INTRODUCTION

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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.

	METHODS

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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.

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.

	RESULTS

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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.

View larger version (14K): [in this window] [in a new window]   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.

View larger version (77K): [in this window] [in a new window]   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.

View larger version (96K): [in this window] [in a new window]   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.

View larger version (25K): [in this window] [in a new window]   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.

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).

View larger version (68K): [in this window] [in a new window]   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.

View larger version (37K): [in this window] [in a new window]   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.

View larger version (60K): [in this window] [in a new window]   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.

	DISCUSSION

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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.