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Endocrinology Vol. 138, No. 5 1959-1963
Copyright © 1997 by The Endocrine Society

ARTICLES

Effect of Carboxypeptidase E Deficiency on Progastrin Processing and Gastrin Messenger Ribonucleic Acid Expression in Mice with the fat Mutation1

Vidyavathi Udupi, Pablo Gomez, Lixin Song, Oleg Varlamov, Jason T. Reed, Edward H. Leiter, Lloyd D. Fricker and George H. Greeley, Jr.

Department of Surgery, University of Texas Medical Branch, Galveston, Texas 77555; the Department of Molecular Pharmacology, Albert Einstein College of Medicine (L.S., O.V., L.D.F.), Bronx, New York 10461; and the Jackson Laboratory (E.H.L.), Bar Harbor, Maine 04609

Address all correspondence and requests for reprints to: George H. Greeley, Jr., Ph.D., Department of Surgery, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0725. E-mail: ggreeley{at}mspo2.med.utmb.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proforms of gastrointestinal peptides are cleaved at paired basic residues into intermediate forms. Paired basic residues at the C-terminal then are excised by carboxypeptidases before the peptide is amidated. An obese mouse, called Cpefat/Cpefat, has a missense mutation in carboxypeptidase E (CPE) with no pancreatic CPE activity and a reduced processing of pancreatic proinsulin to insulin. The purpose of this study was 1) to look for the presence of CPE in the antrum of the stomach, duodenum, and colon in the Cpefat/Cpefat mouse; 2) to determine whether CPE is involved in the processing of progastrin (Pro-G) to its carboxyl-terminal amidated form; and 3) to determine whether a decrease in amidated gastrin results in an up-regulation of stomach gastrin messenger RNA (mRNA) levels. In Cpefat/Cpefat mice, CPE activity was absent in the antrum and colon. In Cpefat/Cpefat mice, amidated gastrin levels were reduced significantly. Levels of the precursor for amidated gastrin (gastrin-Gly-Arg-Arg) were markedly elevated. Gastrin mRNA levels were increased approximately 2-fold over the levels in Cpefat/Cpefat mice. These results indicate that CPE is needed for processing progastrin to gastrin in the stomach and that amidated gastrin exerts an inhibitory feedback effect on gastrin mRNA levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GASTRIN IS an endocrine peptide localized primarily in the antral G cells of the stomach mucosa (1). One of the major functions of gastrin is the regulation of gastric acid secretion by the parietal cells of the stomach (1). Gastrin also has trophic effects on the gastrointestinal tract and pancreas (2, 3, 4, 5, 6); it affects stomach muscle contraction (7), and it stimulates fractional sodium excretion by the kidney while increasing renal plasma flow (8). Gastrin is processed posttranslationally to intermediate forms, gastrin-Gly-Arg-Arg and gastrin-Gly (9). Although amidation of the COOH-terminal of gastrin is thought to be essential for biological activity (10), there is recent evidence to show that gastrin-Gly can stimulate acid secretion (11) and can have a trophic action (12, 13). In addition, although there is no evidence showing that gastrin-Gly-Arg-Arg itself is active biologically, a recent report indicates that elevated levels of progastrin can increase the bromodeoxyuridine labeling index of the colon (14).

An obese mouse model (Cpefat/Cpefat) has been described that has hyperproinsulinemia and late-onset obesity (15). These Cpefat/Cpefat mice have a missense mutation in carboxypeptidase E (CPE), with marginal pancreatic CPE-like activity and a reduced capacity to process pancreatic proinsulin to insulin. The missense mutation converts Ser202 into Pro, a change that eliminates CPE activity (16). CPE is a processing enzyme involved in the excision of paired basic residues remaining at the carboxyl-terminus after cleavage by a prohormone convertase of many endocrine and neuroendocrine peptides (17). Whether CPE is involved in processing progastrin to biologically active amidated gastrin is not known. A deficiency of CPE activity in the stomach may result in the excessive production of intermediate gastrin peptides with C-terminal dibasic residue extensions. In addition, it is conceivable that gastrin messenger RNA (mRNA) expression in the stomach is simultaneously increased as a result of the reduced processing of progastrin to biologically active COOH terminal-amidated gastrin and lower systemic circulatory levels of amidated gastrin. In the present study, we examined the processing of progastrin to gastrin in the antrum of the stomach, gastrointestinal CPE activity, and antral gastrin mRNA levels of Cpefat/Cpefat mice. The specific detection of amidated gastrin and its proforms was facilitated in part by the availability of specific and sensitive antisera for amidated gastrin and gastrin-Gly-Arg-Arg (18, 19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Cpefat/Cpefat and control (Cpefat/+; +/+) mice were produced in a research animal colony by one of the authors (E.L.) at the Jackson Laboratory (Bar Harbor, ME). The mice were weaned on Old Guilford Diet 96WA (Emory Morse Co., Guilford, CT) and maintained on that diet until shipment to University of Texas Medical Branch (Galveston, TX). At the University of Texas Medical Branch, mice were maintained on Teklad rodent diet until death. Mice were acclimated to local housing conditions for at least 1 week and were maintained in an air-conditioned (23 ± 2 C) and light-regulated (lights on 0600–1800 h) vivarium. All mice had free access to chow and tap water. Cpefat/Cpefat mice were identified by genetic markers and, phenotypically, by the development of obesity. Mice were killed by decapitation in the ad libitum-fed condition. The mean body weights ± SEM of control and Cpefat/Cpefat mice were 29 ± 2 and 37 ± 3 g, respectively. The ages of Cpefat/Cpefat and control mice ranged from 2–19 months; Cpefat/Cpefat and control mice were age-matched. The brain, pancreas, antrum of the stomach, duodenum, and colon were removed. Pancreatic islets were prepared by infusion of the common bile duct with a collagenase solution and harvested by hand under a dissecting microscope. The gastrointestinal tract was cleaned carefully of food material or feces. The antrum was bisected; half was frozen for analyses of carboxypeptidases (CPs), and the remaining half was homogenized immediately in distilled water (1:10, wt/vol) and then boiled for 20 min. Boiled extracts were stored frozen until assayed by the various gastrin RIAs. In a separate series of Cpefat/Cpefat and control mice, the entire antrum was removed and homogenized in a RNA extraction solution for preparation of total cellular RNA.

Assays
C-Terminal amidated gastrin and gastrin-Gly-Arg-Arg RIAs.
A double antibody RIA procedure (20) was used to specifically measure C-terminal amidated gastrin. This RIA uses an antiserum (5135, provided by the Center for Ulcer Research and Education, University of California-Los Angeles, Digestive Diseases Center Antibody Core) that recognizes only C-terminally amidated gastrin and cholecystokinin (CCK) and does not cross-react with C-terminally extended forms of either peptide (19). CCK is not present in the antrum (21), so detection of CCK is not a consideration. The sensitivity and ID50 (50% inhibition of bound [125I]gastrin) for amidated gastrin are 6 and 50 pg/tube, respectively. The antiserum was used at an initial dilution of 1:50,000. Gastrin-Gly-Arg-Arg was measured by a double antibody RIA using an antiserum that is highly specific for gastrin-Gly-Arg-Arg (antiserum GL-9, gift from C. Dickinson, University of Michigan, Ann Arbor, MI) (18). This antiserum does not recognize amidated CCK-8, amidated gastrin. or gastrin-Gly; the cross-reactivity of this antiserum with gastrin-Gly-Arg and gastrins beyond the Gly-Arg-Arg residues is less than 10% (18). The sensitivity and ID50 for gastrin-Gly-Arg-Arg are 40 and 300 pg/tube, respectively. A synthetic peptide (YGWMDFGRR), iodinated by the chloramine-T method, was used as the iodinated ligand and standard. The antiserum was used at an initial dilution of 1:3,000.

Assay for CPE-like enzymes.
Gastrointestinal tissue extracts were prepared and analyzed enzymatically for CPD/CPE activities as described previously (22). In brief, tissues were harvested, frozen on dry ice, and stored at -20 C. Tissues later were homogenized in 0.2–2 ml 0.1 M sodium acetate (NaAc; pH 5.5) containing 1 mM phenylmethylsulfonylfluoride. Protein concentrations were determined with the Bradford assay, using BSA as a standard. For the carboxypeptidase assay, 25 µl of the homogenate or 25 ml of a 1:10 dilution were combined with 200 µM dansyl-Phe-Ala-Arg substrate, including 50 mM NaAc (pH 5.0) and 0.01% Triton X-100 (23) in a final volume of 250 µl. Tubes containing either 1 mM CoCl2 or 1 mM guanidino-ethylmercaptosuccinic acid (GEMSA) were included. After incubation at 37 C for 30–60 min, 100 µl 0.5 M HCl and 2 ml chloroform were added, and the tubes were mixed and then centrifuged at 500 x g for 2 min. The amount of product was determined by measuring the fluorescence in the lower (chloroform) layer; excitation was 350 nm, and emission was 500 nm. CP activity is defined as the difference between activity measured in the presence of Co2+, which activates CPE, and that in the presence of GEMSA, which inhibits CPE. These conditions are not specific for CPE because Co2+ activates, and GEMSA inhibits, other metallocarboxypeptidases (24).

The enzymatic properties of affinity-purified gastrointestinal tissue extracts were determined in an assay similar to that described above, except that 50 mM NaAc (pH 5.5) or Tris-Cl (pH 7.4) was used, and the tubes contained various ions or inhibitors instead of Co2+ or GEMSA. Samples were preincubated with inhibitors for 15 min at 4 C, the substrate was added, and the tubes were incubated for an additional 1 h at 37 C. CP activity was calculated as the difference in fluorescence between the tubes containing enzyme and those with only buffer and substrate and was expressed as a percentage of the control tube containing enzyme, buffer, and substrate, but without divalent ions or inhibitors.

Purification of CPE-like enzymes by substrate affinity chromatography.
Gastrointestinal tissue homogenates were combined with Triton X-100 (final concentration, 1%) and NaCl (final concentration, 1 M). Homogenates were mixed and centrifuged for 30 min at 50,000 x g at 4 C, and the supernatants were applied to a p-aminobenzoyl-Arg Sepharose 6B column (23, 24). Columns (0.5-ml bed volume) were washed with 30 ml 1 M NaCl and 1% Triton X-100 in 50 mM NaAc (pH 5.5) and rinsed with 5 ml 10 mM NaAc (pH 5.5). CPE was eluted with 3.6 ml 50 mM Tris-Cl (pH 8.0) including 100 mM NaCl and 0.01% Triton X-100 (elute 1). The column then was eluted with 3.6 ml 5 mM Arg in the same buffer (elute 2). CP activity was determined as described above.

Western blot of purified proteins.
Aliquots of the affinity column elute fractions were dried in a vacuum centrifuge, resuspended in polyacrylamide sample buffer (containing 1% SDS), heated to 95 C for 5 min, and loaded onto a 10% denaturing polyacrylamide gel. Electrophoresed proteins then were transferred to a nitrocellulose membrane and probed with an antiserum generated against a synthetic nine-residue fragment corresponding to the C-terminus of the full-length mouse CPE. This antiserum was used at final dilution of 1:1000.

Measurement of gastrin mRNA.
Total cellular RNA was prepared by homogenization of tissues in 4 M guanidine thiocyanate [including 25 mM sodium citrate (pH 7.0), 0.5% sodium N-lauroylsarcosine, and 0.1 M 2-mecaptoethanol] immediately after dissection. Samples then were centrifuged (18 h, 30,000 rpm) through a CsCl cushion (2 ml, 5.7 M) as described previously (25). RNA concentrations in the samples were quantitated by absorbance at 260 nm. Gastrin mRNA levels were analyzed by means of Northern (30 µg) and slot blot analyses (2.5 µg). For Northern blot analysis, total cellular RNA was separated by electrophoresis on a 1% denaturing agarose gel (25). RNA then was transferred to a nitrocellulose membrane. After an overnight prehybridization at 65 C, hybridization with 32P-labeled gastrin complementary RNA probe was carried out for 24 h. The antisense complementary RNA probe was generated by means of SP6 RNA polymerase-directed transcription of a plasmid containing the rat gastrin with the use of [{alpha}-32P]CTP and the Riboprobe Gemini System (Promega, Madison, WI) according to the directions contained in the kit. The rat gastrin complementary DNA was provided by S. Brand (26). Membranes were hybridized with a rat 18S ribosomal probe to monitor RNA loading. The 18S probe was labeled using a random primed protocol (Promega) with [32P]deoxy-ATP and hybridized at 45 C. Autoradiographs were subjected to densitometric scanning (Visage 60, Bio-Image, Ann Arbor, MI) quantification. The relative amounts of each mRNA species were calculated in arbitrary densitometric units.

Statistical analysis
Results are the mean ± SE. Data were analyzed by either Student’s t test or a one-way classification ANOVA followed by the Newman-Keuls test where pertinent. Differences with P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In the Cpefat/Cpefat antrum, amidated gastrin levels are approximately 3-fold lower (P < 0.05) than those in control mice (Table 1Go). In contrast, gastrin-Gly-Arg-Arg levels are approximately 670-fold higher in Cpefat/Cpefat mice than those in control mice. The ratio of G/gastrin-Gly-Arg-Arg is approximately 3600-fold greater in Cpefat/Cpefat mice than in control mice.


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  		Table 1. Gastrin-Gly-Arg-Arg, amidated gastrin (G) and antral gastrin mRNA levels in Cpefat/Cpefat mice

 
In Cpefat/Cpefat mice, antral gastrin mRNA levels are elevated significantly (P < 0.05; Table 1Go and Fig. 1Go). They are approximately 172% of control levels.



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  		Figure 1. Northern blotting analysis of gastrin mRNA expression in the antrum of control/wild-type (WT) and Cpefat/Cpefat mice. Thirty micrograms of total cellular antral RNA were electrophoresed in a formaldehyde-denaturing gel, transferred onto a nitrocellulose filter, and hybridized with 32P-labeled rat gastrin complementary RNA and rat 18S complementary DNA probes. Only three lanes of each group are shown, although all samples of control (n = 9) and Cpefat/Cpefat (n = 10) were examined by Northern and slot blot analyses.

 
Gastrointestinal tissues contain Co2+-activated and GEMSA-inhibited carboxypeptidase activity (Table 2Go). In control mice, high levels of CPE-like activity are detected in the pancreatic islets, and lower levels are detected in the duodenum, antrum, and colon. The level of CPE-like activity in Cpefat/Cpefat mice is approximately 7% of that in control pancreatic islets. In the colon of Cpefat/Cpefat mice, the levels of CPE-like activity are approximately 50% of those in control colon. In the duodenum and antrum, the levels of CPE-like activity in Cpefat/Cpefat and control mice do not differ statistically (P > 0.05).


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  		Table 2. Carboxypeptidase activity in the pancreas and gastrointestinal tract of Cpefat/Cpefat mice

 
The finding that CPE-like activity is not depressed in some Cpefat/Cpefat mouse tissues compared to those in the corresponding control mouse tissues suggests that either the point mutation affects the enzyme differently in the various tissues or other enzymes contribute to the CPE-like activity. To examine these possibilities, the antral and colonic extracts were fractionated on a substrate affinity column. This had been shown previously to separate CPE from CPD (24). In the Cpefat/Cpefat antrum and colon, the amount of CPE (elute 1) is reduced significantly compared to those in control antrum and colon (Table 3Go). The amounts of CPD activity (elute 2) in control and Cpefat/Cpefat antrum and colon do not differ significantly.


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  		Table 3. Carboxypeptidase activity after purification on a p-aminobenzoyl-Arg Sepharose affinity column

 
Western blot analysis of affinity-purified material from antrum, colon, and duodenum showed (Fig. 2Go) that immunoreactive CPE was present in the affinity column elutes only from control mouse tissues and not from Cpefat/Cpefat mouse tissues. Similar results were obtained when tissues from three control and three Cpefat/Cpefat mice were analyzed; the data from a single control and Cpefat/Cpefat mouse are shown (Fig. 2Go). Immunoreactive CPE is not detectable in the duodenum of control mice.



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  		Figure 2. Western blot analysis of CPE in control (c) and Cpefat/Cpefat (f) mouse tissues after purification on a substrate affinity column. Tissues were extracted, purified on the affinity resin, and analyzed on a Western blot. The blot was probed with an antiserum directed against the C-terminal region of CPE. Antrum and colon from individual male control (+/fat) or Cpefat/Cpefat animals were used. Duodenum was pooled from two male animals per group (either wild type or Cpefat/Cpefat).

 
The small amount of CP activity detected in the elute 1 fraction from the colon of Cpefat/Cpefat mice does not have the same enzymatic properties as purified CPE (Table 4Go). Whereas purified CPE is active optimally at pH 5.5 and is inhibited potently by GEMSA, HgCl2, and para-chloromercuriphenyl sulfonate, the elute 1 activity from Cpefat/Cpefat colon is partially inhibited by these compounds and is more active at pH 7.4 than at pH 5.5 (Table 4Go). The activities in the elute 2 fraction from both control and Cpefat/Cpefat mouse colon are similar, generally, to each other and to purified brain CPD (Table 4Go). The differences between CPE and CPD are similar to those reported previously (24).


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  		Table 4. Effect of inhibitors and pH on enzyme activity (percentage of activity without added inhibitor) in the affinity-purified fractions from the colon of control and Cpefat/Cpefat mice: comparison with purified rat brain CPE and CPD

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study confirm and extend the original report that described the absence of CPE-like activity in the endocrine pancreas of the Cpefat/Cpefat mouse (15). The present study indicates that there is no active CPE in the gastrointestinal tract (stomach, duodenum, and colon) of the Cpefat/Cpefat mouse, as immunoreactive CPE in these tissues of the Cpefat/Cpefat mouse was not detected after purification using a substrate affinity column. CPE does not occur in detectable levels in the duodenum. These tissues show a small amount of CP activity in the affinity column fractions corresponding to CPE; however, the enzymatic properties are not identical to those of CPE. The earlier report indicated that CPE-like activity was marginal, but not eliminated completely, in Cpefat/Cpefat mouse pituitary and pancreatic islets (15). This earlier analysis of CPE-like activity did not consider the possibility that other CPs contributed to the low levels of CPE-like activity in the pancreas and pituitary of Cpefat/Cpefat mice.

Previous to the discovery of CPD (24), CPE was the only described metallocarboxypeptidase with an acidic pH optimum. This is the primary criterion used to distinguish CPE from other CPs (M, N, B, and A) (22). Our results show that a large portion of the CPE-like activity in the antrum and colon is actually CPD, based on the separation of the enzyme activities over the substrate affinity column. Marginal amounts of CPE-like activity are detected in the high pH elutes from the affinity columns of the Cpefat/Cpefat mouse antrum and colon. This activity is not CPE, based on its immunoreactivity and enzyme properties and may represent a novel CP. In any case, this activity is marginal compared to the levels of CPE in control mouse tissues and in control and Cpefat/Cpefat tissues.

The absence of active CPE in the Cpefat/Cpefat mouse results in a substantial reduction in the stomach level of the fully processed gastrin (i.e. COOH-terminal amidated gastrin). As expected, the level of gastrin-Gly-Arg-Arg is elevated dramatically. Whether circulating levels of C-terminal amidated gastrin are reduced with a consequent stomach hypoacidity was not examined in the present study, and it would be intriguing to determine whether Cpefat/Cpefat mice develop hypergastrinemia (C-terminal amidated gastrin) by treatment with acid inhibitory drugs (omeprazole). If omeprazole-treated Cpefat/Cpefat mice develop C-terminal amidated hypergastrinemia not distinguishable from that in control mice, this would suggest that alternate efficient processing pathways can replace the CPE pathway. Progastrin requires a prohormone convertase to cleave at a pair of basic residues, followed by a CP to excise C-terminal basic residues, and then amidation of the C-terminus by peptidylglycine-{alpha}-amidating monooxygenase (9). The levels of the C-terminally extended gastrin precursor were approximately 670-fold higher in the Cpefat/Cpefat mouse stomach than those in control mice. These results are similar to those found previously for proinsulin processing in the pancreatic islets of the Cpefat/Cpefat mouse (15). In islets, C-terminally extended insulin was undetectable in the control mice, but was equal to the level of correctly processed insulin in the Cpefat/Cpefat mouse (15). Recently, CPE activity has been found to be absent in Cpefat/Cpefat mouse brain (27), and the processing of dynorphin (27) and that of neurotensin (28) are altered. Together, these results indicate that the CPE deficiency in the Cpefat/Cpefat mice results in an accumulation of peptides with C-terminal basic residues and a smaller, but significant, decrease in the levels of appropriately processed peptides.

The finding that some C-terminal processing of gastrin continues in the antrum of Cpefat/Cpefat mice suggests an alternative CP exists. CPD may contribute to the processing of these peptides. CPD is in the secretory pathway, based on the studies of the duck homolog of CPD (29, 30) and the recent studies showing that mouse CPD is present in the trans Golgi network (Varlamov, O., L. Song, and L. Fricker, unpublished). It is possible that other CPs present in the secretory pathway may contribute to peptide processing. Alternatively, endopeptidases may excise the peptide precursors to the N-terminal side of the basic residues, giving rise to the C-terminally processed peptide without the need for a CP. Prohormone convertases 1/3 and 2 do not cleave to the N-terminal side of the basic residues within the cleavage site (31, 32); however, other enzyme activities have been reported that can perform this cleavage (33, 34).

Interestingly, gastrin mRNA levels are elevated significantly in the stomach of the Cpefat/Cpefat mouse. This finding suggests that the reduced processing of progastrin to the biologically active form of gastrin, COOH amidated gastrin, triggers an increase in gastrin mRNA transcription in Cpefat/Cpefat mice. The apparent increase in total gastrin-like immunoreactivity in the Cpefat/Cpefat mice (controls, 3409 ng/g; Cpefat/Cpefat, 7903 ng/g) lends support to this idea. In the present study, we did not determine whether the increase in steady state gastrin mRNA levels is due to increased antral gastrin gene transcription rates or to increased mRNA stability. Although speculative, amidated gastrin may regulate its own mRNA expression in a negative fashion by its direct feedback at the stomach on gastrin-producing G cells or on somatostatin-producing D cells. Somatostatin can inhibit gastrin release from the stomach (35), and a decrease in circulating gastrin may decrease the inhibitory input of stomach somatostatin on G cells. Immunoelectron microscopy has shown the presence of the CCK-B (gastrin) receptor on endocrine cells of the guinea pig stomach (36). Parenthetically, the idea that amidated gastrin down-regulates its own mRNA levels can be tested by the administration of exogenous amidated gastrin to Cpefat/Cpefat mice and the monitoring of antral gastrin mRNA levels.


   Acknowledgments
 
The authors acknowledge Karen Martin, Steve Schuenke, Bob Todd, Eileen Figueroa, and Larry Janecka for preparing the manuscript.


   Footnotes
 
1 This work was supported in part by NIDA Grant DA-04494, NIDA Research Scientist Development Award DA-00194, and a grant from the Juvenile Diabetes Foundation (to L.D.F.); a grant from the American Diabetes Foundation and NIH Cancer Center Grant CA-34196 (to E.H.L.); and NIH Grant DK-35608 (to G.H.G.). Back

Received November 20, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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