Dietary induction of angiotensin-converting enzyme in
proximal and distal rat small intestine
Roger H.
Erickson,
Byung-Chul
Yoon,
Danielle Y.
Koh,
Do
Hyong
Kim, and
Young S.
Kim
Gastrointestinal Research Laboratory, Department of Veterans
Affairs Medical Center, San Francisco 94121; and Department of
Medicine, University of California, San Francisco, California 94143
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ABSTRACT |
Induction of angiotensin-converting
enzyme was examined in proximal and distal intestinal segments of rats
fed a low-protein (4%) diet and then switched to a high-protein
(gelatin) diet. Animals were killed at varying time points, and
brush-border membranes and total RNA were prepared from the segments.
In the proximal intestine, there was a fivefold increase in
angiotensin-converting enzyme levels after 14 days but only a twofold
change in mRNA. In the distal intestine, there was no increase in
enzyme activity but mRNA increased 2.4-fold. Organ culture was used to
measure changes in enzyme biosynthesis. There was a 5- to 6-fold
increase in the biosynthesis of angiotensin-converting enzyme in the
proximal intestine 24 h after the switch to the gelatin diet and a
1.6-fold increase in mRNA levels. No change in biosynthesis was
observed in the distal small intestine despite an increase in mRNA.
These results support the conclusion that rapid dietary induction of intestinal angiotensin-converting enzyme is differentially regulated in
proximal and distal segments of the small intestine.
brush-border membrane; peptidase; biosynthesis
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INTRODUCTION |
PREVIOUS STUDIES FROM
OUR laboratory (16, 18) have indicated that the
amount and type of protein in the diet can have a profound effect on
levels of small intestinal brush-border membrane peptidases. Two
enzymes, namely angiotensin-converting enzyme (ACE; EC 3.415.1) and
dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5), are unusually responsive
to dietary modulation, their levels being induced by diets containing
high amounts of proline (24). These two enzymes are
important in that they play a critical role in the terminal digestion
of prolyl peptides, which are generally resistant to hydrolysis by the
peptide-hydrolyzing enzymes of the stomach and pancreas. Proteins high
in proline are important dietary constituents as evidenced by commonly
ingested proteins such as collagen, casein, and gliadin. Thus the
mammalian small intestine is particularly well suited to complete the
digestion of these types of proteins because of the presence of the two aforementioned enzymes in addition to carboxypeptidase P and
aminopeptidase P (5).
Currently, very little is known regarding the regulation of intestinal
brush-border membrane enzymes, though some have been cloned and
sequenced and studies of their gene promoters have commenced (6,
19, 23, 26). A number of these enzymes undergo changes in levels
concomitant with postnatal development and the process of weaning
(9, 11). Many of these changes are thought to
involve transcriptional, translational, and postranslational levels of
control that come into play at various stages of development (4). Also, these maturational events are thought to be
under the influence of various hormonal mediators (11). At
present, knowledge of the factors and biochemical processes that are
important in regulating the expression of enterocytic brush-border
membrane hydrolases in the mature adult small intestine are poorly
understood. The fact that many of these hydrolases have gradients of
activity along the proximal-distal axis of the small intestine points
to the existence of control mechanisms that regulate their expression.
One of the best studied intestinal proteins with respect to
dietary regulation is sucrase-isomaltase. Manipulation of its substrate
sucrose leads to changes in intestinal cellular rates of
sucrase-isomaltase biosynthesis and mRNA levels in only a matter of
hours (1, 2, 28). Currently, the factors and signals responsible for these rapid alterations are unknown. Similar detailed evidence for the effect of diet on intestinal peptidases is generally lacking. Early studies (12, 29) have shown that diets
varying in protein content can affect intestinal peptidase activities along with corticosteroids and hormones. In addition, it has been shown
(22) that the biosynthetic rate of aminopeptidase N can be
rapidly increased by perfusing specific peptide substrates through
segments of rat intestine. Previously, we (24) reported that diets high in protein and proline (gelatin) lead to the five- to
sixfold induction of intestinal levels of DPPIV and ACE after 1 wk of
administration. In addition, these changes are accompanied by a 1.5- to
3.5-fold increase in gene transcription that parallels the observed
changes in mRNA levels in various intestinal segments (25).
In the rat, intestinal ACE displays a pronounced gradient of activity
with the highest levels being observed in the proximal segment. We
(24) have also observed that ACE in the proximal intestine
is much more responsive to diet compared with the distal intestine. To better understand this phenomenon, we looked at early time points during the dietary induction of ACE by monitoring changes in enzyme activity, mRNA, and the biosynthetic rate in proximal
and distal intestinal segments. These results show that 24 h after
the switch to a high-protein diet, there is a 5- to 6-fold increase in
the biosynthetic rate of ACE in proximal intestine with a 1.6-fold
change in mRNA levels. No change in ACE biosynthesis in the distal
intestine was observed despite a 1.3-fold increase in mRNA by 2 days
and 2.4-fold increase by 14 days. These studies indicate that
intestinal ACE is differentially regulated in the proximal and distal
regions of the small intestine.
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METHODS |
Animals and diets.
Male Wistar rats (240-260 g) were purchased from Simonsen
Laboratories (Gilroy, CA) and maintained ad libitum on a low-protein (4% casein) diet for 1 wk. The animals were then switched to a isocaloric diet containing 50% gelatin and maintained on this diet for
varying periods (1, 2, 3, 5, 7, and 14 days) before being killed. The
weight of the animals was monitored during the course of the
experiment. Water was not restricted. Diets were modified formulations
of the rodent AIN 76A diet prepared by Harlan Teklad Research Diets
(Madison, WI). Both diets had the same amount of fat (5.6%), calcium
(0.7%), phosphorous (0.5%), and cornstarch (20%). The amount of
sucrose was varied to make the diets isocaloric. The gelatin was a
general purpose product derived from pork skin (type A, 225 bloom) from
Hormel (Austin, MN). These experiments were carried out according to
ethical guidelines for use of animals in research and were approved by
the Animal Studies Subcommittee at the Department of Veterans Affairs
Medical Center (San Francisco, CA).
Isolation of brush-border membranes, RNA, and assay of enzyme
activity.
Rats (nonfasted) were killed by decapitation under light
CO2 anesthesia. The small intestine was removed and flushed
out with cold saline. A 10-g weight was suspended from one end, and the intestine was divided into three equal-length segments corresponding to
proximal, middle, and distal intestine. The distal two-thirds of each
segment were used for the preparation of brush-border membranes from
mucosal scrapings (13), whereas the proximal one-third was
immediately frozen in liquid nitrogen and used for the preparation of
total RNA (3). In some instances, total RNA was prepared
from scraped mucosa using the TriReagent kit. ACE activity was measured
at 37°C using hippuryl-L-His-L-Leu as substrate (30). Enzyme-specific activity was expressed as
nanomoles of substrate hydrolyzed per minute per milligram of protein.
Protein was measured by the method of Lowry et al. (15).
The purity of brush-border membrane preparations was assessed by
comparing the increase in ACE specific activity to that of other known
brush-border membrane hydrolases such as sucrase-isomaltase and
aminopeptidase N (30). A 10- to 15-fold purification over
cell homogenates was routinely obtained.
Organ culture and biosynthetic labeling.
Intestinal explants were prepared from the proximal or distal segment
of rat small intestine immediately after death. Explants were ~2 × 5 mm and were placed on stainless steel screens in organ culture
dishes after previously described procedures (8, 17). The
center well contained 0.8 ml of methionine and cysteine-free RPMI 1640 medium supplemented with 10% dialyzed fetal bovine serum, penicillin
(100 U/ml), and streptomycin (100 mg/ml). Explants were incubated for
1 h at 37°C in 95% O2-5% CO2 and then
continuously labeled for 1 h with a 35S labeling
mixture (Tran35S-Label, ICN Pharmaceuticals, Costa Mesa,
CA) at 440 mCi [35S]methionine/dish. Labeled explants
were washed on the screens three times with cold PBS and immediately
frozen at
70°C.
Labeled explants were homogenized with a glass-Teflon homogenizer in 10 mM Tris · HCl and 50 mM mannitol, pH 7.2, containing a cocktail
of various protease inhibitors (8). Cellular debris was
removed by a 10-min centrifugation at 2,500 g. The resulting supernatant was then centrifuged at 100,000 g for 1 h
to yield a total cell membrane pellet that was resuspended in
homogenizing buffer (with inhibitors) and stored at
70°C. An
aliquot of labeled membrane containing 106 counts/min (cpm)
of 35S incorporated into TCA-precipitable protein was
solubilized and used in a previously described (10)
immunoprecipitation procedure. Goat anti-rat ACE was used as the
initial antibody and was the generous gift of Dr. Kunio Hiwada (Ehime
University, Ehime, Japan). A rabbit anti-goat secondary antibody was
purchased from Zymed Laboratories (South San Francisco, CA). The final
washed and fixed Staphylococcus aureus immunopellets were
boiled for 5 min in SDS-sample buffer and subjected to SDS-PAGE. After
electrophoresis, the gel was fixed, washed, treated with Autofluor
(National Diagnostics, Atlanta, GA), dried on GelBond PAG film (FMC
Bioproducts, Rockland, ME), and exposed to X-ray film.
Immunobloting, Northern slot-blot analysis, and RT-PCR.
Samples of brush-border membrane (50 µg) were subjected to SDS-PAGE
in 7% acrylamide gels and transferred to either a nitrocellulose or
polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). Briefly, ACE was probed using a rabbit anti-ACE antibody followed
by 106 cpm/ml 125I-labeled protein A. Total RNA
(10 µg) was electrophoresed and transfered to a nylon membrane.
Hybridization analysis was carried out after nick translation and
32P labeling of a mouse ACE cDNA (ACE 0.5) probe that was
kindly provided by Dr. Kenneth Bernstein (Emory University, Atlanta, GA). Blots were erased and then probed with a 32P-labeled
-actin cDNA. ACE protein and mRNA were detected by autoradiography,
and levels were quantified by densitometry. Densitometry was performed
with the Scion Image 1.62c software program after autoradiograms were
scanned into a computer. In some experiments, samples of total RNA were
reverse transcribed and then amplified by PCR using primers for rat ACE
(accession no. U03734; forward, 5'-TGCTGTGGGACTTC-TACAACAGG-3';
reverse, 5'-GGGTTTCATTCCGAGCAACTG-3'; 449 bp) and
-actin (accession
no. M10277; forward, 5'-ACTTGGCACCACACCTTCTACAATGAGCTGCG-3'; reverse, 5'-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3; 838 bp). To attenuate a
strong
-actin signal,
-actin primers blocked at the 3' end were
diluted with normal primers and added to the PCR reactions.
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RESULTS |
Effect of diet on rat weight.
For the dietary induction studies, rats (240-260 g) were initially
maintained on a 4% casein diet for 7 days, by which time they had lost
~5% of their initial weight. They were then switched to the 50%
gelatin diet. Those rats receiving this diet experienced a 3% and 7%
weight gain after 7 and 14 days, respectively. Thus these data are in
line with studies that we (24) have reported previously.
Effect of diet on ACE activity and mRNA.
Figure 1 shows the specific activity of
brush-border membrane ACE in proximal, middle, and distal intestinal
segments at increasing times after the switch to the 50% gelatin diet.
ACE displays a pronounced proximal-distal gradient of activity that was
maintained throughout the induction period. By day 2 there
was a significant increase in ACE activity in the proximal and middle
intestine of 2.2- and 4.5-fold, respectively. This increase continued
in the proximal intestine up to day 14, at which time ACE
activity was approximately fivefold higher than at the start of the
experiment. Activity in the middle intestine registered a ninefold
increase by day 5 with no further increase apparent at
days 7 and 14. In marked contrast, no significant
change in ACE activity was noted in the distal intestinal segment
during the course of the experiment.

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Fig. 1.
Effect of protein diet on rat intestinal brush-border
membrane angiotensin-converting enzyme (ACE) activity. Rats were
maintained for 1 wk on a 4% protein diet and then switched to a 50%
gelatin diet. At the indicated times after the switch, brush-border
membranes were prepared from proximal (filled bars), middle (open
bars), and distal intestine (hatched bars) and ACE was assayed. Data
are means ± SD of 3 animals. The following statistical analysis
was done using the Student's t-test. For proximal
intestine: day 0 vs. day 1, P 0.48;
day 0 vs. day 2, P 0.00008; and
day 0 vs. day 14, P 0.00003. For
middle intestine: day 0 vs. day 1, P 0.15; day 0 vs. day 2, P 0.0023;
and day 0 vs. day 14, P 0.005. For
distal intestine: day 0 vs. day 1, P 0.17; day 0 vs. day 2, P 0.33;
and day 0 vs. day 14, P 0.03.
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Figure 2 shows a Western blot of ACE in
brush-border membranes from the proximal intestine of three different
rats at days 0, 2, and 14. As can be
seen, there was an increase in the amount of ACE protein present at
days 2 and 14 compared with day 0.
Densitometry indicated a 3.3-fold increase in ACE protein by day
2 and a 5- to 6-fold increase by day 14.

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Fig. 2.
Western blot analysis of rat small intestinal ACE.
Samples of brush-border membranes from the proximal intestine of 3 different rats at days 0, 2, and 14 were subjected to SDS-PAGE. ACE was detected with polyclonal antibody
and 125I-labeled protein A.
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Total RNA was isolated from segments of rat intestine at days
0, 2, 7, and 14. Figure
3 shows a representative Northern blot probed with a 32P-labeled mouse ACE cDNA and
-actin as a
control. Blots were quantified by densitometry, and differences in
loading were corrected for by normalizing to the levels of
-actin.
These results are shown in Fig. 4. There
was a 1.6-fold increase in the amount of ACE mRNA in the proximal
intestine by day 2 with similar but lower increases observed
in the middle (1.4-fold) and distal intestine (1.3-fold). By day
14 the mRNA in the proximal and middle intestine had increased
2.1- and 1.2-fold, respectively, compared with day 0.
Interestingly, the distal intestinal segment had a 2.4-fold increase in
ACE mRNA after 14 days. At this point, levels of ACE mRNA in the distal
intestine were ~77% of the levels measured in the proximal
intestine. Also a defining feature is that the longitudinal ACE mRNA
profile (Fig. 4) in the small intestine did not match that of the
enzyme activity profile (Fig. 1). Thus the middle and distal segments
of the small intestine were observed to have relatively high levels of
ACE mRNA compared with levels of ACE activity.

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Fig. 3.
Northern blot analysis of rat intestinal ACE and
-actin mRNA during dietary induction. Total RNA was isolated from
proximal (A), middle (B), and distal intestinal
segments (C). RNA (10 µg) was electrophoresed, transfered
to a nylon membrane, and probed with a 32P-labeled mouse
ACE cDNA. Blots were then erased and probed with a
32P-labeled -actin cDNA. ACE is shown at the
top of each set and -actin at the bottom. Two
individual rats from each time point are shown.
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Fig. 4.
Levels of ACE mRNA in rat intestine during dietary
induction. The blots given in Fig. 3 were scanned into a computer and
quantified by densitometry. The level of -actin in each sample was
used to normalize ACE mRNA levels from proximal (filled bars), middle
(open bars), and distal intestine (hatched bars). Values are means ± SD of 3 animals. The following statistical analysis was done using
the Student's t-test. For proximal intestine: day
0 vs. day 2, P 0.0007, and day
0 vs. day 14, P 0.0006. For middle
intestine: day 0 vs. day 2, P 0.012, and day 0 vs. day 14, P 0.19. For distal intestine: day 0 vs. day 2,
P 0.06, and day 0 vs. day 14,
P 0.00009.
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Biosynthetic labeling of intestinal explants.
Early changes in ACE protein biosynthesis during dietary induction were
examined in the proximal and distal segments of the small intestine.
These two segments were used because of their differences in levels of
ACE activity and response to dietary manipulation. Explants of small
intestine were maintained in organ culture and labeled with
35S, and newly synthesized, labeled ACE was
immunoprecipitated from a total cell membrane preparation. After 1 h of continuous labeling ACE was readily immunoprecipitated with
polyclonal antibody, displaying a single band of ~170 kDa. As shown
in Fig. 5A, the intensity of
labeling in the proximal intestine of the two rats 1 day after the
dietary switch was approximately five- to sixfold higher than the
control animals. No further increase was observed in the animals at 2 days. Conversely, in the distal intestine (Fig. 5B), there was no significant change in the biosynthetic rate of ACE in animals maintained for up to three days on the 50% gelatin diet. Figure 6 shows a RT-PCR of ACE mRNA isolated
from the proximal intestine of the six animals used in the organ
culture experiment (Fig. 5A). When levels of
-actin were
used for normalizing, there was a 1.2-fold increase in the intensity of
the ACE band relative to
-actin by day 1. By day
2 the increase was ~1.6-fold as measured by this procedure. The
2-day change corresponds to the increase as measured by Northern blot
analysis (Fig. 4).

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Fig. 5.
Biosynthetic labeling of ACE during organ culture of
intestinal explants. Explants from the proximal (A) and
distal intestine (B) of rats at days 0,
1, and 2 after the switch to the 50% gelatin
diet were continuously labeled with 35S for 1 h as
described. ACE was immunoprecipitated from a cell membrane fraction
with polyclonal antibody. Immunoprecipitates were subjected to SDS-PAGE
and autoradiography. Each lane represents 1 animal.
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Fig. 6.
RT-PCR of ACE mRNA from proximal small intestine during
dietary induction. Total RNA was isolated from the proximal intestinal
segment of the 6 individual rats used in the organ culture experiments
(Fig. 5A). Samples of RNA (3 µg) were reverse transcribed
and then amplified (PCR) using specific primers for rat ACE (449 bp)
and -actin (847 bp). The left lane contains a series of
standards (100-bp DNA ladder) as indicated.
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After 14 days we observed that the ACE mRNA levels in the distal
intestine were 77% of those observed in the proximal intestine. Therefore we were interested to see if after 14 days of dietary induction, the biosynthetic rate of ACE in the distal intestine had
increased to a level similar to that observed in the proximal intestine. As seen in Fig. 7A,
the ACE biosynthetic rate was considerably higher in the proximal
region of the intestine compared with the distal segment. Thus the
bisoynthesis profile was similar to that observed for ACE protein in
the brush-border membrane as determined by Western blot analysis (Fig.
7B) and the distribution of enzyme activity (Fig. 1).

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Fig. 7.
Biosynthetic labeling and distribution of ACE in various
regions of the rat intestine after 14 days of dietary induction.
A: explants from the proximal (P), middle (M), and distal
(D) intestine were continuously labeled with 35S for 30 min. ACE was immunoprecipitated, subjected to SDS-PAGE, and
autoradiographed. B: brush-border membranes (50 µg
protein) from intestinal segments were subjected to SDS-PAGE. ACE was
detected with antibody and 125I-labeled protein A.
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DISCUSSION |
The brush-border membrane of intestinal epithelial cells contains
a number of peptidases that play an important role in the final phases
of protein digestion. Little is known, however, regarding the factors
and mechanisms that regulate their expression in small intestinal
epithelial cells. In previous studies, we (24) have observed that intestinal levels of ACE are responsive to changes in the
diet and also show a differential response in the proximal and distal
intestinal segments. In the present study, we have examined long- and
short-term changes in ACE activity, mRNA, and biosynthesis to elucidate
the events responsible for dietary induction, rapidity of the response,
and differences in the proximal and distal small intestine.
The long-term response to the high-gelatin diet was a steady increase
in the levels of ACE activity in the small intestine. However, this
occurred differentially with respect to the longitudinal axis of the
intestine. In the proximal intestine, in which ACE is highly expressed,
the levels of activity increased steadily throughout the 14-day period.
In the middle intestine, activity levels peaked at day 5,
thereafter remaining constant up to day 14. In the distal
intestine, in which ACE activity is the lowest, levels remained
constant throughout the entire 14-day period and were generally
unaffected by the change in diet. The effect of diet on ACE mRNA levels
during the induction period was markedly different from that observed
for ACE activity because we observed only a modest increase in mRNA in
the proximal and middle intestine. Therefore, changes in mRNA did not
correlate with the six- to ninefold increases in ACE activity that we
observed during the course of our experiments. It was also apparent
that the mRNA profile along the proximal-distal axis of the small
intestine did not match that of ACE activity in protein-depleted or
-induced animals. In fact, we have consistently noted these same
characteristic segmental patterns of distribution for ACE activity and
mRNA in all rats, regardless of dietary status. Of particular interest is the fact that the distal intestine had the greatest fold increase in
ACE mRNA after 14 days. At this time, the distal mRNA levels were
~77% of those found in the proximal segment, yet ACE activity levels
were only 6% of those in the proximal segment. One possible explanation is that ACE is synthesized in the distal segment at a rate
comparable with that in the proximal intestine and either secreted or
degraded, thereby not reaching the brush-border membrane. However, our
experiments argue against this possibility because the observed low
biosynthetic rate in 14-day animals paralleled that of enzyme activity
(Figs. 1 and 7). A second possibility is that ACE is present in the
distal intestine at levels comparable with those found in the proximal
segment but in an inactive form. Again, our Western blot analysis
studies suggest that this is not the case because there is
substantially less ACE protein in the distal intestine compared with
the proximal segment.
In the short term, we observed that by 24 h there is a 5- to
6-fold increase in the biosynthesis of ACE in the proximal intestine with just a 1.2-fold change in mRNA. By 7 days, mRNA levels had increased 1.8-fold, which is somewhat less than the 3-fold increase reported previously (24). This may be due to the fact that
the earlier data were not normalized to
-actin mRNA levels. In the distal intestine, there was a 1.3-fold increase in mRNA by day 2; however, there was no change in the ACE biosynthetic rate. This
correlates well with the observations on the low ACE activity and the
absence of ACE induction in this region of the intestine. The increased
levels of mRNA measured at 1 and 2 days indicate that this may be due
to increased transcription of the ACE gene as described in our earlier
work (25). Still, the proximal and distal differences in
the biosynthesis and expression of ACE, in light of the comparatively
high levels of distal ACE mRNA, suggest that control of mRNA
translation may be important in these two regions.
For the biosynthetic studies, we did not examine time points earlier
than 24 h because it would have been necessary to employ a
force-feeding technique to ensure that the rats received adequate amounts of the diet during the appropriate time period. This was primarily due to the nature of the gelatin diet, which was impossible to get into a liquid form for force feeding. Because the ACE
biosynthetic rate did not increase from 1 to 2 days, it had apparently
reached maximum levels already at 24 h under the conditions of
these experiments. This suggests that dietary-induced changes in
ACE biosynthesis may occur at even earlier time points. Though we did
not observe measurable changes in ACE activity until 2 days after the
dietary switch, this may be due to the turnover rate of small
intestinal epithelial cells, which takes 5-7 days.
Induction of sucrase-isomaltase has been shown by several groups to
involve rapid increases in mRNA and protein levels by 3 h,
indicating that gene transcription is an important controlling element
for this intestinal protein (1, 2, 28). Using an
intestinal perfusion technique, Reisenauer and Gray (22) have reported that by 30 min aminopeptidase N undergoes
substrate-specific induction. Previously, we (7) have used
a similar perfusion method and did not observe any change in ACE
biosynthesis for periods of up to 3 h, suggesting that the
response time and/or conditions for ACE induction are different from
those reported for aminopeptidase N (22).
The results of this study suggest that dietary induction of intestinal
ACE has elements of control at the level of mRNA translation. This
possibility is indicated by the observations that 1) levels of mRNA do not correspond to ACE protein levels in different intestinal segments, 2) a 1.2- to 1.6-fold increase in ACE mRNA during
dietary induction results in a rapid 5- to 6-fold change in ACE protein biosynthesis, and 3) the greatest increase in ACE mRNA
(2.4-fold) was observed in the distal intestine with no change in
protein biosynthesis. Therefore translational control of intestinal ACE along the longitudinal axis of the small intestine might be important in maintaining its gradient of expression. It has become obvious that
translational control is an important parameter in the regulation of a
number of eukaryotic genes (20, 21). Protein factors important for the initiation of translation are just now being elucidated. In addition, phosphorylation plays a role in regulating the
activities of some of these initiation factors, which in turn is linked
to signal transduction pathways (14). It is also known that the 3'- and 5'-untranslated regions of mRNA contain regions and
sequences that influence circularization of mRNA, binding of factors,
and therefore translation (27). Hence, the picture that is
emerging is that translation of mRNA is an important, highly regulated
process with multiple control points that are intimately involved in
eukaryotic gene expression. At present, the role of these processes in
the regulation of expression of intestinal brush-border membrane
proteins, their importance in maintaining gradients of expression along
the proximal/distal axis, and significance during the differentiation
of intestinal enterocytes are largely unknown and remain subjects for
future investigation.
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ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-17938 and the Department of
Veterans Affairs Medical Research Service.
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FOOTNOTES |
Address for reprint requests and other correspondence: R. H. Erickson, GI Research Laboratory (151M2), VA Medical Center, 4150 Clement St., San Francisco, CA 94121 (E-mail:
neko{at}itsa.ucsf.edu).
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. Section 1734 solely to indicate this fact.
Received 27 October 2000; accepted in final form 4 July 2001.
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