC relative to a low fat-low cholesterol (LF/LC) control diet.
The abundance of hapoA-1 mRNA in these cells was similar between
hepatocytes prepared from the HF/HC and LF/LC diet fed animals,
suggesting a post-transcriptional mechanism that does not involve mRNA
stability. Inhibition of secretion using brefeldin A revealed an
increase in cellular hapoA-1 accumulation. Thus, the HF/HC diet
apparently affects hepatic hapoA-1 production via a mechanism that is
manifest prior to the exit of newly synthesized hapoA-1 from the Golgi.
Pulse-chase experiments revealed a 39% greater peak hapoA-1 synthesis,
with no difference in the degradation of total labeled hapoA-1 protein,
as a result of the HF/HC diet feeding. Finally, resolution of liver S10
extracts via sucrose density sedimentation and metrizamide density
equilibrium gradient centrifugation analyses both revealed similar
increases (31 and 24%, respectively) in the relative percentage of
hapoA-1 mRNA associated with the translating polysomal fractions as a
result of the HF/HC feeding. Together, these data suggest that the
HF/HC diet affects hepatic hapoA-1 production via a specific modulation
in the relative amount of hapoA-1 mRNA in the polysomal pool. These
observations provide an opportunity to explore a new mechanism
regulating apoA-1 production and might lead to the development of novel
therapies to elevate plasma HDL-C levels.
Epidemiological studies have shown a strong inverse correlation
between high density lipoprotein cholesterol (HDL-C) ( The current study explores the cellular
and molecular mechanism(s) for the post-transcriptional regulation of
apoA-1 production by dietary fat and cholesterol. A different line of
human apoA-1 transgenic mouse was used, which expressed the human gene
in both liver and intestine(4) . We confirmed that primary
hepatocytes prepared from these animals on the HF/HC versus the LF/LC diet showed increased hapoA-1 secretion without an
increase in hapoA-1 mRNA levels. Experiments using brefeldin A (BFA)
suggested that the increase in production preceded the exit of newly
synthesized hapoA-1 from the Golgi apparatus. Pulse-chase experiments
in HF/HC hepatocytes revealed increased synthesis and no change in
hapoA-1 degradation. Sucrose density gradient ultracentrifugation of
liver S10 supernatants showed a HF/HC-induced increase in polysomal versus non-polysomal associated hapoA-1 mRNA but no change in
the average number of ribosomes per hapoA-1 mRNA. Metrizamide
equilibrium density gradient ultracentrifugation analysis showed a
dietary fat-induced increase in the fraction of hapoA-1 mRNA in the
ribosomal versus the non-ribosomal associated peaks,
consistent with the sucrose gradient data. Therefore, a HF/HC diet
raises hapoA-1 production, and thereby plasma levels, by increasing the
fraction of the hapoA-1 mRNA in the translating pool of messenger RNA.
Human apoA-1 transgenic mice were fed the HF/HC or the LF/LC
chow diet for 3 weeks, and HDL-C and hapoA-1 levels were measured. The
mice were males of the A-14 line that were 12-14 weeks old at the
start of the experiment. As shown in Table 1, compared to the
LF/LC diet, the HF/HC diet elevated HDL-C by 68%, hapoA-1 by 40% and
mouse apoA-1 by 52%. Primary hepatocytes were prepared from hapoA-1
transgenic mice on the HF/HC or LF/LC diet, and the rate of secretion
of hapoA-1 was assessed by measuring the time course of appearance of
radiolabeled immunoprecipitable hapoA-1 in the culture medium. As shown
in Fig. 1, hapoA-1 appearance was linear for 2 h, and the
secretion rate was 40% greater (p < 0.01) from hepatocytes
cultured from mice fed the HF/HC diet compared to the LF/LC diet. In
contrast, the incorporation of radiolabel into total cellular and
secreted protein, determined by precipitation using trichloroacetic
acid and subsequent scintillation counting, was found to be similar
between the two diet conditions (data not shown). Human apoA-1 mRNA
levels were next quantified by a solution hybridization/RNase
protection assay of total RNA isolated from whole liver and primary
hepatocytes from mice fed either the HF/HC or LF/LC diets. As shown in Table 2, there was no significant difference between the two diet
conditions in hapoA-1 mRNA levels in either whole liver or cultured
hepatocytes. Interestingly, the relative abundance of hapoA-1 mRNA was
significantly elevated in total RNA prepared from the primary
hepatocyte cultures as compared to whole liver. However, this effect
was not associated with the different diets used and therefore might be
the result of a transcriptional enhancement of hapoA-1 gene expression
previously observed when primary cultured hepatocytes were separated
from non-parenchymal cells(10) . Diet also had no effect on
hapoA-1 mRNA levels in the intestine, the only other site of hapoA-1
synthesis. These secretion and mRNA data support the contention that a
HF/HC diet increases hapoA-1 levels and hepatic hapoA-1 synthesis at
the post-transcriptional level and confirm our previous findings using
the hapoA-1 transgenic mouse line 179, which expresses hapoA-1 only in
the liver(2) . Furthermore, the lack of differences in
intestinal mRNA and the similar magnitude increase (40%) in hapoA-1
plasma levels and hepatocyte secretory output suggest that hepatic
production is the primary determinant of the plasma differences, even
in mice with the capacity to express the transgene in the intestine.
Figure 1:
The effect of the high fat-high
cholesterol diet on hapoA-1 secretion from transgenic mouse primary
hepatocytes. Primary hepatocytes were prepared from hapoA-1 transgenic
mice that were fed a high fat-high cholesterol (HF) or a low
fat-low cholesterol (LF) diet for 25 days as described under
``Materials and Methods.'' The cells were incubated with
leucine- and serum-free MEM supplemented with 40 µM cold
leucine and containing 200 µCi/ml [
To further explore the mechanism of the diet-induced increase in
apoA-1 production, primary hepatocytes from hapoA-1 transgenic mice
were incubated with 2 µg/ml BFA, which blocks transport of nascent
proteins from the Golgi apparatus(11) . As shown in Fig. 2, after a 2-h incubation with radiolabel, BFA inhibited
secretion of hapoA-1 from hepatocytes prepared from mice fed either
diet by greater than 95%. Nevertheless, the accumulation of
radiolabeled cellular hapoA-1 was 48% greater in hepatocytes from the
mice fed the HF/HC diet compared to chow. This experiment localizes the
diet-induced increase in liver apoA-1 production to a step prior to the
exit of the newly synthesized protein from the Golgi apparatus. Since
diet did not change steady-state hapoA-1 mRNA levels, the effect
presumably occurs by either increasing translational efficiency (i.e. greater synthesis) or decreasing the degradation of
newly synthesized protein.
Figure 2:
Effect of the high fat-high cholesterol
diet on intracellular hapoA-1 accumulation in the presence of BFA.
Primary hepatocytes were prepared from hapoA-1 transgenic mice that
were fed either the low fat/low cholesterol (LF, openbars) or high fat-high cholesterol (HF, hatchedbars) diet. Cells were pretreated for 30 min
and then incubated with [
To examine these possibilities, a
pulse-chase experiment was performed. Primary hepatocytes were pulsed
with label for 10 min, washed, and chased. As shown in Fig. 3,
the peak incorporation of label into cellular hapoA-1 at the end of the
labeling period was 39% greater (p < 0.01) in primary
hepatocytes derived from transgenic mice fed the HF/HC diet compared to
LF/LC. This was consistent with the increased secretion of newly
synthesized hepatocyte hapoA-1 (see Fig. 1) and strongly
suggested that increased hapoA-1 synthesis, underlying this enhanced
secretion, is not an artifact of reutilization of label induced by
diet. The disappearance of cellular hapoA-1 following the synthesis
peak was apparently different with the two diet conditions when the
slopes of log-transformed cell data were examined (data not shown).
This might result from either an up-regulated degradative pathway in
the low fat and/or an enhanced secretion in the high fat condition. To
explore these possibilities, the recovery of total labeled hapoA-1 was
calculated by adding together the radioactivity (dpm) of both cellular
and medium immunoprecipitable hapoA-1 protein, as resolved by SDS-PAGE.
As shown in Fig. 3B, the rate of disappearance of total
hapoA-1 was similar between the two diet conditions. The calculated
half-lives were therefore also of comparable values (117 min for high
fat and 128 min for low fat). Thus, there is no apparent difference in
the degradative pathway for hapoA-1 between the two diet conditions.
Interestingly, the data in Fig. 3B also suggests that
about 50% of the hapoA-1 protein is degraded and that this is
unaffected by diet. This degradation might occur prior to the secretion
of the newly synthesized protein or possibly upon re-uptake of secreted
hapoA-1 from the medium. Since the rate of accumulation of labeled
hapoA-1 was linear for the entire labeling period (see Fig. 1),
this degradation probably occurs prior to secretion. Together, these
results clearly show that consumption of a HF/HC diet causes a specific
increase in the rate of hepatic hapoA-1 synthesis and secretion, with
no effect on its degradation.
Figure 3:
Pulse-chase analysis of hapoA-1
synthesis and degradation in primary hepatocytes. Primary hepatocytes
prepared from hapoA-1 transgenic mice (line A-14) fed either the high
fat-high cholesterol (HF) or low fat/low cholesterol (LF) diet. Hepatocytes were incubated 10 min with serum- and
leucine-free MEM containing [
Increased hapoA-1 synthesis without a
change in mRNA levels localizes the diet effect to an increase in the
efficiency with which the hapoA-1 message is translated. This could be
due to increased fractional entry of hapoA-1 mRNA into the polysome
pool and/or an increased rate of translation initiation or elongation
of the translated product. The former would be reflected in an increase
in the fraction of hapoA-1 mRNA associated with polyribosomes versus ribonucleoprotein particles (mRNPs), and the latter
with alteration in the size of the polyribosomal complex reflecting a
change in the number of ribosomes per hapoA-1 mRNA. To explore these
possibilities, liver S10 extracts prepared from transgenic mice fed
HF/HC and LF/LC diets were compared using sucrose density gradient
ultracentrifugation. As shown in Fig. 4A, the
distribution of total RNA on the sucrose density gradient was similar
with extracts from transgenic mice fed both types of diets. Based on
previous studies(7, 12) , it was inferred that
fractions 1-3 contained unassembled ribosomal subunits and
non-ribosome-associated mRNA (mRNPs), whereas fractions 4-15
contained the translating polysomal complexes with the lower numbered,
less dense, fractions containing fewer ribosomes per mRNA than the
higher numbered, more dense fractions. Northern blot analysis, shown in Fig. 4B, revealed similar distributions of hapoA-1 mRNA
among the polysomal fractions (fractions 4-15) in liver S10
extracts from mice fed either diet. This showed that diet did not
influence the average number of ribosomes per hapoA-1 message. The
visual impression of these Northern blots might be misleading, since
the hapoA-1 signal in the polysomal fractions of the LF/LC diet-fed
mice appears to be markedly lower than the HF/HC, although the RNase
protection assays of total RNA indicated that the two diet groups were
similar (Table 2). However, it appeared that the relative amount
of hapoA-1 mRNA in the polysomal fractions of the gradient compared to
mouse
Figure 4:
Sedimentation velocity gradient analysis
of hepatic hapoA-1 mRNA in transgenic mouse liver extracts. Cytoplasmic
extracts prepared from A-14 transgenic mice that were fed the HF/HC or
LF/LC diet were subjected to sedimentation velocity centrifugation on a
sucrose gradient (see ``Materials and Methods''). A,
the absorbance at 254 nm of each gradient fraction was recorded during
its collection by continuous monitoring (LF/LC diet, solidline; HF/HC diet, dashedline). The
density of the gradient increases from left to right. B, total RNA was isolated from equal volumes of each fraction
and subjected to Northern blot analysis using
Figure 5:
Abundance of hapoA-1 mRNA in fractions
following metrizamide equilibrium gradient separation of transgenic
mouse hepatic cytoplasmic extracts. Hepatic cytoplasmic extracts
prepared from hapoA-1 transgenic mice fed either the LF/LC (Low
Fat) or HF/HC (High Fat) diet were sedimented to
equilibrium density in metrizamide (see ``Materials and
Methods''). A, total RNA extracted from equal volumes of
each gradient fraction was assayed for hapoA-1 mRNA abundance by
solution hybridization/RNase protection. Fraction densities increase
from left to right. Solidline,
high fat; dashedline, low fat. B, the
effect of diet on the relative (percent) abundance of hapoA-1 mRNA in
the mRNP (blackbars) peak (fractions 3 and 4) and
the ribosome-associated (polysomal) (shadedbars)
peak (fractions 6 and 7) were compared between hapoA-1 transgenic lines
A-14 (left) and 179 (right).
In humans, a HF/HC diet increases HDL-C levels in part by
increasing the transport rate of hapoA-1, but the mechanism is unknown.
To explore this question, a hapoA-1 transgenic mouse model was created
that expresses hapoA-1 only in the liver. In these mice, the HF/HC diet
increased hapoA-1 transport rate by increasing the hepatic secretion of
hapoA-1 without altering hapoA-1 mRNA levels(2) . In the
current study, a new hapoA-1 transgenic mouse model that expresses
hapoA-1 in both liver and intestine was used for dietary investigation.
As in the previous study, associated with the HF/HC diet, there was an
increase in plasma levels of human apoA-I. A comparable increase in
primary hepatocyte hapoA-I production with no change in intestinal
hapoA-I mRNA levels also suggested that dietary induced changes in the
transport rate of hapoA-I could be primarily attributed to the liver.
These findings were next extended using BFA to localize the site of
increased production of hapoA-I prior to its transport from the Golgi
apparatus. Pulse-chase experiments then showed that increased hapoA-1
production was not due to decreased intracellular catabolism but rather
to increased synthesis. Since hepatic apoA-I mRNA levels were not
affected by diet, the increased synthesis represented an increased
translational yield. Using sedimentation velocity (sucrose) and
equilibrium density (metrizamide) gradients, the basis for this was
then shown to be an increased association of hepatic hapoA-1 mRNA with
ribosomes in the HF/HC group. Since the average number of ribosomes per
hapoA-I mRNA did not change (see Fig. 4), the diet-induced
increase in hapoA-1 synthesis and secretion from hepatocytes could be
explained most simply by an increased fraction of the hapoA-1 mRNA in
the polysomal pool rather than by the absolute rate with which
translation was initiated or elongated. Translation of mRNAs broadly
involves three major processes: initiation, elongation, and
termination. Termination is generally thought not to be
rate-limiting(12, 13) , leaving initiation and
elongation as the two major levels of control (see (14) for a
review). Although examples exist of control at the elongation
step(8, 15) , the more common finding is regulation of
the initiation pathway (16, 17) . This can be
accomplished in two basic ways, namely, inhibition of an initiation
factor(s) or sequestration of mRNA. An example of the former is the
reticulocyte system, where it has been shown that inactivation of the
initiation factor eIF2 by phosphorylation in the absence of heme
decreases protein mRNA translation(18) . On sucrose gradients
of the type employed in the present study, however, this would be
detected by a decrease in the average number of ribosomes per message,
so we do not favor this mode of regulation as an explanation for what
we observed. A model system for the specific sequestration of mRNA,
which may be relevant to the present study, is the control of ferritin
translation. In a series of elegant studies (reviewed in (20) )
from primarily the laboratories of Klausner et al.(21) and Munro et al.(22) , it was shown
that in the absence of iron, there is a protein (IREP) bound to a
stem-loop structure in the 5`-untranslated RNA region of the ferritin
mRNA that prevents the assembly of an initiation complex. When
sufficient iron is present, it binds to the IREP and promotes its
disassociation from the mRNA. The message now can associate with
ribosomes and be translated. Thus, on gradient analysis, in the
presence of iron the ferritin mRNA is associated with polysomes,
whereas in its absence it is not. The resemblance of this result with
those in the present study suggests a similar mechanism for the diet
effect on hapoA-1 translation. As a result of consuming a HF/HC diet, a
factor in the liver that regulates the partitioning of human apoA-I
mRNA between the non-translated compartment (free mRNPs) and the
translated compartment (polysomes) may be modified. As we observed,
this would result in the formation of fully loaded polysomes as well as
free mRNPs in the same environment, with the proportion of each varied
according to metabolic conditions. To emphasize further an analogy with
the ferritin model, computer analysis of the 5`-untranslated RNA region
of hapoA-1 mRNA reveals a stable (free energy of -7.4 Kcal/mol)
stem-loop structure. This could represent the cis-acting diet-response
element to which the liver factor hypothesized above binds. Preliminary
results (data not shown) suggest that at least two cytosolic factors
bind to this mRNA sequence. Future studies focusing on these factors
will explore this previously unrecognized mechanism of apolipoprotein
regulation. There are a number of examples of the production of
hepatic apolipoproteins being regulated by lipids at the
post-transcriptional level. The closest one to the present study was
reported by Go et al.(23) . Rats were placed on diets
of varied contents of cholesterol (none or 2% of dry weight) and
propylthiouracil (none or 0.1% of dry weight). The fat content,
however, remained constant (vegetable fat (4.3%) and lard (5%); the
diet containing no cholesterol or propylthiouracil was considered to be
the control). Hepatic apoA-I mRNA levels were not affected by diet, but
in hepatocyte cultures, relative to the control group, there was an
increase in the production of apoA-I in the cholesterol group.
Polysomes were then isolated, and run-off assays were performed to
determine the number of polysomes initiated in vivo. There was
no difference in the results for the two groups. Although the authors
suggest that the data reflected translational control, further
experiments to investigate the mechanism, similar to those in the
present study, were not reported. However, it is unlikely that the
distribution of the apoA-I mRNA between mRNP and polysome fractions was
a determinant; if it were, then in the bulk polysome isolation
procedure, fewer apoA-I mRNA-containing polysomes would have been
harvested from the control group livers, and the run-off results would
have been relatively lower. Regulation of apoA-I mRNA translation may
differ between the two studies because in the present study cholesterol and fat content of the diets were varied. Another example
of post-transcriptional regulation of hepatic apolipoprotein production
by lipids is the effect of fatty acids on the secretion of apoB, the
major apolipoprotein of VLDL and LDL. Dixon, Ginsberg, and their
colleagues have reported in a series of studies (reviewed in (24) ) using HepG2 cells that the provision of oleic acid
increased apoB secretion by decreasing its intracellular degradation.
We have recently shown in both rat primary hepatocytes and McArdle rat
hepatoma cells(5, 25) , that n-3 fatty acids
decrease apoB secretion by inducing intracellular apoB degradation. As
in the HepG2 studies, apoB synthesis and mRNA levels were not changed.
Clearly, though post-transcriptional in nature, regulation in these
systems was fundamentally different from that in the present study,
since synthesis of the apolipoprotein was not affected. It is well
known that HDL-C levels are strongly correlated with plasma levels of
hapoA-1(3, 26) . Experiments with transgenic and
knockout mice have shown that gene dosage effects on hapoA-1 synthesis
can directly affect HDL-C levels and that this can influence
atherosclerosis susceptibility(27, 28, 29) .
In a previous study(3) , we showed that switching humans from
LF/LC to HF/HC diets raised HDL-C levels primarily by increasing the
apoA-1 transport rate but that there was considerable variation among
subjects in responsiveness. This raised the issue of whether humans on
Western-type diets with low HDL-C levels might be atherosclerosis prone
since they fail to increase apoA-1 synthesis in response to the fat and
cholesterol in their diets. A better understanding of the translational
control of apoA-1 synthesis may therefore reveal a biochemical and
genetic basis for the inter-individual variation in atherosclerosis
susceptibility. Finally, it might be possible to design diets or drugs
that increase hapoA-1 translation, thereby elevating HDL-C levels,
without concomitant effects on raising levels of atherogenic
lipoproteins.
Dedicated to the memory of Dr. Shlomo Eisenberg.
Volume 270,
Number 34,
Issue of August 25, pp. 19833-19838, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)levels and coronary heart disease risk. Ironically,
however, diets high in saturated fat and cholesterol, which increase
atherosclerosis risk, raise HDL-C levels. To probe the mechanism
whereby dietary fat influences apoA-1 metabolism, an animal model, the
human apoA-1 transgenic (hapoA-1) mouse, was studied. ApoA-1 is
normally expressed in liver and intestine, but these mice expressed the
hapoA-1 transgene only in liver(1) . Nevertheless, as
summarized in a previous report(2) , increasing dietary fat and
cholesterol in these mice raised HDL-C levels 65% and hapoA-1 levels
41%. Turnover studies revealed a 29% increase in hapoA-1 transport rate
and a 26% decrease in fractional catabolic rate. These results are
similar to a previous study using human volunteers(3) , and
further, they imply that dietary fat can increase liver production of
apoA-1. This was confirmed by experiments showing increased apoA-1
secretion from primary hepatocytes isolated from animals on the HF/HC
compared to the LF/LC diets. The increased hapoA-1 production was not
accompanied by any increase in hepatic or intestinal hapoA-1 mRNA.
Thus, the mechanism of the diet effect on hapoA-1 production appeared
to be post-transcriptional.
Transgenic Mice
Two lines of human
apoA-1 transgenic mice, A-14 and 179, were
used(1, 4) . Each line contains the entire human
apoA-1 gene but differs in flanking sequences. Line A-14 contains human
DNA from 0.3 kb 5` to 1.7 kb 3` of the gene plus the -1.4 to
-0.2-kb region of the promoter of the adjacent apoCIII gene,
which we previously showed (4) controls apoA-1 intestinal
expression. Line 179 contains a continuous 11.5-kb human genomic
segment extending from 5.5 kb 5` to 3.8 kb 3` of the gene. Both the
endogenous mouse apoA-1 gene and the human apoA-1 transgene were found
to be expressed in both the liver and intestine of line A-14 mice. In
contrast, in line 179 animals, the endogenous mouse apoA-1 gene was
found to be expressed in both the liver and intestine, whereas the
human apoA-1 transgene was expressed only in the liver(4) .Diets
Mice were fed two contrasting
diets. The LF/LC diet was rodent chow (no. 5001; Ralston Purina, St.
Louis, MO), containing 4.5% fat, 59.8% carbohydrate, 23.4% protein,
5.0% fiber, added minerals and vitamins, and 0.02% cholesterol; fats
provided 9% of the calories, equally divided among saturated,
monounsaturated, and polyunsaturated. The HF/HC diet was TD 88137
(Teklad Premier Laboratory Diets, Madison, WI), a milk fat-based diet
containing 21.2% fat, 49.1% carbohydrate, 19.8% protein, 5% fiber,
added minerals and vitamins, an antioxidant, and 0.2% cholesterol; fats
provided 41% of the calories, with saturated, monounsaturated, and
polyunsaturated forms contributing 27, 12, and 2%, respectively.Plasma Lipid, Lipoprotein, and Apolipoprotein
Determinations
The mice were anesthetized with
methoxyflurane, and plasma was taken from the retro-orbital plexus and
assayed for total cholesterol, HDL-C, and human and mouse apoA-1, as
previously described(2) .Primary Hepatocyte Preparations
Mice were
fed either the HF/HC or LF/LC diet for 3 weeks. Hepatocyte cultures
were prepared using a modification of a previously reported
procedure(2, 5) . Briefly, mice (two per experimental
condition) were anesthetized intraperitoneally with 5% pentobarbital,
and then the portal vein was cannulated and the liver perfused with a
CO
-equilibrated, calcium-free MEM (Life Technologies, Inc.)
at 37 °C for 10 min. MEM containing 5 mM CaCl and 0.075% collagenase (Boehringer Mannheim) was then perfused
through the liver for 30 min. The livers were then excised from the
animals, minced with a sterile scissors in a Petri plate, and passed
through a nylon mesh filter, which was then rinsed with Hanks'
buffered saline; this served to separate debris from liver cells. The
cells were then centrifuged at 500 g, resuspended in
MEM, and washed twice by centrifugation. Cell viability (range
88-93%) was assessed by trypan blue exclusion, and 1
10
live cells were plated on 60-mm Petri dishes pre-coated
with poly-D-lysine and incubated in 2 ml of MEM at 37 °C
in 95% air, 5% CO.Labeling and Pulse-Chase Experiments
4 h
after plating, the medium was removed, and the cells were washed once
with phosphate-buffered saline (pH 7.4) and again with nonradioactive
labeling medium (leucine-free MEM supplemented with a small amount of
leucine (40 µM)). For labeling experiments, hepatocytes
were then incubated with 2 ml of labeling medium containing 200
µCi/ml [4,5-
H]leucine (141 Ci/mmol, Amersham)
for the indicated times at 37 °C. After incubation, the medium was
collected and centrifuged at 12,000 g at 4 °C for
5 min. The attached cells were washed once with phosphate-buffered
saline at 4 °C. For pulse-chase experiments, hepatocytes were
labeled for 10 min as described above, washed once with
phosphate-buffered saline and once with chase medium (MEM containing
400 µM leucine), and then incubated with 2 ml of chase
medium for the indicated times. In experiments with BFA, the labeling
protocol above was followed except that hepatocytes were exposed to 2
µg/ml BFA (Sigma) for 30 min prior to and during the entire
labeling period. Radiolabeled hapoA-1 was quantitatively isolated from
conditioned medium and cell lysates by immunoprecipitation using a
specific goat anti-human apoA-1 antibody that does not cross-react with
mouse apoA-1, as previously described (2) . The samples were
then solubilized in non-reducing SDS-PAGE sample buffer (62.5 mM Tris, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol), placed in a
boiling water bath for 5 min, and then applied to a 15% SDS-PAGE gel
containing a 5% stacking gel(6) . Purified nonradioactive
hapoA-1 (3 µg/lane) was mixed with each sample prior to
electrophoresis for visual identification of bands. The gels were
stained with Coomassie Blue and then destained. The bands corresponding
to hapoA-1 were excised and placed in 20-ml glass scintillation vials
containing 0.5 ml each of water and solvable tissue and gel solubilizer
(DuPont NEN). The vials were capped, incubated at 50 °C for 18 h,
and cooled to room temperature. 10 ml of Readysafe scintillation fluid
(Beckman) were added, and the samples were counted.
Preparation of Liver S10 Extracts
Mice
were fed either the HF/HC or LF/LC diet for 3 weeks. To prepare the S10
extracts, mice were sacrificed between 9 and 10 a.m. by cervical
dislocation, and livers were quickly isolated and homogenized in 4
volumes of buffer (250 mM sucrose, 20 mM HEPES, pH
7.5, 250 mM KCl, 5 mM MgCl, 2 mM dithiothreitol, 150 µg/ml cycloheximide, and 1 mg/ml sodium
heparin) using a Polytron homogenizer (Brinkman; Luzern, Switzerland)
at setting 7 for 3 10-s bursts at 4 °C. The livers from three mice
were used for each experimental condition. The homogenate was then
centrifuged for 20 min at 10,000 g at 4 °C, and
the resulting supernatant (S10) was carefully separated and stored at
-70 °C.
Sedimentation Velocity and Equilibrium Density
Analyses
Mouse liver S10 extracts were prepared as
described above. Aliquots (0.5 ml) were thawed on ice and then
overlayered onto linear 15-50% (w/v) sucrose gradients, and
sedimentation velocity ultracentrifugation analysis was then
performed(7) . Gradient fractions (850 µl each) were
collected by upward displacement using a 75% (w/v) sucrose solution and
collected in tubes containing SDS and sodium acetate. For the
equilibrium density analyses, aliquots (150 µl) of mouse liver S10
extracts were mixed with 3.85 ml of 40% (w/v) metrizamide (Aldrich)
solution containing 500 mM KCl, 5 mM MgCl, 20 mM HEPES, pH 7.5, and 1 mM
dithiothreitol. The self-forming gradients were resolved by
ultracentrifugation in a Beckman SW55 rotor at 40,000 rpm for 72.5 h at
4 °C, and 0.5-ml fractions were carefully collected from the top.
Fraction densities were determined as previously described(7) .
Total RNA was extracted from the sucrose and metrizamide gradient
fractions using acid phenol/chloroform(8) .mRNA Quantitation and Protein
Determination
mRNA levels were determined by either
Northern blot analysis or by solution hybridization/RNase protection as
previously described(8, 9) . Probes used for mRNA
detection and quantification included a riboprobe specific to hapoA-1
mRNA that does not cross-hybridize to the endogenous mouse apoA-1 mRNA (3) and a riboprobe specific to mouse 
-actin prepared from
pTri--actin/mouse (Ambion). Probes were prepared using the Ribomax
T7 RNA transcription system reagents (Promega). Protein was determined
using DC protein assay reagents (Bio-Rad) with bovine serum albumin as
the standard.Statistical Analyses
The unpaired
Student's t test was employed to compare means of values
between the different diets. Statistical significance was defined as p < 0.05. Where indicated, results are presented as mean
± S.D.
H]leucine
for the indicated times. Human apoA-1 was immunoprecipitated from the
conditioned medium using an antibody specific to hapoA-1. The immune
complex was resolved by SDS-PAGE. Purified hapoA-1 (3 µg) was added
to each well to assist visualization. Gels were stained using Coomassie
Brilliant Blue and then destained. Bands corresponding to hapoA-1 were
excised, solubilized, and then subjected to scintillation counting.
Three different gels, each derived from three different sets of dishes,
were used in each analysis. The experiment shown was repeated twice
with similar results. Data indicate mean ± S.D. (n = 4).
H]leucine for 2 h in the
presence of 2 µg/ml BFA. Secreted (Medium) and
intracellular (Cell) hapoA-1 were immunoprecipitated and
analyzed via SDS-PAGE/scintillation counting, as described under
``Materials and Methods.'' The results represent the means
± S.D. of triplicate determinations.
H]leucine (200
µCi/ml) and then washed and incubated with MEM containing excess
(400 mM) cold leucine. Incorporation of
[H]leucine into cellular hapoA-1 was determined
as described under ``Materials and Methods.'' The recovery of
total (cellular plus medium) labeled hapoA-1 is shown in B (solidline, high fat; dashedline, low fat). The t was
calculated using the equation t
= (log
2)/(-slope). The t
for labeled total
hapoA-1 for the high and low fat conditions were not significantly
different (p > 0.05; 117 ± 14 and 128 ± 15
min, respectively). Data represent mean ± S.D. of triplicate
determinations. These experiments were repeated twice, with similar
results.
-actin mRNA was increased approximately 31% as a result of
consuming the HF/HC diet, as shown in Fig. 4C. This
could suggest two sub-pools of hapoA-1 mRNA: (a) one not being
translated (i.e. as an mRNP) and (b) one associated
with polysomes that undergoes normal rates of initiation and
elongation. Dietary fat, though not affecting the total pool size of
hapoA-1 mRNA, as shown below, changes the distribution of the hapoA-1
mRNA between these two sub-pools. Sucrose gradients do not well resolve
mRNPs from monosomes and ribosome subunits; therefore, liver S10
extracts were also fractionated by metrizamide equilibrium gradient
ultracentrifugation, which provides a clearer resolution of
non-ribosomal from ribosomal associated forms of mRNA(9) . As
shown in Fig. 5A, solution hybridization/RNase
protection quantitation of each fraction revealed hapoA-1 mRNA
principally in the non-ribosomal lower density fractions 3 and 4
(densities 1.16 and 1.19 g/ml, respectively) and the ribosomal higher
density fractions 6 and 7 (densities 1.21 and 1.29 g/ml, respectively).
As predicted from earlier experiments (Table 2), the total amount
of hapoA-1 mRNA in all the fractions was not changed by diet. However,
on the HF/HC diet the percentage of hapoA-1 mRNA in the ribosomal
fractions increased to 89%, and the percentage in the non-ribosomal
fractions decreased to 8% compared to 72 and 23%, respectively, on the
LF/LC (Fig. 5A and leftpanel in B). To confirm these observations, the same experiment was
also performed with the previously studied 179 line that synthesizes
hapoA-1 only in the liver. As shown in Fig. 5B (rightpanel), similar results were obtained. In
these experiments, to show that the dietary influence on the
distribution of hapoA-1 mRNA was relatively specific, the distribution
of mouse -actin mRNA was studied, and no diet effect was found
(data not shown). Interestingly, the relative ratio of
polysome-associated hapoA-1 mRNA (control versus HF/HC diets)
was found to be similar using these two different gradient techniques:
80% (12.18/15.23) using the sucrose density sedimentation (line A-14) versus 81% (72/89; line A-14) and 85% (74/87; line 179) using
metrizamide density equilibrium gradient methods. Thus, data from the
metrizamide equilibrium density gradient ultracentrifugation
experiments are consistent with the sucrose density gradient
ultracentrifugation experiments, with both suggesting that a HF/HC diet
increases the translation efficiency of hapoA-1 mRNA by increasing its
proportion in the polysomal pool.
P-labeled
riboprobes specific to hapoA-1 or
-actin mRNA. Autoradiograms were
scanned using laser densitometry. A typical film is shown. C,
the hapoA-1 signals (normalized to -actin) in the
polysome-containing fractions (fractions 4-15) were summed. Data
represent the mean ± S.D. of three different samples, each run
on two gradients.
)
We thank and acknowledge the late Dr. Shlomo Eisenberg
(Sheba Medical Center, Tel Hashomer, Israel) for helpful discussion,
Dr. Xiaoli Chen (Medical College of Pennsylvania) for running the
sucrose gradients, and Sharlene Chen and Kathlene Roberts for technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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