Department of Nutritional Sciences, University of Arizona, Tucson, Arizona 85721
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ABSTRACT |
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The influence of Zn deficiency on the plasma level as well as the hepatic and intestinal gene expression of apolipoprotein (apo) A-I was examined in rats and hamsters. Male Sprague-Dawley rats (8 wk old) and Golden Syrian hamsters (7 wk old) were assigned to three dietary treatments: Zn adequate (ZA, 30 mg Zn/kg diet), Zn deficient (ZD, <0.5 mg Zn/kg diet), and Zn replete (ZDA, ZD animals fed the ZA diet for the last 2 days). The dietary treatments lasted for 18 days for rats or 6 wk for hamsters. For the measurement of apoA-I mRNA abundance, hamster apoA-I cDNA was cloned from the small intestine. The full-length 905-base pair cDNA shared ~80% similarity with the human, rat, and mouse apoA-I cDNAs. Hepatic and plasma Zn levels were reduced in ZD animals but normalized in ZDA rats and increased in ZDA hamsters compared with ZA animals. Zn deficiency reduced plasma apoA-I and hepatic apoA-I mRNA levels 13 and 38%, respectively, in ZD rats. The 2 days of Zn replenishment raised plasma apoA-I and hepatic apoA-I mRNA levels in ZDA rats by 34 and 28%, respectively, higher than ZA rats. Similarly, these levels were decreased by 18 and 25%, respectively, in ZD hamsters but normalized in ZDA hamsters compared with ZA hamsters. In contrast to the alterations of hepatic apoA-I mRNA levels, neither Zn deficiency nor subsequent Zn repletion produced alterations in the intestinal apoA-I mRNA abundance. Data from this study demonstrated that Zn deficiency specifically decreases hepatic apoA-I gene expression, which may at least be partly responsible for the reduction of plasma apoA-I levels.
atherosclerosis; cholesterol; cardiovascular disease
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INTRODUCTION |
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MARGINAL Zn deficiency may be a common nutritional problem in the United States (22) and may contribute to the development of atherosclerosis (18). Marked reductions in Zn levels in the plasma and aorta have been observed in atherosclerotic patients (32). In addition, long-term clinical studies have established a possible beneficial effect of Zn supplementation to atherosclerotic patients (13). Moreover, the ability of Zn supplementation to prevent atheromatous changes of the aorta normally induced by cholesterol feeding was also demonstrated in rabbits (5). Furthermore, a dyslipidemic lipoprotein profile, mainly in the reduction of high-density-lipoprotein (HDL) cholesterol, has been documented by numerous studies in Zn-deficient animals. When the HDL fraction was examined by compositional and chromatographic analyses, Zn deficiency significantly reduced the total amount of plasma HDL particles, with no influence on the percent composition of total protein, triglycerides, phospholipids, and cholesterol (15). The reduction in the HDL cholesterol was mainly due to a marked decrease in apolipoprotein (apo) E-free HDL, the major subclass of the HDL fraction (16). No alteration in very-low-density-lipoprotein (VLDL) and low-density-lipoprotein (LDL) cholesterol levels was produced by Zn depletion.
In view of the association of low plasma HDL cholesterol and apoA-I levels with increased risk of cardiovascular disease (CVD), the changes in lipoprotein metabolism induced by Zn deficiency may in part contribute to the development of CVD. The present study was designed to establish the influence of dietary Zn status on the plasma apoA-I level and hepatic apoA-I mRNA abundance in rats and hamsters. In the past, the effects of Zn deficiency on the lipoprotein metabolism were investigated mostly in rats, although the metabolism in rats may not fully represent that in humans. In hamsters the lipoprotein metabolism, however, is closer to that in humans. Compared with those of the rat and guinea pig, the rate of hepatic cholesterol synthesis of the hamster resembles more closely that of the human (28). As in humans, a significant proportion of plasma cholesterol is transported in the form of LDL in the hamster. In addition, similar responses to atherogenic diets are observed in both species, indicating that similar regulatory mechanisms are involved (29). Moreover, the development of atherosclerotic lesions in the hamster is similar to that in the early stage of the human disease (19). Furthermore, neither apoB-48 production (4) nor the apoB mRNA editing can be detected in hamster and human livers (unpublished observation). These features make the hamster a potentially useful rodent model for studying the influence of Zn status on lipoprotein metabolism. To compare the effects of Zn deficiency with previous findings in rats, as well as to more closely relate the new findings in animals to humans, rats and hamsters were used as in vivo animal models in this study. We would like to measure the hamster apoA-I mRNA abundance by using RNase protection assay (RPA); however, the cDNA sequence for hamster apoA-I was not available in all databases. Thus we have embarked on cloning the hamster apoA-I cDNA to provide a probe. We first cloned the central portion of hamster apoA-I cDNA by using the RT-coupled PCR (RT-PCR) method, based on the sequence homology between the human, rat, and mouse apoA-I cDNAs. The 5' and 3' ends of the hamster cDNA were further cloned by using the method of rapid amplification of cDNA end (RACE). The full-length hamster apoA-I1 shared ~80 and 85% similarity to apoA-I from known species at the cDNA and protein levels, respectively. The availability of hamster apoA-I cDNA will enhance the use of the hamster as a suitable model for lipoprotein research.
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MATERIALS AND METHODS |
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Animals and diets. The basal diet was custom formulated by Dyets (Bethlehem, PA) according to the specification of the AIN-93M rodent diet (25), except no Zn supplement was included in the mineral mix (Table 1). This basal diet contained <0.5 mg Zn/kg diet and was used as the Zn-deficient (ZD) diet. The Zn-adequate (ZA) diet was also formulated by the same supplier, with the addition of ZnCO3 (30 mg Zn/kg diet) to the ZD diet. The Zn contents in various diets were measured by atomic absorption spectrophotometry before they were fed.
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Plasma HDL isolation and apoA-I quantitation.
At the end of dietary treatments the diets were removed at 1700, and
the animals were fasted overnight. Each animal was placed under
anesthesia with diethyl ether, and the blood was withdrawn by heart
puncture into a syringe containing 10 mg of EDTA (~1 mg/ml). The
plasma was isolated by centrifugation at 1,000 g for 20 min at 4°C. Different
lipoprotein fractions were isolated by a method described by Radding
and Steinberg (23) with some modifications. The plasma sample was mixed
with KBr solution to achieve a density of 1.063, and then 5 ml of
overlaying buffer with the same density were carefully laid over the
top. The sample was centrifuged at 171,000 g for 18 h at 15°C, and then the
top 3 ml were removed by aspiration. This fraction contained all VLDL
and LDL particles. Another 2 ml of solution were also carefully removed
from the top. An equal volume of HDL density-adjusting buffer was added to the remaining sample to obtain a final density of 1.215. Five milliliters of HDL overlaying buffer (density = 1.215) were carefully laid over the top of this mixture, and then the mixture was centrifuged at 171,000 g for 24 h at 15°C.
Again, the top 3 ml were removed by aspiration. This fraction contained
HDL particles, and it was concentrated in an ultrafiltration membrane
cone (model CF25, Amicon, Danvers, MA) by centrifugation at 1,000 g for 15 min at 4°C. Then this
fraction was further washed twice with PBS to remove KBr from the
solution. This method provided >95% protein retention and was found
to be superior to dialysis. The HDL fraction purified from 2 ml of
plasma was reconstituted to 2 ml with PBS and stored at 4°C for
<3 days before further analyses. SDS-PAGE was used to further
separate apolipoproteins associated with the HDL fraction. Twenty
microliters of purified HDL solution were incubated with 80 µl of
SDS-PAGE loading buffer (6 M urea, 1% SDS, 0.05 M
Tris · HCl, pH 6.8, 2% -mercaptoethanol, 0.01%
bromphenol blue) for 2 h at 37°C, and the mixture was placed in a
boiling water bath for 5 min and electrophoresed on 7.5-20%
SDS-polyacrylamide gels. After electrophoresis, gels were stained with
0.25% Coomassie brilliant blue R-250, destained, and scanned with a
laser densitometer (Molecular Dynamics, Sunnyville, CA). Human apoA-I
protein (Calbiochem, La Jolla, CA) was loaded in several concentrations
on each gel to provide a calibration curve for apoA-I quantitation. The
apoA-I band was identified by comparison to the migration of the apoA-I standard and quantified by use of the standard curve generated from
apoA-I standards. Absorbance of an individual band for apoA-I was
calculated after subtracting the background absorbance. A linear
relationship between the amounts of apoA-I standard applied to the gel
and the absorbance determined by this procedure has been previously established.
Measurement of mineral concentrations. The mineral concentrations were measured by flame atomic absorption spectrophotometry. The plasma derived from each animal was diluted threefold with distilled-deionized water. For the liver samples, ~0.5 g of wet liver was placed in an acid-washed and preweighed tube and dried in a 70°C oven for 48 h. After the sample was weighed again to obtain the dry weight, 3 ml of concentrated nitric acid were added. The sample was cold digested overnight on ice, then hot digested for 3 h by incubation in boiling water. The digested sample was directly used for the measurement of Cu concentration or diluted 10-fold with distilled-deionized water for the measurement of Zn and Fe levels. Known amounts of bovine liver standard reference (US Department of Commerce, National Institute of Standards, Gaithersburg, MD) were also analyzed, and resultant mineral concentrations were within the accuracy ranges as specified for the standard. The concentration of Cu was measured in rat or hamster liver samples, whereas the Fe level was measured only in the diluted rat liver samples. A series of mineral standard solutions (0.05-1.0 ppm for Zn and Cu and 0.05-5.0 ppm for Fe) were used to generate a linear standard curve for each mineral. Appropriate blanks were employed for all measurements.
Cloning of the full-length hamster apoA-I cDNA. The apoA-I mRNA sequences for the human, rat, and mouse were obtained from the GenBank database and analyzed. A pair of primer Fa and Ra were designed on the basis of the 5' and 3' conserved regions, which spanned the entire open reading frame. Five hundred nanograms of DNase I-digested hamster intestinal RNA were mixed with 50 pmol of Ra and 4 µl of 5× first-strand synthesis buffer, 1 µl of 10 mM dNTP mixture, and 2 µl of 100 mM dithiothreitol, and diethyl pyrocarbonate-water was added to adjust the final volume to 19 µl. The mixture was incubated at 70°C for 10 min and then at 37°C for 10 min. One microliter of Moloney's murine leukemia virus RT (Life Technologies, Grand Island, NY) was added. The RT reaction was carried out at 42°C for 60 min and stopped by incubation at 99°C for 5 min. To the 20 µl of the RT reaction mixture, 10 µl of 10× PCR buffer, 10 µl of 25 mM MgCl2, 50 pmol of the primer Fa, 1 µl of 10 mM dNTP mixture, and 1 µl of Taq DNA polymerase (Promega, Madison, WI) were added, and distilled water was added to provide a total volume of 100 µl. After 3 min at 95°C, PCR was performed for 30 cycles in a Perkin-Elmer thermocycler as follows: 30 s at 95°C, 1 min at 42°C, and 2 min at 72°C, with a 10-min final extension at 72°C. The PCR product was analyzed by agarose electrophoresis, purified, digested by BamH I and Kpn I, and cloned into pKS vector. Correct clones were screened and verified by sequencing analysis.
To obtain the full-length cDNA, the 5' and 3' ends were further cloned by the RACE method. In 5' RACE the RT reaction was performed as described above with the primer Rb. After RT the reaction mixture was purified to remove excessive dNTP and primer Rb, and the poly(A) tail was added to the first-strand cDNA by incubating the purified RT reaction mixture with 0.1 mM dATP and 3 µl of terminal deoxynucleotidyl transferase (Promega, Madison, WI) at 37°C for 30 min. The PCR was then performed with primers Rb and P1 for 40 cycles of 30 s at 94°C, 2 min at 42°C, and 2 min at 72°C, with a final extension for 10 min at 72°C. In 3' RACE the RT-PCR was performed with the primers P1 and Fb under the same condition as that for the 5' RACE. Both RACE products were directly cloned into the PCR2.1 TA cloning vector (Invitrogen) by following the manufacturer's protocol. The correct clones were selected and confirmed by sequencing analysis: 5'-cggaattcTTTTTTTTTTTTTTTTTT-3' (P1), 5'-cgggatCCTTCAGGATGAAAGCTGC-3' (Fa; human apoA-I cDNA, nt 79-97), 5'-ggggtaCCACTTTGGAAACGTTTATT-3' (Ra; human apoA-I cDNA, nt 925-944), 5'-TTGGTCGCCTGCAGGAACAG-3' (Fb; hamster apoA-I cDNA, nt 288-307), and 5'-CACGAAGATCCTCGCCCAGA-3' (Rb; hamster apoA-I cDNA, nt 566-547).Isolation of total cellular RNA and determination of apoA-I mRNA
abundance.
Total cellular RNA was isolated by using the TRIzol reagent (Life
Technologies). To isolate liver RNA, 200 mg of fresh liver sample were
homogenized in TRIzol. To isolate the intestinal RNA, the small
intestine was excised from each animal and immediately rinsed with
ice-cold diethyl pyrocarbonate-treated PBS. Then the intestine was cut
into four pieces and opened, and a disposable cell scraper was used to
scrape out the mucosal layer. The resulting intestinal mucosa from the
same animal was pooled and immediately homogenized in TRIzol. Then
tissue RNA was isolated according to the manufacturer's protocol.
Suitable amounts of the RNase inhibitor RNasin (Promega) were added to
each RNA sample. Aliquots of each sample were stored at 80°C
until further analysis. The hepatic and intestinal apoA-I mRNA
abundances were further analyzed by RPA by using the RPA-II kit
(Ambion, Austin, TX) and following the manufacturer's protocol. For
the RNA derived from rat liver and intestine, rat metallothionein
(MT)-II, apoA-I,
-actin, cyclophilin, and 18S rRNA probes were used
in the same RPA reaction. In contrast, hamster MT-II, apoA-I,
cyclophilin, and 18S rRNA probes were used for the hamster RNA samples.
All RNA probes and the RNA century size marker were labeled at
predetermined specific activities by using the MAXIscript in vitro
transcription kit (Ambion), so that the band intensities in the final
RPA gel were comparable. Band intensities of autoradiographs were
quantified by a laser densitometer (Molecular Dynamics). The relative
mRNA abundance in the samples was expressed as the arbitrary units of
the apoA-I band per arbitrary unit of the internal reference in the
same RPA reaction.
Data analysis. Data were analyzed by one-way ANOVA, and the treatment means were further ranked by Duncan's new multiple range test.
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RESULTS |
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A significant reduction in body weight and a small but significant increase in relative liver weight were observed in the ZD rats compared with the ZA rats (Table 2). The 2-day repletion in the ZDA rats did not improve body weight; however, the relative liver weight was normalized to that of the ZA rats. The average daily food intake of the ZD rats was significantly lower than that of the ZA rats (data not shown), suggesting that the observed reduction in weight gain was related to the reduction in food intake. A 21% reduction in the plasma Zn level and a 15% reduction in the hepatic Zn concentration were observed in the ZD rats compared with the ZA rats (Table 2). In the ZDA rats, plasma and hepatic Zn levels were restored to the levels observed in the ZA rats, suggesting that the 2-day repletion was sufficient to normalize Zn homeostasis in these tissues. No difference was found in the hepatic Cu concentration in all three groups. However, hepatic Fe was significantly higher in ZD than in ZA rats, and 2-day repletion failed to normalize this elevated hepatic Fe level.
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MT-II mRNA abundance was also measured in rat hepatic and intestinal samples by RPA to serve as another index for cellular Zn status and to provide a positive control for Zn-regulated gene expression. Hepatic MT-II mRNA abundance was reduced by 32% in ZD rats compared with ZA rats, whereas Zn replenishment restored it to the control level (Table 2). However, the intestinal expression of MT-II mRNA in rats was unexpectedly low, regardless of the treatment group. Although we could not accurately estimate the relative intestinal MT-II mRNA abundance, even after prolonged exposure of the autoradiographs (data not shown), changes showed similar trends among the treatment groups compared with hepatic samples.
Plasma HDL apoA-I was different among the three groups of rats. Representative sample lanes of SDS-PAGE for all three dietary groups are shown in Fig. 1A. The intensity of the apoA-I band was lower for ZD than for ZA rats, whereas a higher band intensity of apoA-I was observed for ZDA than for ZA rats. ZD rats demonstrated a reduction in plasma HDL apoA-I level (87% of the ZA rats), whereas the Zn repletion resulted in a higher level (134% of the ZA rats) of HDL apoA-I (Fig. 1B). An aliquot of the pre-HDL fraction representing the VLDL plus the LDL fraction and an aliquot of the remaining fraction with a density of 1.21 after removal of the HDL fraction, obtained from the same sequential ultracentrifugation, were also analyzed by SDS-PAGE. No visible band at the position of apoA-I was detected in the pre- or post-HDL fractions (data not shown), indicating that the apoA-I quantitated in the HDL fraction represented the majority of the plasma apoA-I pool.
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The reduction in the plasma apoA-I may be due to a decrease in the
apoA-I synthesis by the small intestine and liver, which are the major
sites of apoA-I production. Thus the intestinal and hepatic mRNA
abundances of apoA-I were measured by RPA to provide estimates of the
capacities of these tissues to produce apoA-I. Representative hepatic
sample lanes are shown in Fig. 2A, and
quantitated values of hepatic apoA-I mRNA abundance (after normalization with 18S reference) are depicted in Fig.
2B. The hepatic apoA-I mRNA level was
reduced by 38% in the ZD rats but elevated by 28% in the ZDA rats
compared with the ZA rats. In contrast to the alterations of hepatic
apoA-I mRNA levels, neither Zn deficiency nor subsequent Zn repletion
produced any alteration in the intestinal apoA-I mRNA abundance (Fig.
3). Similar results were also obtained when
-actin or cyclophilin was used as the internal reference. However,
the expression levels of
-actin and cyclophilin were quite low in
the rat intestine, whereas those for 18S were constant and comparable
in both tissues. Thus 18S was used as the reference in our
determination of relative apoA-I mRNA levels between rat liver and
intestine.
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The hamster has been considered an appropriate rodent model for lipoprotein research, because its lipoprotein profile is closer to that of humans. Zn deficiency was also induced in hamsters by feeding the ZD diet. During the 7 wk of treatment the average weekly body weight gain was significantly reduced in hamsters fed the ZD diet (ZD and ZDA groups; Table 3). The 2-day repletion did not change the body weight gain (data not shown). Food intakes in the last week of dietary treatment were not significantly different among all three groups, and the food intake for the ZDA group was not increased during the 2-day repletion (data not shown). In contrast to rats, no significant difference was detected in the relative liver weight (Table 3). Plasma and hepatic Zn concentrations in the ZD hamsters were reduced to 76 and 87%, respectively, of the values for the ZA hamsters. Plasma and hepatic Zn concentrations were higher in the ZDA than in the ZA hamsters. Similar to the findings observed in rats, the hepatic Cu concentration was not affected by the dietary Zn status in hamsters, whereas the Fe concentration was not measured.
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Moreover, hepatic and intestinal MT-II mRNA levels in the ZD hamsters were markedly reduced by 98 and 71%, respectively, compared with those in the ZA hamsters (Table 3). Furthermore, normalized hepatic and elevated (259% of ZA) intestinal MT-II mRNA levels were observed in ZDA compared with ZA hamsters (Table 3).
In hamsters, plasma levels of HDL apoA-I were affected by the dietary Zn status in a manner similar to that observed in rats. The plasma HDL apoA-I concentration was reduced by 18% in ZD hamsters compared with ZA hamsters, whereas the subsequent repletion with the ZA diet completely restored the plasma apoA-I level to the normal value (Fig. 4). No apoA-I was detected in the post-HDL plasma fraction, whereas a very small amount (<1%, compared with that in the HDL fraction) of apoA-I was detected in the pre-HDL fraction (data not shown). Nevertheless, the amount of apoA-I quantitated in the HDL fraction represented the majority of apoA-I in the plasma pool.
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The influence of dietary Zn status on the hepatic and intestinal apoA-I mRNA abundances was also examined in hamsters. To measure the mRNA abundance by RPA, hamster apoA-I cDNA has to be cloned. On the basis of the sequence homology between known species, we have cloned the entire coding region of hamster apoA-I cDNA, which was successfully used as the probe for Northern blot analysis and RPA. Similar to what was found for other species, the apoA-I gene was expressed only in hamster liver and small intestine, as revealed by Northern blot analysis (data not shown). Subsequently, we also cloned the 5' and 3' ends of the cDNA. The primer extension assay demonstrated that liver and small intestine utilized the same transcription start site, which was the first nucleotide of the 5' RACE product (data not shown). The full-length 905-bp cDNA (Fig. 5) shares ~80.2, 82.6, and 79.8% similarity with human, mouse, and rat apoA-I cDNA, respectively. This sequence contains a single long open reading frame, which encodes a protein with 264 amino acid residues, which shares 85.6% similarity and 73.9% identity with the human apoA-I, 85.2% similarity and 70.5% identity with the mouse apoA-I, and 85.3% similarity and 72.6% identity with the rat apoA-I, respectively (Fig. 5). Identical to the mouse and rat apoA-I, the Phe27, Gly209, and Gly210 residues of the human apoA-I are skipped in the hamster apoA-I.
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Similar to findings observed in rats, the hepatic apoA-I mRNA relative abundance in the ZD hamsters was reduced to 75% of that of the ZA hamsters (Fig. 6A). The subsequent repletion with the ZA diet restored this value to the normal level observed in ZA hamsters (Fig. 6A). Unlike the data observed in rats, such repletion did not induce a higher hepatic apoA-I mRNA level than that of ZA hamsters. Also similar to findings observed in rats, the intestinal apoA-I mRNA abundance was not different among all three dietary treatments (Fig. 6B). Because the expression levels of cyclophilin were found to be similar in hamster liver and intestine, cyclophilin was selected as the internal reference here.
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DISCUSSION |
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In this study, marked reductions in plasma total HDL apoA-I levels were observed in ZD rats and hamsters. These results confirmed the previous findings of reductions in apoA-I protein level of the plasma apoE-free HDL subfraction in ZD rats (16). Lipoprotein profiles of rats and hamsters are known to be different. The rat has a higher level of HDL than LDL, whereas the HDL-to-LDL ratio in the hamster is lower and is closer to that of humans. The different profiles may suggest that the overall lipoprotein metabolism in these two species may vary. However, animals from both species exhibited lower HDL apoA-I levels in response to Zn deficiency, indicating that Zn deficiency may influence apoA-I metabolism and gene expression via a common mechanism.
Zn deficiency is known to reduce appetite, food intake, and growth in animals, as also observed in the present study. Thus the observed changes in the plasma apoA-I levels may not be solely due to the Zn depletion and may be partially related to the reductions in body weight and food consumption. Pair feeding is a common technique used to address reductions in food intake and body weight gain. However, the pair-fed animal is continuously under the stress of semistarvation, and the whole body metabolism may be altered compared with that of the ad libitum-fed animal. Therefore, the restriction of food intake may not correctly represent the reduction in food intake associated with Zn deficiency. Instead of using pair-fed animals, a group of ZD animals that were subsequently replenished with the ZA diet was included in the present study. During the repletion period, neither the body weight nor the daily food intake was increased in the replenished animals (data not shown) compared with the same animals before repletion, as well as unreplenished ZD animals. However, the plasma apoA-I level was rapidly restored to the same level as that of ZA hamsters or increased above the level of ZA rats after only 2 days of repletion. These observations indicated that the alterations in plasma apoA-I levels appeared to be specifically related to the dietary Zn status. Similarly, the pair-feeding approach used by Koo and Lee (16) also demonstrated that the reduction in food intake per se had no effect on the plasma apoA-I level in apoE-free HDL.
Liver and small intestine are the major sites for apoA-I production. A decrease in the apoA-I production by both organs and/or an increase in the clearance of plasma apoA-I may contribute to the decrease in plasma apoA-I in Zn deficiency. In the case of Cu deficiency in rats, the increase in plasma apoA-I level was mainly due to an increased hepatic output, rather than a decreased clearance (6). Thus it was decided to study the regulation of apoA-I production. Zn deficiency depressed the hepatic apoA-I mRNA abundance (Figs. 2 and 6A) without affecting the intestinal apoA-I mRNA abundance (Figs. 3 and 6B) in rats and hamsters. The short-term repletion completely reversed the effect of Zn deficiency on the hepatic apoA-I mRNA level for both species (Figs. 2 and 6A), and an overcompensatory effect was observed in the replenished rat liver (Fig. 2).
Although the small intestine and liver are actively involved in apoA-I
production, the intestinal regulatory mechanisms appeared to be less
responsive to dietary and hormonal changes. Similar to the observations
of this study, the administration of fibrate to rats resulted in
decreases in plasma and hepatic apoA-I mRNA levels, but the intestinal
apoA-I mRNA abundance remained constant (30). In addition, thyroid
hormone (triiodothyronine) administration has been reported to increase
the hepatic apoA-I mRNA abundance (3); however, triiodothyronine
appeared not to alter the intestinal apoA-I mRNA level (3, 11).
Furthermore, diets rich in polyunsaturated fatty acids were able to
lower the plasma apoA-I and reduce the hepatic but not the intestinal
apoA-I mRNA levels in African green monkeys (27). These studies
indicated that the apoA-I gene expression was regulated in a
tissue-specific manner. Recently, the human apoA-I promoter has been
cloned (26). In Hep G2 cells, a human hepatoblastoma cell line, the
proximal apoA-I promoter (starting at 256 bp) was
found to be sufficient for the optimal promoter activity (26). In
contrast, in Caco-2 cells, a human intestinal cell line, a larger
promoter region extending further upstream was found to be essential
for the optimal promoter activity (26). Thus the utilization of
different promoter regions by the small intestine and liver for their
tissue-specific expression of the apoA-I gene may provide a molecular
mechanism for the differential regulations observed in the present and
previous studies.
Recently, the regulation of gene expression by metals has received
considerable attention. The model most extensively studied is MT.
Expressions of MT are induced primarily by Zn and, to a lesser extent,
by various other metals. Such inductions are mediated by the presence
of multiple copies of metal-responsive elements (MREs) in their
promoters (12). These MRE sequences are small imperfect motifs, with a
consensus MRE sequence of CGGCC (core sequence is underlined) in either orientation. Several nuclear factors are able to bind to MREs, and such bindings are enhanced by
metal induction (2). One of these factors (MTF-1) was recently cloned
and characterized as a transcription activator (21, 24). The increase
in cellular Zn level appears to release an unidentified inhibitor from
the MTF-1-inhibitor complex and allow the binding of MTF-1 to MREs
(21). In addition, the influx of dietary Zn into cell nuclei has been
found to be influenced by dietary Zn status (9). Thus dietary Zn status
is capable of altering the expression of the MT genes, as demonstrated
in the present study (Tables 2 and 3), via the MRE sequences in their promoters.
Metal-responsive transcription is common among eukaryotes. The MRE
mechanism may be a common mechanism for these regulations. Recently, a
number of potential candidate genes responsive to metal regulation have
been reviewed (8, 31). The initial search of the rat apoA-I promoter
revealed a DNA sequence with high homology to the MRE consensus
sequence (8). In addition, our laboratory has searched for the MRE-like
sequences in the human apoA-I promoter with sequences up to
2,500 bp and identified seven potential MRE-like
sequences (unpublished data). Most of these MREs were located within
the proximal promoter (starting from
256 bp), which may be more
important for the hepatic expression of apoA-I. These findings
suggested that the expression of the apoA-I gene may be regulated by
cellular Zn status via the MRE-mediated mechanism, and the small
intestine may be less sensitive, since fewer MREs are located further
upstream in the apoA-I promoter. Future studies are required to test
such hypotheses.
On the basis of the hypothesis that the regulation of apoA-I gene expression can be mediated by MRE, an elevated hepatic Zn level should be able to induce a higher level of apoA-I mRNA and, possibly, a higher level of plasma apoA-I and HDL. Such a perspective may not always be true. On the one hand, the supplementation of Zn to Zn-deficient rats, hamsters, or Hep G2 cells did evoke a higher level of apoA-I mRNA in this study. Several earlier reports also indirectly agreed with these findings. Abnormally low levels of Zn were found in the plasma or serum as well as the aortas of patients with atherosclerosis (32). Long-term clinical studies demonstrated the possible beneficial effect of Zn supplementation in such patients (13). In addition, supplementation with Zn prevented atheromatous changes of the aorta and elevated plasma HDL in cholesterol-fed rabbits (5). On the other hand, excessive supplementation of Zn appeared not to induce apoA-I gene expression to any higher level. In healthy humans, excessive intakes of Zn actually decreased the plasma HDL levels (7), whereas the relatively lower doses of Zn supplementation did not alter the HDL levels (10).
Recently, the influence of Cu deficiency on lipoprotein metabolism has been intensively studied by Lei and associates as well as by other investigators. In contrast to Zn deficiency, Cu deficiency resulted in hypercholesterolemia (17). Cu deficiency selectively increases the plasma apoA-I levels (17) as well as hepatic apoA-I production and secretion (14). Similarly, the depletion of Cu from Hep G2 cells resulted in increases in the synthesis of apoA-I (34) and gene transcription (33). So far, the studies that addressed the influence of Zn deficiency on apoA-I gene expression have yielded results exactly opposite to those observed in Cu-depletion studies. In addition, the hepatic Zn concentration was significantly elevated in Cu-deficient rats (1), although the hepatic Cu concentration was not altered by Zn deficiency in the present study. These observations suggested that the regulations of apoA-I gene expression by dietary Zn and Cu, and perhaps by other minerals, may share a common mechanism. One potential candidate is the MRE-mediated regulatory system. Future studies will be designed to establish the contribution of the MRE, within the apoA-I promoter, in the regulation of apoA-I gene expression.
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ACKNOWLEDGEMENTS |
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This work was partially supported by US Department of Agriculture National Research Initiative Competitive Grant 96-35200-3248 and funds from the University of Arizona Agricultural Experiment Station.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1 The cDNA and deduced protein sequences have been submitted to GenBank (accession no. AF046919).
Address for reprint requests: K. Y. Lei, Dept. of Nutritional Sciences, 309 Shantz Bldg., University of Arizona, Tucson, AZ 85721.
Received 5 February 1998; accepted in final form 27 August 1998.
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