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Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85721
3To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • rats • dietary zinc • apolipoprotein B • apobec-1 • apolipoprotein B mRNA editing
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Powell et al. 1987
). Editing results in
changing the first base of the codon CAA, encoding glutamine 2153, to
UAA, an in-frame translational stop codon. Consequently, apoB-48 is
approximately half the size of apoB-100 and is identical to the
amino-terminal half of apoB-100. Because the apoB-48 protein does
not contain the carboxy-terminal half of the apoB-100, which is
recognized by the LDL receptor, apoB-48 is not taken up by LDL
receptor–mediated endocytosis. Instead, lipoproteins containing
apoB-48 are cleared more quickly than those containing apoB-100 by a
distinct receptor-mediated pathway involving apolipoprotein E.
The editing reaction is an intranuclear event (Lau et al. 1991
), catalyzed by an editing complex or "editosome"
(Harris et al. 1993
) consisting of a 27-kDa protein
termed apoB mRNA editing catalytic
subunit 1 (apobec-1) and other complementary protein factors
(Lau et al. 1990
, Navaratnam et al. 1993b
). Assembly of the editing complex is directed by an
11-nucleotide "mooring" sequence, beginning five nucleotides
downstream of the editing site in the apoB mRNA (Shah et al. 1991
, Smith et al. 1991
). Although apobec-1
represents the catalytic component of the reaction, several studies
have shown that both apobec-1 and the complementary factors must be
present for editing to occur (Anant et al. 1995
,
Teng et al. 1993
). Apobec-1 is an RNA-specific
cytidine deaminase that exists as a homodimer (Lau et al. 1994
) and has sequence homology with other cytidine deaminase
family members (Navaratnam et al. 1995
). Homology is
highly conserved within the zinc-coordinating region, and in vitro
studies have shown apoB mRNA editing to be zinc dependent
(Barnes and Smith 1993
).
Although apoB metabolism and plasma cholesterol have been linked to the
development of atherosclerosis, zinc may also play a role; however, the
cause-and-effect relationship has not been established. In
atherosclerotic patients, the abnormally low levels of zinc in the
plasma or serum (Halstead and Smith 1970
, Netsky et al. 1969
) as well as in the aorta (Volkov 1963
) indicate that zinc metabolism has been altered. Zinc
deficiency can affect the level of plasma lipoproteins, particularly
those containing apolipoprotein B. In zinc-deficient rats, the HDL
cholesterol level was decreased (Koo and Williams 1981
), and there was a decrease in apoA-I and apoC
of HDL (Koo and Lee 1988
). Lipid malabsorption and
defective chylomicron formation have also been reported in
zinc-deficient rats (Koo and Turk 1977
). Moreover, a
reduction in the apoB content of chylomicrons was observed in the lymph
of zinc-deficient rats (Koo et al. 1987
).
Because of the adverse changes in lipoprotein metabolism detected in
zinc-deficient rats and the prevalence of marginal zinc deficiency
in certain subpopulations of the U.S. (Sandstead 1995
),
these studies were performed to examine how zinc deficiency affects
specific aspects of apoB metabolism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eight-week-old male Sprague-Dawley rats were randomly assigned to
one of the following three dietary treatment groups: zinc-adequate
(ZA; n = 8), zinc-deficient (ZD;
n = 8), or zinc-replenished (ZDA;
n = 4). The replenished group was fed the ZD diet
until the last 2 d of treatment; then they were fed the ZA diet.
Dietary treatment was for 18 d; rats consumed their respective
diet ad libitum and had free access to distilled demineralized water.
Food was removed from the cages 10 h before the rats were killed.
Each rat was housed separately in stainless steel wire cages with a
room temperature of 22–24°C and a 12-h light:dark
cycle. Body weights and daily food intake were measured once per week
before the last week. During the last week, body weights and food
intake were measured three times. All procedures were approved by the
Animal Care and Use Committee of the University of Arizona. The basal
diet was purchased from Dyets (Bethlehem, PA) and was formulated
according to the AIN-93-M rodent diet (Reeves et al. 1993
) recommendations. The composition of the diet was modified
specifically for the egg white–based diet (Reeves et al. 1993
) and included the omission of zinc from the mineral mix.
The zinc-adequate diet contained 30 mg Zn/kg diet added in the form
of ZnCO3, whereas the zinc-deficient diet contained
<0.5 mg Zn/kg diet as determined by HNO3 digestion and
subsequent flame atomic absorption spectrophotometry (model 180–70,
Hitachi, San Jose, CA).
Isolation of plasma lipoproteins and apolipoprotein quantitation.
On the day of killing, the rats were anesthetized with diethyl ether,
and blood was collected by cardiac puncture using a syringe containing
10 mg EDTA. Plasma was centrifuged at 1000 x g for
20 min at 4°C. Two milliliters of plasma was used for lipoprotein
analysis; the remainder was used to determine plasma zinc levels.
Lipoprotein fractions were isolated by ultracentrifugation
followed by delipidation and quantitation of apoB-100 and apoB-48 as
previously described (Reaves et al. 1996
) with slight
modifications. Briefly, after delipidation, samples were quantitated by
the method of Bradford (1976)
, then electrophoresed on
3.5–15% SDS-polyacrylamide gels. Standard curves were generated
by loading purified apoB onto each gel. Each band on the
Coomassie-stained gels was quantitated by laser densitometry
(Molecular Dynamics, Sunnyvale, CA), and a linear relationship existed
between the amount of apoB loaded onto the gel and the laser
densitometric absorbances.
Quantitation of mineral content in liver and plasma.
The mineral concentrations in liver and plasma were quantitated by flame atomic absorption spectrophotometry. The plasma was diluted with distilled-deionized water before zinc analysis. Liver samples were dried and then digested in concentrated HNO3 before zinc, copper and iron analyses. Copper was measured directly in the digested samples, whereas zinc and iron were measured after samples were diluted 10-fold in distilled-deionized water. Certified reference solutions (Fisher Scientific, Fair Lawn, NJ) for zinc, copper and iron were used to generate linear standard curves for quantitation of each mineral. In addition, bovine Liver Standard Reference (U.S. Department of Commerce, National Institute of Standards, Gaithersburg, MD) was also analyzed to validate experimental findings.
Isolation of total cellular RNA from liver and intestine.
Total cellular RNA was isolated from rat liver and small intestine using TRIzol Reagent (Life Technologies, Grand Island, NY). At the end of the dietary treatment, rats were killed, and each rat liver was immediately perfused with 40 mL of ice-cold diethyl pyrocarbonate (DEPC)-treated PBS through the portal vein. The liver was then quickly removed and 0.2 g tissue was placed in 2 mL TRIzol for homogenization. To isolate intestinal RNA, the small intestine was removed and rinsed with DEPC-treated ice-cold PBS. Mucosal cells were scraped using a rubber cell scraper, collected and transferred to a tube before the addition of 2 mL of TRIzol Reagent. Isolation of RNA was performed as recommended by the manufacturer's protocol.
Ribonuclease protection assays.
Plasmids containing rat apoB and apobec-1 cDNA were kindly provided by
Dr. Teng (Baylor College of Medicine, Houston, TX). A 470-bp region of
apoB cDNA (nucleotides 6512–6982) was inserted into the
SmaI site of pGEM-3Zf (+) vector (Promega, Madison, WI).
The plasmid was used as a template to produce a 307-bp fragment by
polymerase chain reaction (PCR) using the primers M13R
(5'-AGGAAACAGCTATGACCATG-3') and forward primer JW1
(5'-CAGTATCATATCCGTGTAAATCT-3'). Antisense radiolabeled apoB RNA probe
was synthesized using T7 RNA polymerase. The protected fragment was 211
bases in length and corresponded to nucleotides 6771–6982. For
apobec-1, a 423-bp SmaI/KpnI fragment was subcloned into
pBluescript II (KS+) (Stratagene, La Jolla, CA). PCR was performed
using the primers M13R (5'-AGGAAACAGCTATGACCATG-3'), forward primer EP1
(5'-TTCCTGTCCTGGAGTCCCTG-3') and plasmid as template. T3 RNA polymerase
was used to synthesize an antisense radiolabeled apobec-1 RNA probe
that yielded a 258-bp protected fragment during RNase protection.
Before preparing the RNA probes for apoB and apobec-1, sequences were
confirmed by both restriction enzyme analysis and direct DNA
sequencing. For cyclophilin, a rat cyclophilin antisense template was
purchased from Ambion (Austin, TX) and was used as an internal control.
Labeling of RNA probes was performed using a MAXIscript In Vitro
Transcription Kit (Ambion) and (
-32P)-UTP (NEN Life
Science Products, Boston, MA) according to the manufacturer's
protocol. Transcription products were electrophoresed on 5%
polyacrylamide, 8 mol/L urea gels, and full-length transcripts were
localized by autoradiography. Gel slices containing full-length
transcripts were excised and the RNA probe was eluted from each gel
slice. RNase protection assays were performed using the RPA II kit
(Ambion) essentially as described by the manufacturer. Total cellular
RNA (10 µg) was combined with the apobec-1 or apoB
probe, and the cyclophilin probe. RNase protection assay products were
fractionated on 6% polyacrylamide, 8 mol/L urea gels at 300 V for
1.5 h. Gels were dried for 1 h, subjected to autoradiography
at -80°C for several different times to ensure linearity and then
analyzed by laser densitometry (Molecular Dynamics). The size of the
protected fragments was compared with [32P]-labeled RNA
markers (Ambion) and a full-length undigested probe.
ApoB mRNA editing assay.
Reverse transcriptase (RT)-PCR was performed essentially as described
by Giannoni et al. (1994)
. RNA samples used to
measure apoB mRNA editing were digested with DNase I to remove any DNA
contamination. Each digestion reaction mixture contained ~5
µg of RNA, 2 units of DNase I (2
U/µL; Worthington Biochemical, Freehold,
NJ), 10 mmol/L DTT, 50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L
CaCl2, 10 mmol/L MgCl2, and 1
µL RNasin (40 U/µL;
Promega) and was digested at 37°C for 90 min. RNA was then extracted
with phenol/chloroform and precipitated with ethanol. Pellets were
washed with ethanol, resolubilized in DEPC H2O, and RNA
concentrations were determined by measuring absorbance at 260 nm. For
RT-PCR, 200 µmol/L of each dNTP, 10X RT buffer
(Perkin-Elmer, Norwalk, CT), 5 U rTth DNA Polymerase
(Perkin-Elmer), 1 mmol/L MnCl2, and 50 pmol of downstream
primer (5'-AGCATTTTTAGCTTTTCAATGATT-3') were combined with DNase
I-digested RNA (~125 ng). RNA samples were then heated to 70°C
for 7 min; annealing was at 55°C for 5 min, and reverse transcription
was performed at 70°C for 10 min. After reverse transcription, 80
µL of PCR buffer, which contained 6
µL of 25 mmol/L MgCl2, 50 pmol of upstream
primer (5'- TCCTCAGCAGATTCATGATTATCT-3') and 8 µL of
10x chelating buffer (Perkin-Elmer) was added. The reaction was
incubated for 3 min at 95°C followed by 30 cycles of 95°C/30 s,
55°C/1 min and 72°C/1.5 min. For the last cycle, extension was
performed for 10 min. PCR with these rat primers resulted in the
amplification of a 281-bp fragment flanking the editing site and was
verified by sequencing. Oligonucleotides were purchased from NBI
(National Biosciences, Plymouth, MN) and PCR products were purified
with QIAquick PCR purification kit (Qiagen, Valencia, CA) after size
verification by agarose gel electrophoresis. A negative control for
each sample was kept on ice during reverse transcription to check for
DNA contamination; in addition, PCR was performed using Taq DNA
Polymerase (Perkin-Elmer) as another means to test for the presence of
DNA. For primer extension analysis, the annealing primer was 5'-end
labeled with (
-32P)-ATP (Amersham, Piscataway, NJ) and
then purified by a QIAquick Nucleotide Removal Kit (Qiagen). The rat
primer, (5'-AGTCCTGTGCATCATAATTATCTCTAATATACTGA-3') spanning nt
6674 to 6708 of rat apoB mRNA was purchased gel-purified from NBI.
Labeled annealing primer, 2 µL 250 mmol/L NaCl, 2
µL 5x 1st Strand Buffer (Life Technologies) were
combined with 2 µL of diluted purified PCR product.
Tubes were heated at 95°C for 5 min; after annealing at 70°C for 10
min, the mixture was cooled to 37°C over 2 min. Thereafter, 20
U of SuperScript II Reverse Transcriptase (Life
Technologies), 1 µL of 100 mmol/L DTT, 1 µL of 5
mmol/L ddGTP, and 0.5 µL of a 10 mmol/L mixture of
dATP, dCTP and dTTP were added. Extension was performed at 42°C for
10 min. Samples were then placed on ice, and the products were
ethanol-precipitated and separated on 6% polyacrylamide, 8 mol/L
urea gels at 55 W for 2 h. Gels were dried, subjected to
autoradiography and analyzed by laser densitometry (Molecular
Dynamics). Primer extension of the unedited apoB-100 mRNA resulted in a
product of 43 bases in length, whereas the edited apoB-48 mRNA product
was 48 bases in length.
Data analysis.
The data were analyzed using one-way ANOVA and Duncan's new
multiple range test (Jaccard and Becker 1990
). Values
are means ± SEM and differences were considered
significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rats fed the ZD diet had significantly lower body weight and relative
liver weight compared with those fed the ZA diet (Table 1
). In contrast, the body weight of the ZDA rats was not significantly
different from the ZD rats after consumption of the ZA diet for the
last 2 d before killing (Table 1)
. However, the relative liver
weight of the ZDA rats was not different from that of the ZA rats.
Daily food intake of the rats fed a ZD diet was significantly lower
than that of rats fed the ZA diet, suggesting that a reduction in food
intake in rats fed the ZD diet may have contributed in part to the
depressed weight gain (Table 1)
. Furthermore, the 2-d supplementation
with the ZA diet did not increase food consumption of the ZDA compared
with the ZD rats (Table 1)
.
|
). No differences in hepatic copper levels were observed among the
treatment groups. However, hepatic iron levels were significantly
greater in ZD and ZDA rats compared with ZA rats (Table 2)
. Thus, the
plasma and hepatic mineral analyses confirmed that the ZD rats were
indeed deficient in zinc compared with controls.
|
Samples of each of the apoB-100 and apoB-48 were chosen from each
dietary treatment group and are shown in Figure 1A
. Plasma apoB-48, apoB-100 and total apoB levels were not significantly
different among the three dietary treatment groups (Fig. 1
B). A ratio was calculated as the percentage of plasma
apoB-48 to total apoB [apoB-48/(apoB-48 + apoB-100) x 100] to
examine how the subtle differences in apoB-100 and apoB-48
concentrations among groups may affect the overall plasma apoB profile
(Fig. 1
C). The percentage of plasma apoB-48 to total apoB in
the ZD rats was significantly reduced to nearly half the level observed
in the ZA rats. In the ZDA group, zinc replenishment restored the
percentage of plasma apoB-48 to a level comparable to that observed in
the ZA rats (Fig. 1
C).
|
Representative samples of primer extension products from the three
treatments are shown in Figure 2
A. Hepatic editing was significantly lower in ZD than in ZA rats.
However, hepatic editing in the ZDA rats was significantly different
only from the ZD rats (Fig. 2
B). Analysis of intestinal
editing levels was also performed; however, there was no significant
difference in intestinal editing among the ZA, ZD and ZDA groups (data
not shown). Mean value for intestinal editing for the three treatment
groups was 86.9 ± 1.57%, which is similar to previously reported
rat intestinal editing levels.
|
In view of our findings that hepatic apoB mRNA editing was altered,
hepatic apoB and apobec-1 mRNA levels were also measured.
Representative samples of apoB mRNA from the three treatments are shown
in Figure 3
A and illustrate that there was no difference in hepatic apoB mRNA
abundance among the three treatment groups. Analysis of apoB mRNA bands
by laser densitometry, using cyclophilin mRNA as the internal standard,
indicated that there was no difference in apoB mRNA abundance among the
treatment groups (Fig. 3
B). These data support the
observation that total apoB plasma protein levels were not
significantly different among groups. Because the plasma apoB-48 to
total apoB ratio and hepatic apoB mRNA editing data showed differences
among treatment groups, hepatic apobec-1 mRNA abundance was measured.
Bands corresponding to apobec-1 mRNA among the experimental animals
suggested that the highest abundance of apobec-1 mRNA was present in ZA
rats (Fig. 4
A). The hepatic apobec-1 mRNA abundance was significantly higher in ZA
rats compared with ZD or ZDA rats (Fig. 4
B). Apobec-1 mRNA
abundance was reduced by 46 and 37% for the ZD and ZDA rats,
respectively, compared with the ZA rats.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
were also observed in this study. We also included a third group that
was comprised of ZD rats fed the ZA diet for the last 2 d before
killing. This group (ZDA rats) was used as an attempt to evaluate the
specific effect of zinc replenishment. Although plasma and hepatic zinc
returned to control levels after the 2-d replenishment with ZA diet,
the ZDA rats did not consume significantly more food than ZD rats
during that period. Two days may simply not be enough time for intake
to increase to normal levels. Perhaps there is a lag time before
zinc-deficient rats resume normal growth after receiving a
zinc-adequate diet. Nevertheless, 2 d of zinc replenishment
did specifically cause hepatic zinc, the percentage in plasma of
apoB-48 compared with total apoB and hepatic apoB mRNA editing levels
to revert to those observed in ZA rats. In addition, all rats were food
deprived for 10 h before being killed so that the presence or
absence of food in the gastrointestinal tract would not have been a
factor or potential difference among animals. We chose not to use a
pair-fed group because this technique tends to inflict the constant
stress of semistarvation, which may induce metabolic alterations as
well as result in a meal-eating food consumption pattern compared
with animals consuming food ad libitum. Therefore, the restriction in
food intake imposed by pair-feeding may have limitations in
mimicking the reduction in food intake associated with zinc deficiency.
Although it can be argued that both pair-fed and zinc-repletion
controls have certain shortfalls, we believe that zinc replenishment
offered an intriguing option in determining whether the effects were
specific to zinc.
Moreover, in this study, the lack of change in hepatic copper
concentrations among the three dietary treatment groups suggests that
copper-zinc antagonism was not important. In the ZD rats, the
stable hepatic copper level may indicate that the zinc deficiency was
not drastic enough to alter hepatic zinc-copper equilibrium. Thus,
the ZD rats may be only moderately deficient in zinc, which would more
closely resemble the chronic marginally zinc-deficient status often
encountered in certain human populations (Hambidge et al. 1985
). Nevertheless, the hepatic iron concentrations were
markedly higher in ZD rats than in ZA rats and remained elevated for
the ZDA rats. Because iron as well as zinc are transported by
transferrin in the portal circulation (Evans and Winter 1975
), a reduction in dietary zinc may enhance the amount of
iron bound to transferrin, leading to hepatic iron accumulation.
However, normalized hepatic zinc levels in the zinc-repleted group
did not result in a reduction in hepatic iron.
Impaired absorption of dietary lipids (Koo et al. 1986
)
as well as alterations in lymphatic chylomicron apoB content have been
reported in zinc-deficient rats (Koo et al. 1987
).
In contrast, in this study, the plasma total apoB, apoB-100 and apoB-48
levels were not significantly changed by dietary treatment.
Nevertheless, we did observe a markedly reduced plasma apoB-48 to total
apoB ratio in ZD rats. We chose to measure editing because apoB mRNA
editing has been shown to be zinc dependent in vitro (Barnes and Smith 1993
, Navaratnam et al. 1993a
). We
suspected that zinc status may have affected editing in these rats and
that this may be related to the ratio of plasma apoB-48 to total apoB.
Indeed, we first reported that hepatic editing was altered in
zinc-deficient rats using the direct primer extension
method.1 These findings were reconfirmed using the
RT-PCR primer extension method reported in the present studies.
Both assays revealed that the percentage of hepatic apoB mRNA edited
was decreased significantly in the ZD rats. Interestingly, in the ZDA
rats, the percentage of plasma apoB-48 to total apoB as well as hepatic
apoB mRNA editing returned to control levels.
However, because differences in plasma apoB-48 ratios between
zinc-adequate and zinc-deficient rats were relatively large
compared with the modest but significant differences in hepatic
editing, we suspect that other facets of apoB metabolism may also be
affected. Processes such as synthesis, degradation and peripheral
uptake may also be altered by zinc status, but we chose to focus our
efforts on editing for the current studies because other studies have
also demonstrated that dietary factors can influence hepatic apoB mRNA
editing. For example, when rats were food deprived for 24 h, the
proportion of hepatic apoB mRNA editing was decreased to 30–40%. Yet,
when the rats were refed a high carbohydrate diet for 24–48 h, editing
was markedly increased to 80–90% (Baum et al. 1990
).
Similarly, chronic ethanol consumption in rats increased hepatic apoB
mRNA editing to 90–100% (Lau et al. 1995
). Moreover,
hepatic apoB mRNA editing was increased when rats were fed a diet
deficient in copper (Reaves et al. 1996
). Even though
dietary factors can alter hepatic apoB mRNA editing, intestinal apoB
mRNA editing appears to be more resistant to modulation as indicated by
these as well as numerous other studies. In addition, hepatic apoB mRNA
abundance was not affected by the zinc status of the rats. Other
studies have also shown that dietary modulation does not affect the
hepatic total apoB mRNA abundance (Lau et al. 1995
,
Leighton et al. 1990
, Nassir et al. 1996
,
Reaves et al. 1996
).
A significant decrease in hepatic apobec-1 mRNA abundance in the ZD
rats was observed in this study. In previous studies, changes in
hepatic editing were found to correlate with changes in hepatic
apobec-1 mRNA abundance. Rats that were food deprived for 24 or 48 h demonstrated a decrease in hepatic apobec-1 mRNA abundance and a
concomitant decrease in hepatic apoB mRNA editing. After the refeeding
of a high carbohydrate diet for 24 or 48 h, hepatic apobec-1 mRNA
abundance was increased 2.1- to 2.8-fold, and hepatic editing of apoB
mRNA was also increased (Funahashi et al. 1995
). In
contrast, cholesteryl ester supplementation depressed hepatic apoB mRNA
editing and reduced apobec-1 mRNA abundance in rats (Inui et al. 1994
). However, changes in editing are not always "coupled"
with changes in apobec-1 mRNA abundance, illustrating that several
mechanisms may play a role in regulating apoB editing. For example, in
our ZDA rats, the hepatic apoB mRNA editing level was normalized to
that of the ZA rats, whereas apobec-1 mRNA remained depressed. These
data suggest that a post-transcriptional regulatory mechanism could
be responsible for the rapid normalization of editing levels in the ZDA
rats before the restoration of the apobec-1 mRNA abundance to the level
of controls. Obviously, the same mechanism could be responsible for the
observed decrease in hepatic editing of the ZD rats, and it may not be
simply transcriptional regulation of apobec-1 that is responsible for
the alteration in editing.
It must be noted that data from our study differ from those recently
reported by Nassir et al. (1996)
. There are many
differences in the experimental design between the two studies, which
may account for our incongruent observations. For example, our rats
were slightly older and were sexually mature animals. We fed our rats
AIN-96M rodent diet as opposed to their use of AIN-76A formulations.
Although the duration of treatment was similar, our zinc-deficient
diet was lower in zinc (<0.5 vs. 1 mg Zn/kg diet). Food deprivation or
food deprivation and refeeding have both been shown to affect hepatic
apoB mRNA editing levels in rats (Baum et al. 1990
,
Harris and Smith 1992
, Leighton et al. 1990
). These data suggest that there is a critical period of
time just before killing in which dietary intake can influence editing
levels; therefore, dietary intake should be very similar in animals of
each treatment group during this time period. As an attempt to minimize
variations, we chose to remove food from the cages 10 h before
killing. In the other study, food was not removed before killing, which
potentially could be a key difference between the two studies. Also,
instead of using pair-fed animals we opted for a
zinc-replenishment group in an effort to show the effects of adding
back zinc to the zinc-depleted rats. However, there were some
similarities between studies; both studies found a reduction in plasma
apoB-48 to total apoB in zinc-deficient vs. zinc-adequate rats.
In addition, in their study, there appeared to be a trend for hepatic
editing to be lower in ZD than ZA rats, but the differences were not
significant. Also, both studies found that hepatic apoB mRNA abundance
was not affected by the dietary treatments. Exactly how these
differences in experimental design relate to the different observations
between studies is uncertain and remains open to speculation.
In summary, we found that rats consuming a zinc-deficient diet exhibited a significantly reduced percentage of plasma apoB-48 to total apoB compared with zinc-adequate and zinc-repleted rats. A modest yet significant reduction in hepatic apoB mRNA editing was observed in zinc-deficient rats, but editing levels returned to control values in rats of the zinc-replenished group. Hepatic apobec-1 mRNA abundance was reduced in zinc-deficient rats and was not restored by zinc replenishment. These data suggest that several aspects of apoB metabolism may be affected when utilizing a diet low in zinc to induce zinc deficiency.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Supported by the U.S. Department of Agriculture
Human Nutrition Competitive Grants Program (95–37200-1672) and funds
from the University of Arizona Agricultural Experiment Station.
4 Abbreviations used: apoB, apolipoprotein B;
apobec-1, apolipoprotein B mRNA editing catalytic subunit 1; DEPC,
diethyl pyrocarbonate; RT-PCR, reverse transcriptase-polymerase
chain reaction; ZA, zinc-adequate diet; ZD, zinc-deficient
diet; ZDA, zinc-replenished diet.
Manuscript received March 3, 1999. Initial review completed May 11, 1999. Revision accepted July 6, 1999.
|
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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