Zinc deficiency decreases plasma level and hepatic mRNA abundance of apolipoprotein A-I in rats and hamsters

John Y. J. Wu, Scott K. Reaves, Yi Ran Wang, Yan Wu, Polin P. Lei, and Kai Y. Lei

Department of Nutritional Sciences, University of Arizona, Tucson, Arizona 85721

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Table 1.   Diet composition

Golden Syrian hamsters (7 wk old) or male Sprague-Dawley rats (8 wk old) were randomly assigned to three experimental treatments: ZD, ZA, and Zn deficient replenished with Zn (ZDA). The ZDA groups of animals were fed the ZD diet throughout the dietary treatment period, except on the last 2 days, when they were fed the ZA diet. Eight animals were included in ZA and ZD treatments (n = 8), and four animals were included in the ZDA treatment (n = 4). The dietary treatment lasted for 18 days for rats and 49 days for hamsters. Animals were housed individually in suspended stainless steel wire cages in a laboratory maintained at 22-24°C with a 12:12-h light-dark cycle. They were provided free access to their respective diets and distilled water. The body weight and daily food intake were measured once a week before the last week and three times a week within the last week of treatment.

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% beta -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, beta -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.

The antisense RPA probes for 18S rRNA, rat beta -actin, and rat cyclophilin, as well as the RNA century size marker, were purchased from Ambion. The antisense probes for rat apoA-I, rat MT-II, hamster MT-II, and hamster cyclophilin were cloned by using RT-PCR, as described above. In addition, hamster apoA-I RPA probe was directly prepared from cloned hamster apoA-I cDNA. The protected size for each probe is as follows: 201 nt for rat and hamster MT-II, 167 nt for rat and hamster apoA-I, 125 nt for rat beta -actin, 103 nt for rat cyclophilin, 113 nt for hamster cyclophilin, and 80 nt for 18S. All primers used in probe preparation are listed below. The MT-II primer pair was used for rat and hamster MT-II probe preparation: 5'-ATGGACCCCAACTGCTCCTGTG-3' (MT-II, forward), 5'-GAGGGGAATCCCACTTCAGGC-3' (MT-II, reverse), 5'-AGGAGTTCTGGGATAACCTGG-3' (hamster apoA-I, forward), 5'-GGCTCCATCTTCTGGCGGTA-3' (hamster apoA-I, reverse), 5'-AGGAGTTCTGGGCTAACCTGGA-3' (rat apoA-I, forward), 5'-GGCTCCAGCTTCTGGCGGTA-3' (rat apoA-I, reverse), 5'-CAGGGTGGTGACTTCACAC-3' (hamster cyclophilin, forward), and 5'-ATGGACAAGATGCCAGGACC-3' (hamster cyclophilin, reverse).

Data analysis. Data were analyzed by one-way ANOVA, and the treatment means were further ranked by Duncan's new multiple range test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Table 2.   Effect of dietary Zn status on body weight, relative liver weight, plasma Zn, and liver mineral levels in rats

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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Fig. 1.   Effect of dietary Zn status on level of plasma high-density-lipoprotein (HDL) apolipoprotein A-I (apoA-I) in rats. Plasma was isolated, and HDL fraction was purified by sequential ultracentrifugation. Apolipoproteins associated with HDL from known amount of plasma were separated by SDS-PAGE. A: representative sample lanes of SDS-PAGE gel. B: amount of apoA-I quantitated by laser densitometry using apoA-I standard. Values are means ± SE from 8 animals for Zn-adequate (ZA) or Zn-deficient (ZD) group and 4 animals for Zn-replenished (ZDA) group. Means with different letters (a-c) are significantly different (P < 0.05 by 1-way ANOVA).

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 beta -actin or cyclophilin was used as the internal reference. However, the expression levels of beta -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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Fig. 2.   Effect of dietary Zn status on hepatic total cellular apoA-I mRNA abundance in rats. Total cellular RNA was isolated from liver. ApoA-I mRNA abundance was determined by RNase protection assay, with 18S as reference. A: representative sample lanes. B: autoradiographic signals quantitated by laser densitometer. MT, metallothionein. AU, arbitrary units. Values are means ± SE from 8 animals for ZA or ZD group and 4 animals for ZDA group. Means with different letters (a-c) are significantly different (P < 0.05 by 1-way ANOVA).


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Fig. 3.   Effect of dietary Zn status on intestinal total cellular apoA-I mRNA abundance in rats. Total cellular RNA was isolated from entire small intestine. ApoA-I mRNA abundance was determined by RNase protection assay, with 18S as reference. Values are means ± SE from 8 animals for ZA or ZD group and 4 animals for ZDA group. No treatment difference was detected.

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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Table 3.   Effect of dietary Zn status on average weekly body weight gain, relative liver weight, plasma Zn, and liver mineral levels in hamsters

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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Fig. 4.   Effect of dietary Zn status on level of plasma HDL apoA-I in hamsters. Plasma was isolated, and HDL fraction was purified by sequential ultracentrifugation. Apolipoproteins associated with HDL from known amount of plasma were separated by SDS-PAGE. Amount of apoA-I was quantitated by laser densitometry, with apoA-I as standard. Values are means ± SE from 8 animals for ZA or ZD group and 4 animals for ZDA group. Means with different letters (a and b) are significantly different (P < 0.05 by 1-way ANOVA).

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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Fig. 5.   Complete cDNA and deduced amino acid sequence of hamster apoA-I and alignment with other mammalian apoA-I sequences. <UNL>ATG</UNL> and <UNL>TGA</UNL> (*) refer to start and stop codons, respectively. For amino acid sequences from other mammals, only residues that are different from hamster sequence are shown.

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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Fig. 6.   Effect of dietary Zn status on hepatic and intestinal total cellular apoA-I mRNA abundance in hamsters. Total cellular RNA was isolated from liver (A) and small intestine (B). ApoA-I mRNA abundance was determined by RNase protection assay, with cyclophilin as reference. Values are means ± SE from 8 animals for ZA or ZD group and 4 animals for ZDA group. Means with different letters (a and b) are significantly different (P < 0.05 by 1-way ANOVA).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 CTC<UNL>TGCRCNC</UNL>GGCC (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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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