©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Dietary Fat Elevates Hepatic ApoA-I Production by Increasing the Fraction of Apolipoprotein A-I mRNA in the Translating Pool (*)

(Received for publication, March 24, 1995; and in revised form, June 12, 1995)

Neal Azrolan (1)(§) Hiroyuki Odaka (1) Jan L. Breslow (1) Edward A. Fisher (2)

From the  (1)Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, New York 10021 and the (2)Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129

ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Epidemiological studies have shown a strong inverse correlation between high density lipoprotein cholesterol (HDL-C) (^1)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.

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.


MATERIALS and METHODS

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(2)-equilibrated, calcium-free MEM (Life Technologies, Inc.) at 37 °C for 10 min. MEM containing 5 mM CaCl(2) 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 10^6 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(2).

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-^3H]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), 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(2), 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 beta-actin prepared from pTri-beta-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.


RESULTS

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 [^3H]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).





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 [^3H]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.



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 [^3H]leucine (200 µCi/ml) and then washed and incubated with MEM containing excess (400 mM) cold leucine. Incorporation of [^3H]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.



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 beta-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 beta-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.


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 P-labeled riboprobes specific to hapoA-1 or beta-actin mRNA. Autoradiograms were scanned using laser densitometry. A typical film is shown. C, the hapoA-1 signals (normalized to beta-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.




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).




DISCUSSION

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL32435 and HL33714 (to J. L. B.) and HL22633 (to E. A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to the memory of Dr. Shlomo Eisenberg.

§
To whom all correspondence should be addressed: Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543-8000. Tel.: 908-274-4304; Fax: 908-274-4129.

(^1)
The abbreviations used are: HDL-C, high density lipoprotein cholesterol; LDL, low density lipoprotein; hapoA-1, human apolipoprotein A-I; mRNP, messenger ribonucleoprotein; BFA, brefeldin A; HF/HC, high fat-high cholesterol; LF/LC, low fat-low cholesterol; MEM, minimum essential medium; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s).


ACKNOWLEDGEMENTS

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.


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